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An Electron Paramagnetic Resonance Study of HIV-1 Protease and the Development of a Soluble Expression System for Prorenin

Permanent Link: http://ufdc.ufl.edu/UFE0041834/00001

Material Information

Title: An Electron Paramagnetic Resonance Study of HIV-1 Protease and the Development of a Soluble Expression System for Prorenin
Physical Description: 1 online resource (291 p.)
Language: english
Creator: Kear, Jamie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: circular, conformation, dichroism, directed, double, electron, hiv, inhibitor, label, mass, paramagnetic, prorenin, protease, protein, resonance, site, spectrometry, spin, thioredoxin
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF HIV-1 PROTEASE AND THE DEVELOPMENT OF A SOLUBLE EXPRESSION SYSTEM FOR PRORENIN By Jamie Laura Kear August 2010 Chair: Gail E. Fanucci Major: Chemistry All work performed for this dissertation dealt with, in general, the expression, purification, and biophysical characterization of aspartic proteases, specifically HIV-1 protease and the activatable renin zymogen called prorenin. Chapters 1 and 2 describe the relevant biology and methodologies, including pulsed and continuous wave electron paramagnetic resonance spectroscopy. HIV-1 protease is a viral aspartic protease that functions in regulating post-translational processing of the viral polyproteins gag and gag-pol. The enzyme is a dimer comprised of 99 amino acid monomeric subunits. Accessibility of substrate to the active site is mediated by two ?-hairpins called the flaps (one belonging to each monomer). The flaps have been shown to undergo a large conformational change during substrate binding and catalysis; molecular dynamics simulations have captured three distinct conformations of the flaps in HIV-1 protease, namely the closed, semi-open, and wide-open conformations. Reported in this work are results of continuous wave and pulsed electron paramagnetic resonance studies of HIV-1 protease. Continuous wave EPR, though it does not report on the flap motions of the protease, was used to examine the autoproteolytic activity of the protease. The EPR spectral line shape is highly sensitive to mobility in the environment of the spin label, thus it changes dramatically with changes in correlation time. Autoproteolysis affects the rate of global protein tumbling by decreasing rotational correlation time of the spin-labeled protein as a smaller spin-labeled peptide fragment is liberated. As total correlation time decreases, the derivative EPR spectra decrease in breadth and resonance line shapes become sharper and increasingly narrow. The appearance of a sharp component in the high field, proportional to the amount of degraded protein in the sample, was monitored. The intensity of the high field line was quantitatively analyzed to give a term proportional to the amount of uncleaved peptide remaining in the sample. The pulsed technique, called double electron-electron resonance, was utilized to examine the differential flap conformations and flexibility of various HIV-1 protease constructs under various conditions. DEER experiments provided a means to determine distance profiles between two spin-labeled sites in the flaps (sites K55C and K55Caccent acute), which were used to describe and quantify conformational sampling of protease constructs. DEER echo curves were analyzed via Tikhonov Regularization methods and the resulting distance profiles were regenerated using a series of Gaussian-shaped functions, each representative of a distinct flap conformation. Distance profiles from spin-labeled constructs of Subtypes B, C, and F, CRF01_A/E, and drug-resistant patient isolates V6 and MDR769, without ligand were analyzed in order to identify what effect natural and drug-induced polymorphisms have on the conformational ensemble of the protease. The dipolar modulated echo data and resulting distance distribution profiles differed greatly among the apo protease constructs. These results demonstrated that natural and drug-induced polymorphisms in the amino acid sequence of various subtypes and patient isolates alter the average flap conformations and flexibility of the flaps. Additionally, in order to monitor differences in flap conformations upon inhibitor binding between Subtype B and CRF01_A/E proteases, constructs were analyzed upon addition of inhibitors and a non-hydrolysable substrate mimic, CA-p2. These studies yielded interesting results in that the conformational ensembles of the protease differ drastically with the various inhibitors. Renin, also known as angiotensinogenase, is an aspartic protease that plays a vital role in blood pressure regulation by catalyzing the first and rate-limiting step in the activation pathway of its substrate angiotensinogen. The protease cleaves angiotensinogen to form angiotensin I, which is then converted into angiotensin II by angiotensin I converting enzyme in a process known as the renin-angiotensin cascade, which has an important effect on aldosterone release, vasoconstriction, electrolyte imbalance, congestive heart failure, and an increase in blood pressure leading to hypertension. Many hypertension drugs function by regulating blood pressure at various points in the renin-angiotensin system. Prorenin is an inactive zymogen of renin that circulates through the plasma until it reaches the secretory granules, where the pro-segment is cleaved and active renin is released. Currently, very little structural data on prorenin is available in the literature, likely because current methods for recombinant bacterial expression of multiply disulfide bonded aspartic proteases from Escherichia coli have been plagued by difficulty due to expression as inclusion bodies that require denaturation and refolding in order to obtain properly folded, functional protein. Refolding, however, does not ensure that the protein will be both properly folded and active. A bacterial expression system to circumvent these difficulties was developed and work presented herein. Thioredoxin fusion methodology was employed in order to maintain protein solubility and avoid inclusion body formation. The resultant protein was shown to have secondary structure consistent with aspartic protease zymogens and a FRET-based assay was used to demonstrate pH-dependent activation; however, the system was only minimally successful due to low yields and protein instability upon cleavage from the fusion partner thioredoxin.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jamie Kear.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Fanucci, Gail E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041834:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041834/00001

Material Information

Title: An Electron Paramagnetic Resonance Study of HIV-1 Protease and the Development of a Soluble Expression System for Prorenin
Physical Description: 1 online resource (291 p.)
Language: english
Creator: Kear, Jamie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: circular, conformation, dichroism, directed, double, electron, hiv, inhibitor, label, mass, paramagnetic, prorenin, protease, protein, resonance, site, spectrometry, spin, thioredoxin
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF HIV-1 PROTEASE AND THE DEVELOPMENT OF A SOLUBLE EXPRESSION SYSTEM FOR PRORENIN By Jamie Laura Kear August 2010 Chair: Gail E. Fanucci Major: Chemistry All work performed for this dissertation dealt with, in general, the expression, purification, and biophysical characterization of aspartic proteases, specifically HIV-1 protease and the activatable renin zymogen called prorenin. Chapters 1 and 2 describe the relevant biology and methodologies, including pulsed and continuous wave electron paramagnetic resonance spectroscopy. HIV-1 protease is a viral aspartic protease that functions in regulating post-translational processing of the viral polyproteins gag and gag-pol. The enzyme is a dimer comprised of 99 amino acid monomeric subunits. Accessibility of substrate to the active site is mediated by two ?-hairpins called the flaps (one belonging to each monomer). The flaps have been shown to undergo a large conformational change during substrate binding and catalysis; molecular dynamics simulations have captured three distinct conformations of the flaps in HIV-1 protease, namely the closed, semi-open, and wide-open conformations. Reported in this work are results of continuous wave and pulsed electron paramagnetic resonance studies of HIV-1 protease. Continuous wave EPR, though it does not report on the flap motions of the protease, was used to examine the autoproteolytic activity of the protease. The EPR spectral line shape is highly sensitive to mobility in the environment of the spin label, thus it changes dramatically with changes in correlation time. Autoproteolysis affects the rate of global protein tumbling by decreasing rotational correlation time of the spin-labeled protein as a smaller spin-labeled peptide fragment is liberated. As total correlation time decreases, the derivative EPR spectra decrease in breadth and resonance line shapes become sharper and increasingly narrow. The appearance of a sharp component in the high field, proportional to the amount of degraded protein in the sample, was monitored. The intensity of the high field line was quantitatively analyzed to give a term proportional to the amount of uncleaved peptide remaining in the sample. The pulsed technique, called double electron-electron resonance, was utilized to examine the differential flap conformations and flexibility of various HIV-1 protease constructs under various conditions. DEER experiments provided a means to determine distance profiles between two spin-labeled sites in the flaps (sites K55C and K55Caccent acute), which were used to describe and quantify conformational sampling of protease constructs. DEER echo curves were analyzed via Tikhonov Regularization methods and the resulting distance profiles were regenerated using a series of Gaussian-shaped functions, each representative of a distinct flap conformation. Distance profiles from spin-labeled constructs of Subtypes B, C, and F, CRF01_A/E, and drug-resistant patient isolates V6 and MDR769, without ligand were analyzed in order to identify what effect natural and drug-induced polymorphisms have on the conformational ensemble of the protease. The dipolar modulated echo data and resulting distance distribution profiles differed greatly among the apo protease constructs. These results demonstrated that natural and drug-induced polymorphisms in the amino acid sequence of various subtypes and patient isolates alter the average flap conformations and flexibility of the flaps. Additionally, in order to monitor differences in flap conformations upon inhibitor binding between Subtype B and CRF01_A/E proteases, constructs were analyzed upon addition of inhibitors and a non-hydrolysable substrate mimic, CA-p2. These studies yielded interesting results in that the conformational ensembles of the protease differ drastically with the various inhibitors. Renin, also known as angiotensinogenase, is an aspartic protease that plays a vital role in blood pressure regulation by catalyzing the first and rate-limiting step in the activation pathway of its substrate angiotensinogen. The protease cleaves angiotensinogen to form angiotensin I, which is then converted into angiotensin II by angiotensin I converting enzyme in a process known as the renin-angiotensin cascade, which has an important effect on aldosterone release, vasoconstriction, electrolyte imbalance, congestive heart failure, and an increase in blood pressure leading to hypertension. Many hypertension drugs function by regulating blood pressure at various points in the renin-angiotensin system. Prorenin is an inactive zymogen of renin that circulates through the plasma until it reaches the secretory granules, where the pro-segment is cleaved and active renin is released. Currently, very little structural data on prorenin is available in the literature, likely because current methods for recombinant bacterial expression of multiply disulfide bonded aspartic proteases from Escherichia coli have been plagued by difficulty due to expression as inclusion bodies that require denaturation and refolding in order to obtain properly folded, functional protein. Refolding, however, does not ensure that the protein will be both properly folded and active. A bacterial expression system to circumvent these difficulties was developed and work presented herein. Thioredoxin fusion methodology was employed in order to maintain protein solubility and avoid inclusion body formation. The resultant protein was shown to have secondary structure consistent with aspartic protease zymogens and a FRET-based assay was used to demonstrate pH-dependent activation; however, the system was only minimally successful due to low yields and protein instability upon cleavage from the fusion partner thioredoxin.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jamie Kear.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Fanucci, Gail E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041834:00001


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AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF HIV-1 PROTEASE AND
THE DEVELOPMENT OF A SOLUBLE EXPRESSION SYSTEM FOR PRORENIN



















By

JAMIE LAURA KEAR


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010

































2010 Jamie Laura Kear



























To my parents David and Gail Kear and to my brother Sergeant Daniel (Danny) Kear
With a special dedication to my grandfather Norman Kear









ACKNOWLEDGMENTS

I would first and foremost like to thank my parents David and Gail Kear for their endless

encouragement, love, and support, my brother Sergeant Daniel Kear for his services to our

country and just for being the wonderful friend that he is. I would also like to offer a special

thanks to my grandfather and friend Norman Kear for his unconditional love and so many words

of wisdom which I will carry with me forever. Secondly, I need to express my most sincere

gratitude to my advisor and mentor, Doctor Gail E. Fanucci for her patience and encouragement,

for countless opportunities to present my research at national conferences, and for her guidance

and support. I would also like to thank all the other members of my doctoral committee,

Doctors Jon Stewart, Nicole Horenstein, Alex Angerhofer, Distinguished Professor Ben Dunn of

the Department of Biochemistry and Molecular Biology within the University of Florida College

of Medicine, and Maureen Goodenow of the Department of Pathology, Immunology, and

Laboratory Medicine at the University of Florida College of Medicine. I would like to thank

Ben Dunn and Maureen Goodenow for many helpful discussions and for their collaborations

with the HIV-1 Protease project, Alex Angerhofer for the time on the E-580 instrument by which

a substantial portion of the data in this dissertation was obtained, Nicole Horenstein for always

being available when I wanted to chat about science, and Jon Stewart for the opportunity to teach

alongside him for many semesters of Biochemistry Lab, during which time I was given the

University of Florida Graduate Teaching Award. I owe this award, in part, to him.

I would like to thank all past and present members of the Fanucci group for their

friendship, motivation, help and support, particularly Austin Turner, Natasha Pirman, Jeffrey

Carter, Stacey-Ann Benjamin, Ian Mitchelle de Vera, Star Gonzales, Mike Veloro, and former

members Doctors Luis Galiano, Jordan Mathias and Mandy Blackburn. Each of you played a

part in making graduate school much more enjoyable for me and provided me with memories









that will last forever. In addition, thanks go to several undergraduates who studied under me and

helped perform experiments while doing undergraduate research in the Fanucci group, including

Justin Hewlett, Lisette Fred, and Alvancin Louis.

I would like to thank the Department of Chemistry and Biochemistry and the Department

of Biological Sciences at Southern Illinois University Carbondale, where I worked to obtain my

Bachelor of Science (B.S.) degrees in both chemistry and biological Sciences. In particular, I

want to express my deepest gratitude to Doctors Matthew McCarroll and Boyd Goodson. Doctor

McCarroll provided me with the opportunity to do research in his lab as an undergraduate, an

invaluable experience that was greatly appreciated. Doctor Goodson, an absolutely amazing

teacher, cemented my love of chemistry. The two of them were ultimately the reason I chose I

pursue a Ph.D. in Chemistry. I would like to thank the American Heart Association for support

via a 2-year pre-doctoral fellowship, the National Institutes of Health (NIH) Acquired

Immunodeficiency Syndrome (AIDS) Reagents Program for free access to human

immunodeficiency virus type 1 (HIV-1) protease inhibitors, and the National High Magnetic

Field Lab (NHMFL) in-house research proposal (IHRP), the NIH and National Science

Foundation (NSF), and University of Florida (UF) Startup for funding. This work was supported

by NSF MBC-0746533 and ARI DMR-9601864, NIH R37 AI28571, AHA 0815102E, the UF

Center for AIDS Research and NHMFL-IHRP.









TABLE OF CONTENTS


page

A C K N O W L E D G M E N T S ..............................................................................................................4

LIST O F TA BLE S ......... ................. .............. ............................ 11

L IS T O F F IG U R E S ....................................................................................................... .... 15

LIST OF AMINO ACIDS AND AMINO ACID ABBREVIATIONS ......................................23

LIST OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 (HIV-1) PROTEASE (HIV-
1PR) INHIBITORS AND ABBREVIATIONS...................... ... ......................... 24

L IST O F A B B R E V IA T IO N S ............................ ................................................... ...................25

CHAPTER

1 INTRODUCTION TO ASPARTIC PROTEASES.......................................................34

Introdu action to P roteases ............................................................................. .....................34
Introduction to A spartic Proteases............................................ ........................... 35
General Mechanism of Catalysis by Aspartic Proteases ...........................................36
Introduction to H IV -1 P protease ..................................................................... ...................37
H IV as a W world P andem ic .............................................................................. ........ 37
Introduction to H IV -1 ............................................... ... ...... .............. .. 39
H IV -1 V iral L ife C ycle ................................................................. .. .....42
T he H IV -1 V iral G enom e..................................................................... ............... 44
Structure and Function of HIV-1 Protease ................................................. .......... 45
Conformational Sampling and the Conformational Ensemble of the HIV-1 Protease
F lap s ......... .. ........................................................................ .........................50
H ydrophobic Sliding M echanism ...................... ................................. ............... 50
HIV-1 Protease Construct Terminology .................................................................51
H IV -1 Subtype B P protease ..................................................................... ...................52
H IV -1 Subtype F Protease......................................... .. ......................... ............... 53
H IV -1 Subtype C Protease ................................ ...................... ............... 54
HIV-1 Protease Recombinant Form CRF01 A/E ..................................................55
Therapeutic Approaches to HIV-1 Infection and Inhibition of HIV-1 Protease............56
Drug Resistance in Protease Inhibitor-Exposed Patient Isolates of HIV-1 Protease ......62
Multi-drug Resistant Patient Isolate MDR769..................................... ...............64
D rug R esistant Patient Isolate V 6......................................................... ............... 65
Introduction to Prorenin ................. ........................................ .. ....... ... 65
Hypertension and Its Im pact on Society ......... .. ................. ......... .....................65
Renin, Prorenin, and the Renin-Angiotensin System.....................................................66
B iosynthesis and Intercellular Processing ......... ................. ......................................67
Structure and Function of Prorenin ................................. .. .................................69


6









Difficulties in Expression and Purification of Prorenin ...............................................70
Scope of the D issertation ........................................................................................ .......7 1

2 BACKGROUND FOR TECHNIQUES AND METHODOLOGIES ..................................75

Introdu action ........................... ............ ..... ......... ....................... ................. 75
Setting up a Protein Expression System for Escherichia coli .............................................75
Inclusion Body Isolation and Protein R folding ........................................ .....................81
Common Methods of Protein Separation ................................................... ..................83
In tro d u ctio n ..............................................................................8 3
Separation B asked U pon Size ..................... ........................................... ............... 83
Separation Based Upon Charge or Isoelectric Point ................................................. 84
Separation Based Upon Binding Affinity.................................................................85
Circular D ichroism Spectroscopy .............................................. ............................... 86
Site-D directed Spin-Labeling (SD SL)........................................................... ............... 88
In tro d u ctio n ..............................................................................8 8
C choice of Spin L ab el ................... .... ........... .... .............. ................... .................90
Spin Label Conformations and theX4/5 Model for (1-oxyl-2,2,5,5-tetramethyl-A3-
pyrroline-3-methyl)methanethiosulfonate (MTSL).................................................91
Continuous-Wave Electron Paramagnetic Resonance (CW EPR) Spectroscopy...................92
In tro d u ctio n ............................................................................................................... 9 2
N itroxide Spectral Line Shapes ............................................... ............................ 95
Protein R equirem ents for CW EPR ..................................................................... ...... 96
CW EPR D ata A nalysis........................... ............................................................ 96
Pulsed Electron Paramagnetic Resonance (EPR) Spectroscopy ........................................98
In tro d u ctio n ..............................................................................9 8
Phase M em ory Tim e, Tm .......................... .. .... ............................................... 102
Protein Requirements for Pulsed EPR Experiments ............................................. 104
Analysis of Double Electron Electron Resonance (DEER) Data...............................104
D irect F ourier tran sform .............................................................. ..................... 105
Curve fitting and Monte Carlo analysis ...................................... ............... 105
T ikhonov regularization ........................................... ........................................ 106
Z ero-tim e selection ......... ............................................................ .. .... ..... .. 108
Self-consistent analysis ................. .......................... ................ ............. 109
Interpretation of distance distribution profiles ...................................................... 110
Gaussian reconstruction process ................................................... .................111
Error analysis by population suppression and validation...................................112

3 CONTINUOUS WAVE ELECTRON PARAMAGNETIC RESONANCE STUDIES
O F H IV -1 P R O T E A SE .............................................................................. ..................... 114

In tro d u ctio n ........... ...... ... ............. ... .............................................................1 14
M materials and M methods ................ ........ ......................................................... 119
M materials ........................................................................................ ... .....................119
M methods .................... ..................................................................... ................... 120
Cloning of H IV -1 protease .......................... .................... .... ................... 120
Site-directed mutagenesis of HIV-1 protease constructs ............ ... ................121


7









Expression of HIV-1 protease constructs................ .......... ...................125
D details of protease constructs...................................... ......... ............ ... 125
H IV -1 protease purification buffers ............................................ ............... 126
H IV -1 protease purification .............................................................................128
S p in -la b e lin g .................................................................................................... 1 3 2
Circular dichroism spectroscopy ........................................... ......... ... ............... 133
Sample preparation for EPR data collection .................................. ............... 134
C W E P R m easurem ents ........................................... ........................................ 135
Mass spectrometry experiments ................................. ............... 136
R results and D discussion ............................. .......... .. ............ ........ ............... 137
Affect of Inhibitors on CW EPR Line Shapes of HIV-1PR Subtype F and
C R F 0 1 A /E .................................... ......... .... ................... ....................... 137
Monitoring the Autoproteolysis of HIV-1 Protease by SDSL EPR and Mass
S p e ctro m e try ........................................................................................................ 1 3 9
Conclusions........................ ....... ..................... ...............146
Affect of Inhibitors on CW EPR Line Shapes of HIV-1PR Subtype F and
C R F 0 1 A /E .................................... ......... .... ................... ....................... 146
Monitoring the Autoproteolysis of HIV-1 Protease by SDSL EPR and Mass
Sp ectrom etry ...................................... .............................................. 14 7

4 PULSED ELECTRON PARAMAGNETIC RESONANCE STUDIES OF HIV-1
P R O T E A S E ................................ .......................................................15 4

In tro d u ctio n ........................ ................... .. ...................................................................... 1 5 4
P reviou s W ork ................................156.............................
M materials and M methods ........................... .......................... .... .... ......... ......... 161
M materials ................................161.............................
M eth o d s ............ .... ........................ .. ...................................................1 6 2
D details of protein constructs ........................................................ ............. 162
Expression of H IV -1 protease ..................................................... ...... ......... 164
Purification of H IV -1 protease ......... ............................................ ................. 164
S p in -la b e lin g .................................................................................................... 1 6 4
B uffer require ents .. ........................................................... ..................... 165
Circular dichroism spectroscopy ............................ ........................ .............. 165
D E E R experim ents ......... ............................. .......... .......... ......... 166
D E E R data analysis ........... .......................................................... .. .... ..... .. 166
P population v alidation ..................................................................... ....... ............ 167
Results and D discussion ................... ..................... ............... ......................... 167
Subtype Polymorphisms Found Among Subtypes B, C, F, CRF01_A/E and Patient
Isolates V6 and MDR769 Confer Altered Flap Conformations and Flexibility in
the Apo Protease .................................... ..... .. ...... .. ........... 167
Introduction ............... ......... .................. 167
Zero-tim e selection.............. ................................................ .................... ......168
Background subtracted dipolar modulated echo curves..................... ........169
Data analysis and population validation process: Subtype Bsi.............................171
Data analysis and population validation process: Subtype Csi............ ...............173
Data analysis and population validation process: Subtype Fsi .............................175


8









Data analysis and population validation: CRF01 A/E .......................................177
Data analysis and population validation: V6i ........... ......... .. ......... ..179
Data analysis and population validation: MDR769i........................... ...........181
Polymorphism-induced shifts in the conformational ensemble ........................183
Inhibitor-Induced Flap Closure in CRF01_A/E Constructs.............................. 188
CRF01_A/Esi dipolar modulation zero-time determination ..............................188
CRF01_A/Esi with CA-p2 substrate.................... ............... 190
CRF01_A/Esi with Nelfinavir (NFV)...... ...................... ...............192
CRF01_A/Esi with Tipranavir (TPV) ........................ ............... 194
CRF01_A/Esi with Lopinavir (LPV) .......................... ...... ................. 196
CRF01_A/Esi with Saquinavir (SQV)........................... ................................198
CRF01_A/Esi with Atazanavir (ATV)...................... ............................. ............ 200
CRF01_A/Esi with Darunavir (DRV) ........ ............................. 202
CRF01_A/Esi with Amprenavir (APV).........................................................204
CRF01_A/Esi with Ritonavir (RTV) ......................... ......... ............... 206
CRF01_A/Esi w ith Indinavir (ID V)......................................... ......................... 208
A comparison of distance profiles from CRF01_A/Esi with various inhibitors.....209
Inhibitor-Induced Flap Closure in Subtype Fsi ................................................ 211
Subtype Fsi dipolar modulation zero-time determination ............... ...............211
Subtype Fsi w ith R TV .................................................. ............................... 213
Subtype Fsi w ith ID V ....................... ................ ................... ........ 215
Subtype F si w ith LPV ................................ ................ ................ ............. 2 17
Subtype Fsi w ith TPV .................. .......................... .... .... ... ........ .... 219
Subtype F si w ith SQ V ............. ...................................................... 22 1
Subtype F si w ith D R V ........................................ .............................................223
Subtype Fsi w ith N FV .......................................................................... 225
Subtype Fsi w ith A TV .......................................................................... 227
Subtype Fsi w ith A PV ......... ................. ................... .................. ............... 229
Subtype Fsi w ith CA -p2 ...... ........ ..... ....... ...... .... ....... ............................. 231
A comparison of distance profiles from Subtype F with various inhibitors ..........233
C onclu sions..... ..................................... .............................................233

5 SOLUBLE EXPRESSION AND PURIFICATION OF MULTIPLY DISULFIDE
BONDED PROTEINS FROM ESCHERICHIA COLI..................................................... 235

In tro du ctio n ................... ...................2.............................5
Materials and Methods ...............................239
M materials and M methods .......... ................................................................239
M eth o d s ...............................................................................2 4 0
Cloning of prorenin ...... ....... ........... ...... ............... .... ...... .. ...... .. 240
Expression of prorenin-thioredoxin fusion construct...........................................243
Harvesting of cells and collection of soluble protein...........................................244
Purification of fusion construct, gel electrophoresis, and protein concentration
estim ates ........................................... ................... ..................... 2 4 4
HiTrapTM Chelating HP affinity chromatography................................................244
HiTrapTM Q HP anion exchange chromatography ...........................................245
Buffer exchange by HiPrepTM desalting column................................................. 245


9









Concentrating of protein sam ples...................................... ......................... 245
Cleavage of Fusion Construct ............ ........................ .. .. ... ............ 246
Quenching Factor Xa reaction with Novagen Xarrest agarose ...........................246
Purification of prorenin from Factor Xa protease and thioredoxin........................247
Circular dichroism spectroscopy .................................... .................................. 247
A ctivation of prorenin .................................... ............ ................ .....................247
P rorenin activ ity assay ........................................ .............................................24 7
R results and D discussion .............. .................................................... ............ .... .. 248
Sub-cloning of Prorenin Gene into pET32a Expression Vector ................................248
Over-expression and Purification of Prorenin-thioredoxin Fusion Construct.............248
Enzym atic Rem oval of Thioredoxin ........................................ ........................ 252
Evidence of Proper Folding ........................ ......... ......................... ............... 253
Renin Activity Measurements of pH-activated Prorenin ...........................................254
Cysteine Mutagenesis of Prorenin Deoxyribonucleic Acid (DNA) for Possible EPR
Studies......................... ... ............ ............... ............ 255
Expression and Purification of V28C Mutant Prorenin ............................ ..............257
C onclu sions..... ................................... ..............................................257

6 CONCLUSIONS AND FUTURE DIRECTIONS .................................... ...............259

C o n clu sio n s............................................................................. .2 5 9
Future D directions ............................................. ............. ............................. ......... 261
A Site-Directed Spin-labeling Approach to Studying HIV-1 Protease.........................261
Recombinant Bacterial Expression and Biophysical Characterization of the
A spartic A cid Zym ogen Prorenin ........................................ ......................... 262

APPENDIX

A PRORENIN DNA AND AMINO ACID SEQUENCE............................. ..................264

B YEAST PROTEINASE A DNA AND AMINO ACID SEQUENCES .............................270

C HIV-1 PROTEASE DNA AND AMINO ACID SEQUENCES .............. ... ................272

Subtype F C construct Sequences................................................................. .....................272
CRF01_A/E Construct Sequences ......................................................... ............... 275

D A SOLUBLE EXPRESSION SYSTEM FOR GM2 ACTIVATOR PROTEIN ..................277

L IST O F R E F E R E N C E S ......... ..... ............ ................. ..........................................................2 80

B IO G R A PH IC A L SK E T C H ............................................................................. ....................291









LIST OF TABLES


Table page

1-1 Representative aspartic proteases by name, function, source, size, and structure..................36

1-2 Primary geographic prevalence of subgroups of human immunodeficiency virus type 1
(H IV -1) ......................................................... ..................................4 1

1-3 HIV-1 protease (HIV-1PR) polyprotein processing sites .................................................46

1-4 HIV-1PR construct abbreviations and descriptions...................... ...................... 52

1-5 Food and Drug Administration (FDA)-approved drugs for the treatment of HIV.................57

1-6 FDA approved protease inhibitors for HIV-1 highly active antiretroviral therapy
(H A A R T) treatm ent. .......................... ........................... .... ........ ......... 58

1-7 Classifications of hypertension and respective systolic and diastolic pressure ranges ..........66

1-8 Pro-peptide sequences of several aspartic protease zymogens ............................... ....68

2-1 Differential codon usage in Homo sapiens and Escherichia coli (E. col)i cells. .................76

2-2 Common microwave bands and frequencies used in continuous wave (CW) electron
paramagnetic resonance (EPR) spectroscopy. ...................................... ............... 95

2-3 Standard pulse table used for double electron electron resonance (DEER) experiments. ...100

2-4 Standard pulsed EPR parameters used in this study ...................................................... 101

2-5 Typical parameters used for the echo decay experiment for determination of Tm...............103

3-1 Polymerase chain reaction (PCR) primers utilized to introduce mutations to HIV-1PR
C R F 0 1 A /E ......................................................................... 12 2

3-2 PCR primers utilized to introduce mutations to HIV-1PR subtype F. .............................122

3-3 Thermal cycling parameters for HIV-1PR site-directed mutagenesis reactions ................122

3-4 E. coli codon-optimized HIV-1PR Subtype Fsi K45C deoxyribonucleic acid (DNA) and
am ino acid sequences .................. ......................................... ... ...... .... 122

3-5 E. coli codon-optimized HIV-1PR Subtype FsiK55C DNA and amino acid sequences......123

3-6 E. coli codon-optimized HIV-1PR Subtype FsK55C DNA and amino acid sequences. .....123

3-7 E. coli codon-optimized HIV-1PR CRF01 A/Esi K55C DNA and amino acid
sequ en ces. ............................................................................ 12 4









3-8 E. coli codon-optimized HIV-1PR A/E, K55C DNA and amino acid sequences...............124

3-9 L uria B ertani (L B ) M edia .............................................................................................. 125

3-10 HIV-1PR purification buffers ....................................................................... 127

3-11 Standard parameters used for circular dichroism (CD) experiments ..............................133

3-12 Standard CW EPR parameters used in this study.................................136

3-13 CW EPR data analysis for Subtype Fsi K55MTSL.......................... ... ............... 138

3-14 CW EPR data analysis for A/Esi K55MTSL ............................. ....................... 139

4-1 Comparison of relative percentage of closed flap conformation of HIV-1PR subtype B
to published values of Ki, KD, and the number of non-water-mediated hydrogen ..........160

4-2 HIV-1PR variant sequence alignment residues 1-50.................................... ............... 163

4-3 HIV-1PR variant sequence alignment residues 51-99 ........................................................ 163

4-4 Zero-tim es chosen for apo data analysis........................................ ........................... 169

4-5 Values of signal:noise ratios (SNRs) for background subtracted echo data.......................170

4-6 Results of Gaussian reconstruction and population validation procedures for Bsi. .............172

4-7 Results of Gaussian reconstruction and population validation procedures for Csi. .............174

4-8 Distance distribution profile for apo HIV-1PR Fsi. .................................. ............... 176

4-9 Distance distribution profile for HIV-1PR A/Esi ........................................... ...............178

4-10 Distance distribution profile for HIV-1PR V6i. ...................................... ............... 180

4-11 Distance distribution profile for HIV-1PR MDR769i................................................... 183

4-12 Summary of distance parameters obtained from DEER distance profiles of HIV-1PR.....185

4-13 Zero-times chosen for CRF01_A/Esi data analysis............................................... 189

4-14 Distance distribution profile for CRF01_A/E with CA-p2. .............................................191

4-15 Distance distribution profile for CRF01_A/E Nelfinavir (NFV). ......................................193

4-16 Distance distribution profile for CRF01_A/E with Tipranavir (TPV) .............................195

4-17 Distance distribution profile for CRFO1_A/E Lopinavir (LPV). .......................................197

4-1 Distance distribution profile for CRFO1_A/E Saquinavir (SQV). .......................................199









4-19 Distance distribution profile for CRFO1_A/E Atazanavir (ATV). .................................201

4-20 Distance distribution profile for CRF01_A/E Darunavir (DRV).............. .................203

4-21 Distance distribution profile for CRF01_A/E Amprenavir (APV). ..................................205

4-22 Distance distribution profile for CRF01_A/Esi with Ritonavir (RTV)........................207

4-23 Distance distribution profile for CRF01_A/Esi with Indinavir (IDV). ............................209

4-24 Zero-tim es chosen for F i data analysis. ........................................................ ........... 211

4-25 Distance distribution profile for Subtype Fsi with RTV. .........................................214

4-26 Distance distribution profile for Subtype Fsi with IDV. ..........................................216

4-27 Distance distribution profile for Subtype Fsi with LPV..................................218

4-28 Distance distribution profile for Subtype Fsi with TPV.......................220

4-29 Distance distribution profile for Subtype Fsi with SQV. .........................................222

4-30 Distance distribution profile for Subtype Fsi with DRV ..........................................224

4-31 Distance distribution profile for Subtype Fsi with NFV. .........................................226

4-32 Distance distribution profile for Subtype Fsi with ATV. .........................................228

4-33 Distance distribution profile for Subtype Fsi APV. .......................................230

4-34 Distance distribution profile for Subtype Fsi with CA-p2. ..............................................232

5-1 Prorenin DNA sequence with restriction sites, stop codons, and Factor Xa cutsite.............240

5-2 Prorenin am ino acid sequence. ..................................................................... ..................246

5-3 Secondary structural data for prorenin ...........................................................................253

5-4 PCR Primers utilized to introduce mutations to prorenin.................................256

A-1 E. coli codon-optimized prorenin sequence with Factor Xa cutsite ..................................264

A-2 E. coli codon-optimized prorenin T7C sequence with Factor Xa cutsite..........................265

A-3 E. coli codon-optimized prorenin F8C sequence with Factor Xa cutsite ..........................266

A-4 E. coli codon-optimized prorenin L13C sequence with Factor Xa cutsite..........................267

A-5 E. coli codon-optimized prorenin V28C sequence with Factor Xa cutsite..........................268









A-6 E. coli codon-optimized prorenin G35C sequence with Factor Xa cutsite..........................269

B-1 E. coli codon-optimized Pro-YPRA D215N sequence with Factor Xa cutsite...................270

C-1 E. coli codon-optimized HIV-1 Protease subtype Fsi K45C sequence .............................272

C-2 E. coli codon-optimized HIV-1 Protease subtype Fs K45C sequence.............................273

C-3 E. coli codon-optimized HIV-1 Protease subtype Fsi K55C sequence .............................273

C-4 E. coli codon-optimized HIV-1 Protease subtype Fs K55C sequence...............................273

C-5 E. coli codon-optimized HIV-1 Protease subtype Fsi T74C sequence .........................273

C-6 E. coli codon-optimized HIV-1 Protease subtype Fs T74C sequence..............................274

C-7 E. coli codon-optimized HIV-1 Protease subtype Fsi sequence........................................274

C-8 E. coli codon-optimized HIV-1 Protease subtype Fs sequence...................................274

C-9 E. coli codon-optimized HIV-1 Protease subtype A/Esi K55C sequence...........................275

C-10 E. coli codon-optimized HIV-1 Protease subtype A/Es PMPR K55C sequence .............275

C-11 E. coli codon-optimized HIV-1 Protease subtype A/Esi T74C sequence.........................275

C-12 E. coli codon-optimized HIV-1 Protease subtype A/Es T74C sequence .........................276

C-13 E. coli codon-optimized HIV-1 Protease subtype A/Esi sequence..............................276

C-14 E. coli codon-optimized HIV-1 Protease subtype A/Es sequence ...................................276









LIST OF FIGURES


Figure page

1-1 Scheme of hydrolysis of a polypeptide chain............... .. .......................... 34

1-2 Generally accepted mechanism of catalysis by aspartic proteases ......................................36

1-3 Map showing percent of adult population living with human immunodeficiency virus
(H IV ) infection in 2007. .......................... ...... ................................... .. .....38

1-4 Cartoon representation of the structure of the HIV-1 virus............................... ...............39

1-5 Phylogenic tree for HIV-1 and HIV-2, groups, subtypes, and CRFs ...................................40

1-6 Cartoon representation of the HIV-1 life cycle. ........................................ ............... 43

1-7 Cartoon representation of the HIV-1 viral genome..................................... .................44

1-8 Structure of Subtype B HIV-1 Protease. ........................................ .......................... 45

1-9 Ribbon diagram of HIV-1 protease (HIV-1PR) bound to the non-hydrolyzable substrate
m im ic C A -p2. .............................................................................47

1-10 Structures of norleucine and methionine. ........................................ ......................... 47

1-11 HIV-1PR nuclear Overhauser effect (NOE) values of liganded and unliganded HIV-
1P R ................... .............................................................. ................ 4 8

1-12 Flap conformations of HIV-1PR captured by molecular dynamics (MD) simulations........49

1-13 Crystal structure of H IV -1PR Subtype F....................................................................... 53

1-14 Crystal structure of Subtype C HIV-1PR. ........................................ ........................ 54

1-15 Graphical description of the circulating recombinant form CRF01_A/E .............................55

1-16 Points of inhibition within the HIV-1 viral life cycle ............................... .....................56

1-17 Regions of amino acid conservation in protease inhibitor naive exposed HIV-1PR. ..........63

1-18 Crystal structure of HIV-1PR multi-drug resistant patient isolate MDR769 ....................64

1-19 Renin-angiotensin system (RAS) and the sites of inhibition within the RAS ....................67

1-20 Crystal structures of aspartic proteases renin, pepsin, and pepsinogen ............................ 69

2-1 Example vector map highlighting necessary features of plasmid. .........................................80









2-2 Structures of lactose and the lactose analog isopropyl-P-D-1-thiogalactopyranoside
(IPTG) ....................................................... 81

2-3 Internal structures of prokaryotic and eukaryotic cells. ................................. ............... 82

2-4 Structure of the detergent sodium dodecyl sulfate (SDS). ...........................................84

2-5 Anion exchange Q-resin bound to a positively charged quaternary amine..........................85

2-6 Structures of nitrilotriacetic acid and iminodiacetic acid. ................... ................... .......... 85

2 -7 E elliptical p olarized light.. ............................................................................ ....................86

2-8 Sample circular dichroism spectra for a-helix, P-sheet, and random coil.............................87

2-9 Site-directed spin-labeling (SDSL) scheme for (1-oxyl-2,2,5,5-tetramethyl-A3-
pyrroline-3-methyl) methanethiosulfonate (MTSL) addition to a thiol group of a CYS
residue ........................................................ 89

2-10 Structures of MTSL and other common nitroxide spin labels...........................................90

2-11 Electron paramagnetic resonance (EPR) spectral line shapes of HIV-1PR Subtype Bsi
w ith various s spin labels........... .... .......................................... .............. .......... .. 9 1

2-12 Graphical representation of the X4/X5 model for the MTSL label............................ 92

2-13 Energy level diagram for a free electron in an applied magnetic field and
corresponding spectral line shape. ............................................. ............................ 94

2-14 Energy diagram for a system with a free electron undergoing hyperfine interaction
with the nucleus of nitrogen, and a representative spectral line shape. ...........................94

2-15 Dependence of EPR spectral line shape on motion................................... .................95

2-16 Common spectral parameters AHpp, ILF, ICF, and IHF, and second moment........................98

2-17 The 4-pulse double electron electron resonance (DEER) sequence...................................99

2-18 Absorption spectra for a nitroxide spin-label with positions of the low field transition
marked as the "observe" frequency and the center field transition marked as the
"pum p" frequency.. ......................... .......................... .. .... ...................100

2-19 Sample dipolar evolution curve showing locations of raw dipolar modulation,
background subtraction, and background-subtracted echo curve with fit .....................102

2-20 Typical results of an echo decay experiment using a 2-pulse Hahn echo sequence for
determ in action of T m ....... ........................................................... ........................... 103

2-21 Pictorial representation of the direct Fourier transform method of analysis .................... 105









2-22 Pictorial representation of the M onte Carlo analysis ........................................................ 106

2-23 Selection of regularization parameter in Tikhonov regularization (TKR) DEER
an aly sis m eth od .................................. ................................................... ............... 107

2-24 Example of zero-time selection for dipolar modulated echo data............... ................. 108

2-25 Self-consistent analysis scheme e................................................................................... 110

2-26 Semi-quantitative analysis of distance distribution profiles.................... ................111

2-27 Quantitative description of distance distribution profiles using Gaussian reconstruction
p ro ced u re ...................................... .................................................... 1 12

2-28 Example of population suppression error analysis ................................. .... .................. 113

3-1 Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gel of self-
cleavage products for Subtype B with stabilizing mutations and V6 and MDR769
w ith no stabilizing m stations. ....................... ....... ........ ................................. 115

3-2 R ibbon diagram of H IV -1PR structure ............................ ...............................................116

3-3 Overlay of day 1 and day 47 CW EPR spectra for Subtype B HIV-1 protease. ................ 118

3-4 X-band CW EPR spectra of 100 tM HIV-1PR as a function of salt concentration............19

3-5 Vector maps of pJ201:24237 and pJ201:24236. ...................................... ............... 121

3-6 Decomposition of urea; with heat and time, urea degrades into ammonium and cyanate...128

3-7 Thermo brand 35 mL French pressure cell and Fisher Scientific brand tip sonicator..........128

3-8 Typical anion exchange chromatogram for purification of HIV-1PR..............................130

3-9 Typical size exclusion chromatogram of HIV-1PR on S-100 column.............................131

3-10 SDS PAGE gel showing purity of HIV-1PR at various steps in the purification..............132

3-11 Temperature control set-up for CW EPR experiments.................................. ... ............... 135

3-12 CW EPR nitroxide spectral line shapes for HIV-1PR Subtype Fsi K55MTSL ................137

3-13 CW EPR nitroxide spectral line shapes for HIV-1PR CRF_01A/Esi K55MTSL ...............138

3-14 Circular dichroism spectra for spin labeled HIV-1PR, subtypes B, F, CRF01_A/E..........141

3-15 Overlay of day 1 and day 30 CW EPR spectra for subtype Fs HIV-1PR stored at 37 C,
25 C and 4 C, and CRF01 A/Es HIV-1PR stored at 37 C, 25 C, and 4 C. ............142









3-16 Mole fraction of degraded protein (Xb) vs. time (days) for CRF01_A/E and Subtype F
protease for samples stored at 37 C, 25 C, and 4 C. ................................................144

3-17 CW EPR spectra of HIV-1PR subtype F with tipranavir over the course of 30 days........145

3-18 CW EPR spectra of HIV-1PR CRF01_A/E with CA-p2 over the course of 30 days. .......145

3-19 Mass spectrum for HIV-1PR CRF01_A/E K55MSL.................................... ...............149

3-20 Mass spectrum for HIV-1PR CRFO1_A/E K55MSL ................................... ...............150

3-21 Mass spectrum for HIV-1PR subtype Fs K55MSL...................................151

3-22 Mass spectrum for HIV-1PR CRF01 A/E K55MSL.................................... ...............152

3-23 Sites of autoproteolytic cleavage in HIV-1PR subtype Fs K55MSL...............................153

3-24 Sites of autoproteolytic cleavage in HIV-1PR CRF01 A/E K55MSL ..............................153

4-1 Ribbon diagrams showing HIV-1PR in the closed and semi-open flap conformations ......154

4-2 DEER results of subtype B HIV-1PR with and without Ritonavir ................................ 156

4-3 Distance distribution profiles of subtype B HIV-1PR with inhibitors ..............................159

4-4 Ribbon diagrams of HIV-1PR with amino acid differences relative to subtype B. ..............163

4-5 Circular dichroism spectra for spin labeled HIV-1 PR ............... .............. ..................... 166

4-6 Dipolar modulated echo curves used for zero-time selection of Subtype Bsi, Csi,Fsi,
CRF01 A/Esi, V6i, and M DR769i. ........................................... ........................... 168

4-7 Background subtracted time-domain echo data for Subtypes B, C, F, CRF01_A/E and
M DR769 and V6, with fits generated by TKR.......................................................... 170

4-8 Data analysis for HIV-1PR subtype Bsi apo.. ................................................................171

4-9 Population validation process for HIV-1PR subtype Bsi apo.. .........................................172

4-10 Data analysis for HIV-1PR Subtype Csi apo........................................... ............... 173

4-11 Population validation process for HIV-1PR subtype Csi apo.. .......................................174

4-12 Data analysis for HIV-1PR Subtype Fsi apo......................................... ............... 175

4-13 Population validation process for HIV-1PR subtype Fsi apo ..........................................176

4-14 Data analysis for HIV-1PR CRF01_A/Esi apo.. ..................................... ............... 177









4-15 Population validation process for HIV-1PR CRF01_A/Esi apo.. ...................................178

4-16 Data analysis for HIV-1PR patient isolate V6i apo..................................... ............... 179

4-17 Population validation process for HIV-1PR V6i apo.. ................................................. 180

4-18 Data analysis for HIV-1PR patient isolate MDR769i apo.. ...........................................181

4-19 Population validation process for HIV-1PR MDR769i apo .................... ................182

4-20 DEER results for apo HIV-1PR subtypes B, C, F, CRF01_A/E, V6, MDR769 ...............184

4-21 Relative percentage of conformations of protease constructs in the apo form ................... 184

4-22 Relative percentage of conformations for apo protease constructs ..................................185

4-23 Distance distribution profile overlays of subtype B with each other proteae construct.....186

4-24 Derivative spectra and second derivative spectra of distance profiles for apo protease. ...187

4-25 Truncated dipolar modulated echo curves used for zero-time selection of CRF01_A/E
w ith inhibitors. .................................................................189

4-26 Data analysis for HIV-1PR CRF01_A/E with CA-p2................................... ................190

4-27 Population validation process for HIV-1PR CRF01_A/E with CA-p2.............................191

4-28 Data analysis for HIV-1PR CRF01_A/E NFV........................... ..................... 192

4-29 Population validation process for HIV-1PR CRF01_A/E NFV............... ...............193

4-30 Data analysis for HIV-1PR CRF01_A/E TPV .............................................. 194

4-31 Population validation process for HIV-1PR CRF01_A/E TPV. .............. ................... 195

4-32 Data analysis for HIV-1PR CRF01_A/E LPV................ ..................196

4-33 Population validation process for HIV-1PR CRF01_A/E LPV.. .............................197

4-34 Data analysis for HIV-1PR CRF01_A/E SQV......... .......................................198

4-35 Population validation process for HIV-1PR CRF01_A/E SQV......................................199

4-36 Data analysis for HIV-1PR CRF01_A/E ATV........................................ ............... 200

4-37 Population validation process for HIV-1PR CRF01_A/E ATV .............. ...... ..........201

4-38 Data analysis for HIV-1PR CRF01_A/E DRV.. ..............................................................202

4-39 Population validation process for HIV-1PR CRF01_A/E DRV.....................................203









4-40 Data analysis for HIV-1PR CRF01_A/E APV..... ...................... ............204

4-41 Population validation process for HIV-1PR CRF01_A/E APV .............................205

4-42 Data analysis for HIV-1PR CRF01_A/Esi with RTV. ................ .. ............. 206

4-43 Population validation process for HIV-1PR CRF01_A/Esi with RTV..............................207

4-44 Data analysis for HIV-1PR CRF01_A/Esi with IDV.............. ..... ..................208

4-45 Population validation process for HIV-1PR CRF01_A/E........................................209

4-46 Overlay of distance distribution profiles for apo HIV-1PR CRF01_A/Esi and with
inhibitors ...............................................................................2 10

4-47 Distance distribution profiles of HIV-1PR CRF0 _A/Esi with inhibitors .........................210

4-48 Population analysis for HIV-1PR CRF01_A/E....................................... ............... 210

4-49 Truncated dipolar modulated echo curves used for zero time selection of HIV-1PR
CRF01 A/E with inhibitors. ...... ........................... .........................................212

4-50 Data analysis for HIV-1PR subtype Fsi with RTV ......................... ...............213

4-51 Population validation process for HIV-1PR Fsi with RTV.....................................214

4-52 Data analysis for HIV-1PR subtype Fsi with IDV. ...................................... ...........215

4-53 Population validation process for HIV-1PR subtype Fsi with IDV .............................216

4-54 Data analysis for HIV-1PR subtype Fsi with LPV........................ ...... ..............217

4-55 Population validation process for HIV-1PR Fsi with LPV ...........................................218

4-56 Data analysis for HIV-1PR subtype Fsi with TPV........................................................219

4-57 Population validation process for HIV-1PR Fsi with TPV.. ................. ......................220

4-58 Data analysis for HIV-1PR subtype Fsi with SQV.. ............................... ....................221

4-59 Population validation process for HIV-1PR Fsi with SQV ..................... ....................222

4-60 Data analysis for HIV-1PR subtype Fsi with DRV .............................. .....................223

4-61 Population validation process for HIV-1PR subtype F with DRV..................................224

4-62 Data analysis for HIV-1PR subtype Fsi with NFV.. ........................................ ...............225

4-63 Population validation process for HIV-1PR Fsi with NFV.............................................226









4-64 Data analysis for HIV-1PR subtype Fsi with ATV............ .................... .................227

4-65 Population validation process for HIV-1PR Fsi with ATV .............................................228

4-66 D ata analysis for H IV -1PR Fsi w ith APV ............... ...... .........................................229

4-67 Population validation process for HIV-1PR Fsi with APV ......................... ............230

4-68 Data analysis for HIV-1PR subtype Fsi with CA-p2.. .................................................231

4-69 Population validation process for HIV-1PR subtype Fsi with CA-p2. ............................232

4-70 Population analysis for subtype Fsi. ......................................................... .....................233

5-1 Crystal structure of hum an renin. ............................................... .............................. 236

5-2 Ribbon diagram showing x-ray structure of oxidized thioredoxin.............................. 239

5-3 pJ2:G02057 storage vector in which prorenin DNA was obtained............... .................241

5-4 pE T -32a(+ ) vector m ap .. .......................................................................... ......................242

5-5 1% agarose gel used to confirm purity of pET32a XaPR plasmid................................ 243

5-6 Domain diagram of the prorenin-thioredoxin fusion construct........................................243

5-7 Pilot expression of prorenin-thioredoxin fusion construct in BL21(DE3) strain
E scherichia coli cells .. ........................ ...... .........................................249

5-8 Pilot expression of prorenin-thioredoxin fusion construct in OrigamiB(DE3) strain E.
co li cells. ....................................................................................... 2 4 9

5-9 Typical chromatogram from HiTrapTM Chelating HP affinity column.............................250

5-10 Typical chromatogram from HiTrapTM Q HP anion exchange column.. ........................251

5-11 SDS-PAGE gel demonstrating purity of prorenin-thioredoxin fusion construct
following sequential chromatographic steps .... ........... ........ ........................... 251

5-12 SDS-PAGE gel demonstrating purity of prorenin-thioredoxin fusion construct and
prorenin after separation from thioredoxin......................................... ............... 252

5-13 Circular dichroism spectrum of prorenin after liberation from thioredoxin.......................253

5-14 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL) absorption overlaps
with the 5-((2-aminoethyl)amino)naphthalene-l-sulfonic acid (EDANS) fluorescence
thereby quenching the fluorescence through fluorescence resonance energy transfer.. ..254

5-15 Renin Substrate I Activity screening resuts.................................. ......................... 255









5-16 SDS-PAGE gel showing purity of V28C mutant following anion exchange
chromatography.. ...................... ........ .. ... .. .. ...... ............ 257

B-l Map of pJ201 :19864 with D215N Pro-Yeast proteinase A insert (red) ........................271

D-1 Pilot expression of prorenin GM2 activator protein (GM2AP)-thioredoxin fusion
construct in OrigamiB(DE3) strain E. coli cells.. ..................................................... 277

D-2 Typical chromatogram for GM2AP-thioredoxin fusion construct using 5 mL Ni-
charged HiTrap HP affinity column. ........................................ ......................... 278

D-3 SDS-PAGE gel showing in purity of GM2AP-trx fusion construct following
p u rific atio n ........................................................................... 2 7 8

D-4 Results of a fluorescence resonance energy transfer (FRET)-based functional assay for
GM 2AP function ............... ................. ........................ ............. 279









LIST OF AMINO ACIDS AND AMINO ACID ABBREVIATIONS

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic Acid Asp D

Cysteine Cys C

Glutamic Acid Glu E

Glutamine Gin Q

Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V









LIST OF HIV-1 PROTEASE INHIBITORS AND ABBREVIATIONS

APV Amprenavir

ATV Atazanavir

CA-p2 Capsid-p2 substrate mimic

DRV Darunavir

FPV Fosamprenavir

IDV Indinavir

LPV Lopinavir

NFV Nelfinavir

RTV Ritonavir

SQV Saquinavir

TPV Tipranavir









LIST OF ABBREVIATIONS

(+)mRNA plus-sense messenger RNA

ACE Angiotensin Converting Enzyme

AIDS Acquired Immunodeficiency Syndrome

All Angiotensin II

AmpR ampicillin resistance

AP post-acidification pellet

ARB Angiotensin Receptor Blockers

ARV antiretroviral drugs

AS post-acidification supernatant

AT adenine-thymine

BEV baculovirus

BME P-Mercaptoethanol, 2-Mercaptoethanol

bp base pair

B.S. bachelor of science

BSA bovine serum albumin

CD circular dichroism

CD4 Cluster of Differentiation 4

CHO Chinese hamster ovary

CID collision induced dissociation

CRF circulating recombinant form

CW continuous wave

DABCYL 4-(dimethylaminoazo)benzene-4-carboxylic acid

dB decibel










DEER double electron-electron resonance

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid

Dr. doctor

E. coli Escherichia coli

EDANS 5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid

El entry inhibitor

Env envelope

EPR electron paramagnetic resonance

ER endoplasmic reticulum

ESEEM electron spin echo envelope modulation

ESI electrospray ionization

ESR electron spin resonance

FDA Food and Drug Administration

FI fusion inhibitors

FIV feline immunodeficiency virus

FRET fluorescence resonance energy transfer

FWHM full-width at half-maximum

G Gauss

GHz gigahertz

gp 120 glycoprotein 120

gp 41 glycoprotein 41

HAART Highly Active Antiretroviral Therapy










HEK

HIV

HIV-1

HIV-1PR

HIV-2

IAP

IASL

B1

IDA

II

IHRP

IMAC

Int

IPTG

ITC

kD

LB

LP

LS

LTR

M

MALDI-TOF

MC


human embryonic kidney

Human Immunodeficiency Virus

Human immunodeficiency virus type 1

HIV-1 protease

Human immunodeficiency virus type 2

3-(2-Iodoacetamido)-PROXYL

4-(2-Iodoacetamido)-TEMPO

inclusion body

iminodiacetic acid

integrase inhibitor

In-house research program

immobilized metal ion affinity chromatography

integrase

Isopropyl-P-D-1-thiogalactopyranoside

isothermal titration calorimetry

kilodalton

Luria-Bertani media

lysed cell pellet

lysed cell supernatant

long terminal repeat

major group

matrix-assisted laser desorption ionization time of flight

Monte Carlo










MCS multiple cloning site

MD molecular dynamics

MDR769 Multi-drug resistant variant 769

mmHg millimeters of mercury

Mol. wt. molecular weight

mS milli-Siemens

MSL 4-Maleimido-TEMPO

MTSL (1-oxyl-2,2,5,5-tetramethyl-A3-pyrroline-3-methyl)methanethiosulfonate

MW molecular weight

NHLBI National Heart, Lung, and Blood Institute

NHMFL National High Magnetic Field Lab

NIH National Institute of Health

NK natural killer

Nle norleucine

NMR nuclear magnetic resonance

NNRTI non-nucleoside reverse transcriptase inhibitor

NOE Nuclear Overhauser Effect

NRTI nucleoside reverse transcriptase inhibitor

NSF National Science Foundation

NTA nitrilotriacetic acid

O outlier group

OD optical density

OI Opportunistic infection











ORF

PDB

PEG

Ph.D.

PI

PMPR

RAS

RNA

RP

rpm

RS

RT

SDS

SDSL

SDS-PAGE

SIV

SIVcpz

SIVsm

SNR

SPR

SU

TKR

Tm


open reading frame

Protein Data Bank

polyethylene glycol

Doctor of Philosophy

protease inhibitors

pentamutated protease

Renin-Angiotensin System

ribonucleic acid

resuspension pellet

rotations per minute

resuspension supernatant

reverse transcriptase

sodium dodecyl sulfate

site-directed spin-labeling

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Simian Immunodeficiency virus

Simian Immunodeficiency virus, chimpanzee

Simian Immunodeficiency virus, sooty mangabey

signal to noise ratio

surface plasmon resonance

surface

Tikhonov Regularization

melting temperature










TM phase memory time

TM transmembrane

tRNA transfer RNA

trxA Thioredoxin A

UF University of Florida

UNAIDS Joint United Nations Programme on HIV/AIDS

UV ultraviolet

VMD visual molecular dynamics

WHO World Health Organization

WP wash pellet

WS wash supernatant

YPRA yeast proteinase A









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF HIV-1 PROTEASE AND
THE DEVELOPMENT OF A SOLUBLE EXPRESSION SYSTEM FOR PRORENIN

By

Jamie Laura Kear

August 2010

Chair: Gail E. Fanucci
Major: Chemistry

All work performed for this dissertation dealt with, in general, the expression, purification,

and biophysical characterization of aspartic proteases, specifically human immunodeficiency

virus type 1 (HIV-1) protease (HIV-1PR) and the activatable renin zymogen called prorenin.

Chapters 1 and 2 describe the relevant biology and methodologies, including pulsed and

continuous wave electron paramagnetic resonance spectroscopy. HIV-1PR is a viral aspartic

protease that functions in regulating post-translational processing of the viral polyproteins gag

and gag-pol. The enzyme is a dimer comprised of 99 amino acid monomeric subunits.

Accessibility of substrate to the active site is mediated by two P-hairpins called the flaps (one

belonging to each monomer). The flaps have been shown to undergo a large conformational

change during substrate binding and catalysis; molecular dynamics simulations have captured

three distinct conformations of the flaps in HIV-1 protease, namely the closed, semi-open, and

wide-open conformations. Reported in this work are results of continuous wave and pulsed

electron paramagnetic resonance studies of HIV-1 protease. Continuous wave electron

paramagnetc resonance (CW EPR) spectroscopy, though it does not report on the flap motions of

the protease, was used to examine the autoproteolytic activity of the protease. The EPR spectral

line shape is highly sensitive to mobility in the environment of the spin label, thus it changes









dramatically with changes in correlation time. Autoproteolysis affects the rate of global protein

tumbling by decreasing rotational correlation time of the spin-labeled protein as a smaller spin-

labeled peptide fragment is liberated. As total correlation time decreases, the derivative EPR

spectra decrease in breadth and resonance line shapes become sharper and increasingly narrow.

The appearance of a sharp component in the high field, proportional to the amount of degraded

protein in the sample, was monitored. The intensity of the high field line was quantitatively

analyzed to give a term proportional to the amount of uncleaved peptide remaining in the sample.

The pulsed technique double electron-electron resonance (DEER) was utilized to examine

the differential flap conformations and flexibility of various HIV-1PR constructs under various

conditions. DEER experiments provided a means to determine distance profiles between two

spin-labeled sites in the flaps (sites K55C and K55C'), which were used to describe and quantify

conformational sampling of protease constructs. DEER echo curves were analyzed via Tikhonov

Regularization methods and the resulting distance profiles were regenerated using a series of

Gaussian-shaped functions, each representative of a distinct flap conformation. Distance profiles

from spin-labeled constructs of subtypes B, C, and F, CRF01_A/E, and drug-resistant patient

isolates V6 and MDR769, without ligand were analyzed in order to identify what effect natural

and drug-induced polymorphisms have on the conformational ensemble of the protease. The

dipolar modulated echo data and resulting distance distribution profiles differed greatly among

the apo protease constructs. These results demonstrated that natural and drug-induced

polymorphisms in the amino acid sequence of various subtypes and patient isolates alter the

average flap conformations and flexibility of the flaps. Additionally, in order to monitor

differences in flap conformations upon inhibitor binding between Subtype B and CRF01_A/E

proteases, constructs were analyzed upon addition of inhibitors and a non-hydrolysable substrate









mimic, CA-p2. These studies yielded interesting results in that the conformational ensembles of

the protease differ drastically with the various inhibitors.

Renin, also known as angiotensinogenase, is an aspartic protease that plays a vital role in

blood pressure regulation by catalyzing the first and rate-limiting step in the activation pathway

of its substrate angiotensinogen. The protease cleaves angiotensinogen to form angiotensin I,

which is then converted into angiotensin II by angiotensin I converting enzyme in a process

known as the renin-angiotensin cascade, which has an important effect on aldosterone release,

vasoconstriction, electrolyte imbalance, congestive heart failure, and an increase in blood

pressure leading to hypertension. Many hypertension drugs function by regulating blood

pressure at various points in the renin-angiotensin system. Prorenin is an inactive zymogen of

renin that circulates through the plasma until it reaches the secretary granules, where the pro-

segment is cleaved and active renin is released. Currently, very little structural data on prorenin

is available in the literature, likely because current methods for recombinant bacterial expression

of multiply disulfide bonded aspartic proteases from Escherichia coli have been plagued by

difficulty due to expression as inclusion bodies that require denaturation and refolding in order to

obtain properly folded, functional protein. Refolding, however, does not ensure that the protein

will be both properly folded and active. A bacterial expression system to circumvent these

difficulties was developed and work presented herein. Thioredoxin fusion methodology was

employed in order to maintain protein solubility and avoid inclusion body formation. The

resultant protein was shown to have secondary structure consistent with aspartic protease

zymogens and a fluorescence resonance energy transfer (FRET)-based assay was used to

demonstrate pH-dependent activation; however, the system was only minimally successful due to

low yields and protein instability upon cleavage from the fusion partner thioredoxin.









CHAPTER 1
INTRODUCTION TO ASPARTIC PROTEASES

Introduction to Proteases

Proteases are defined as enzymes that hydrolyze a polypeptide chain. Also called

proteinases or proteolytic enzymes, proteases are a class of hydrolases that are necessary for life

and are thus naturally occurring in all organisms. Protein hydrolysis is defined by the lysis of a

polypeptide chain by water, which is broken down into a hydrogen cation and a hydroxide anion

during the course of the reaction (McKee and McKee 2003). The general reaction scheme is

given in Figure 1-1.

R H R' R R'
I I I I I
NH2 CH C N CH COOH + H20 + 2H+ +NH3 CH COOH + +NH3-CH COOH
II
0

Figure 1-1. Scheme of hydrolysis of a polypeptide chain. Note charge states will vary
depending on pH.

Proteases can either exhibit limited proteolysis, that is they target hydrolysis on specific

peptide bonds, or unlimited proteolysis in which they carry out complete digestion of a peptide

into its amino acid constituents. Proteases can be classified as either exoproteases or

endoproteases based upon the region of the target protein or peptide in which proteolysis is

carried out. Exoproteases are a subclass of proteases that exhibit hydrolytic activity only near

the end of a peptide chain, while endoproteases can cleave near the center of the peptide

sequence. Proteases often contain a highly-conserved catalytic triad which is involved in

hydrolysis of the substrate. A catalytic triad refers to a series of three amino acids that function

together and are directly involved in catalysis. Proteases are active in a specific pH range, and

based on the particular active pH range of the enzyme, they can be classified as acidic, neutral, or

basic proteases. Due to the varying nature of this class of enzyme, proteases are involved in a









wide variety of different physiological reactions (McKee and McKee 2003). Proteases are

crucial to the peptide hydrolysis process. Uncatalyzed peptide bond hydrolysis reactions at 25

C and pH 7 are carried out with half-times of approximately 500 years for the C-terminal bond

of acetylglycylglycine, 600 years for the internal peptide bond of acetylglycylglycine N-

methylamide, and 350 years for the dipeptide glycylglycine (Radzicka and Wolfenden 1996).

Introduction to Aspartic Proteases

There exist six classes of proteases; namely, serine proteases, cysteine proteases,

metalloproteases, threonine proteases, glutamic proteases, and aspartic proteases. Each class

functions by providing a nucleophile, usually a water molecule or an amino acid, for attack on

the peptide carboxyl group. All of the key proteins investigated throughout this dissertation are

aspartic proteases.

Aspartic proteases are a subclass of endoproteases that often play an important role in

health and disease. Found in a range of different organisms, from mammals and fungi to viruses

and plants, aspartic proteases utilize aspartate residues to render catalytic activity. Generally

speaking, aspartic proteases have two highly-conserved aspartic acid residues in the active site of

the enzyme and are optimally active at acidic (or sometimes neutral) pH. Some aspartic

proteases function as monomers while others function as dimers; however, all aspartic proteases,

regardless of whether they function as a monomer or dimer, generally always have a tertiary

structure composed of two similar lobes, thereby creating a two-fold symmetry. Aspartic

proteases carry out a wide variety of reactions to regulate physiological phenomenon, including

but not limited to regulation of blood pressure, protein catabolism, digestion, and lysosomal

protein degradation, as illustrated by the examples given in Table 1-1.










Table 1-1. Representative aspartic proteases by name, function, source, size, and structure.
Name Function Source Size (kDa) Structure
Renin Blood pressure regulation Kidney 37 Monomer
HIV-1 protease Maturation of HIV Virus HIV-1 21 Dimer
Pepsin Digestion Stomach 35 Monomer
Cathepsin D Cellular turnover Lysosomes 48 Dimer
*kDa = kilodalton: 1 kDa is equal to approximately the weight of 1000 hydrogen atoms, or the
weight of 1/16th of the weight of 1000 Oxygen16 atoms or 1.66 x 10-21 grams.

General Mechanism of Catalysis by Aspartic Proteases

Aspartic proteases are a subclass of endoproteases that cleave specific dipeptide bonds

within their target substrate. Most often these dipeptide bonds are in close proximity to

hydrophobic residues and a P-methylene group. The most widely accepted mechanism of

catalysis by aspartic proteases, shown in arrow-pushing format in Figure 1-2, is based upon a

general acid-base catalysis mechanism that involves coordination of a water molecule, which

acts as a nucleophile, between the two active site catalytic aspartate residues.






H. H~0 ..
e e


H R N R N R

1 H1 'H H N".H. H
co.c c OH. /CH
-ee
RNH3

H20
H R



,O---H---O C H.
e 0e 0 1


/ ----- H--/0 C
N R H N o


O H H H





Figure 1-2. Generally accepted mechanism of catalysis by aspartic proteases. Figure generated
in ChemDraw and modified from Brik et al. (Brik and Wong 2003).









A unique characteristic of the two aspartate residues is that one has a relatively low pKa

(usually between 1 and 3.5), while the other has a relatively high pKa (usually between 4 and

5.5). The deprotonated residue acts as the general base, accepting a proton from the coordinated

H20, rendering it available to make a nucleophilic attack on the carbonyl group of the cleavable

peptide bond. The protonated aspartate residue acts as a general acid, donating a proton for

formation and rearrangement of the tetrahedral oxyanion intermediate and protonation of the

amide group.

Introduction to HIV-1 Protease

HIV as a World Pandemic

Much of today's federally and privately funded medical research focuses on Acquired

Immune Deficiency Syndrome (AIDS), which is a devastating worldwide health crisis caused by

the Human Immunodeficiency Virus (HIV). This disease of the human immune system is

currently spreading at frightening rates, though epidemiological data indicate that the spread of

HIV appears to have peaked in 1996 when 3.5 million new infections occurred; in 2008, the

estimated number of new HIV infections was approximately 30% lower (2009). Nonetheless,

the World Health Organization (WHO) has reported that the number of people living with HIV

or AIDS as of December 2008 was approximately 33.4 million, 31.3 million of which were

adults (WHO 2009). This number is approximately 20% higher than the number in 2000, and

roughly three times the number in 1990 (WHO 2009). Approximately 2.7 million people were

newly infected worldwide and approximately 2 million people died from AIDS-related deaths in

2008 alone (WHO 2009). The Joint United Nations Programme on HIV/AIDS (UNAIDS)

reported that approximately three-quarters of those deaths took place in Sub-Saharan Africa,

where an estimated 15-30% of the population was living with HIV or AIDS in 2007. This region

accounted for approximately 72% of AIDS-related deaths worldwide in 2008 (WHO 2009). By









comparison, the United States had approximately 0.5% 1% of its population living with HIV or

AIDS in 2007 (Figure 1-3).





i -



-- : *'1r...
o ... ..;
Ol *t '



Figure 1-3. Map showing percent of adult population living with HIV infection in 2007
according to country. Figure modified from UNAIDS.

HIV infection is characterized by a suppression of the immune system, and is said to have

four stages. The first stage of HIV infection is the period following infection and is generally

referred to as the "window." During this stage, a person will likely not test positive for HIV

because most HIV tests look for antibodies to the virus rather than for the virus itself. The

"window" phase is characterized as the period of time after which a person has become infected

but has not yet developed antibodies to the virus. The second stage is called serconversion and is

characterized by production of antibodies; a person is highly infectious during this stage. The

third stage is the often symptom-free stage and can last anywhere from a few months to more

than ten years. Stage four is called AIDS. A person is said to have AIDS when his/her CD4

(Cluster of Differentiation 4) cell count falls below 200 per cubic millimeter of blood (normal

CD4 cell count is between 500 and 1500 per cubic millimeter of blood) and/or the patient has

one or more opportunistic infections (OIs). OIs are typically normal infections from which a

person with a healthy immune system could usually recover. Though there are many drugs

aimed at slowing the course of the disease, there is currently no vaccine and no cure.









Introduction to HIV-1

Viruses are small, non-living infectious particles comprised of at least two main

components, the viral genome composed of either deoxyribonucleic acid or ribonucleic acid

(DNA or RNA) and a protein coat designed to house the genetic material and insert it into a host

cell. Additionally, certain viruses also contain an exterior lipid coat. There exist two distinct

major strains of HIV, called HIV-1 and HIV-2. Epidemiologic data suggest that HIV-1

originated from the simian immunodeficiency virus (SIV), called SIVcpz, of the chimpanzee,

which is highly homologous with HIV-1. Similarly, a simian immunodeficiency virus strain

called SIVsm from the sooty mangabey is highly homologous with HIV-2, thus it is likely that

HIV-2 originated from that strain. HIV-1 and HIV-2 possess similar modes of transmission, but

HIV-2 is more difficult to spread. HIV-2 is characterized by a longer incubation period and

lower infectivity than HIV-1 due to a lower viral density in the bloodstream. In addition, HIV-2

has a rather isolated geographic pattern; it is primarily found in the Western portions of Africa

and it is estimated that less than one hundred people are infected by HIV-2 in the United States.




















from NIAID.
Figure 1-4. Cartoon representation of the structure of the HIV-1 virus, showing the locations of
the host cell-derived outer lipid membrane, the Env protein comprised of gpl20 and
pg41 glycoprotein subunits, the matrix, and the capsid, which houses two strands of
viral RNA, plus viral proteins integrase, protease, and reverse transcriptase Adapted
from NIAID.









HIV is roughly spherical in shape and has a diameter of approximately 1000 Angstroms

(X). The virus is encompassed by a viral envelope of lipids derived from a budding event from

the human host cell. Also found in the viral envelope are various host cell proteins and

numerous copies of the viral protein called Envelope (Env), which is comprised of glycoproteins

called glycoprotein 120 (gpl20) and glycoprotein 41 (gp41). Within the viral envelope is the

capsid, which surrounds two strands of HIV-1 RNA and several viral proteins, including

Integrase (Int) and Reverse Transcriptase (RT). The structure of HIV-1 is shown in Figure 1-4.

Because HIV is a virus, it depends on a host cell for reproduction. HIV is in the family of

viruses called retroviruses, which are characterized by possessing a single-stranded plus sense

(+) mRNA genome, which must be reverse transcribed to give a DNA intermediate. The enzyme

responsible for this process is called reverse transcriptase and it lacks proofreading activity; the

mutation rate is estimated to be approximately 3.4 x 10-5 mutations/bp/replication, or one every

three rounds of replication (Mansky and Temin 1995). Most eukaryotic polymerases possess

proofreading activity and as a result have very low mutation rate. For example, human DNA

polymerase replicates DNA with an estimated 5x10-11 mutations/bp/replication (Drake,

Charlesworth et al. 1998). Some of these mutations will be silent (DNA mutations that do not

change the amino acid sequence of the protein), but those that are not silent can result in proteins

containing a polymorphism.

HIV-1 HIV-2


Group M Group N Group O Group P


A B C D F G H J K CRF

Figure 1-5. Phylogenic tree for HIV-1 and HIV-2 groups, subtypes and circulating recombinant
forms.









Table 1-2. Primary geographic prevalence of subgroups of HIV-1.
Group/Subtype/CRF Primary Geographic Prevalence
Group M
Subtype A East and Central Africa; Central Asia; Eastern Europe
CRF01 A/E Southeast Asia
CRF01 A/G West Africa
Subtype B North and South America; Western Europe; East Asia; Oceania
Subtype C India; Southern and Eastern Africa
Subtype D East Africa
Subtype F Africa; South America; Eastern Europe
Subtype G West Africa
Subtype H Central Africa
Subtype J Central America
Subtype K Cameroon and Democratic Republic of Congo
Group N Cameroon
Group O West Africa
Group P Cameroon

HIV-1 is categorized into different groups, subtypes (or clades), and circulating

recombinant forms (CRFs). Groups refer to viral lineages, subtypes to taxonomic groups within

a particular lineage, and CRFs to recombinant forms of the virus comprised of different viral

strains; Figure 1-5 shows a rough phylogenic tree for HIV-1 (Kantor, Shafer et al. 2005). Within

HIV-1, there are four groups, namely group M (major), group O (outlier), and groups N and P.

Groups N and P are found primarily in Cameroon and are very rare. Group O is slightly more

common and is found primarily in west-central Africa. Group M is by far the most common

with at least nine distinct subtypes and several CRFs, which are recombinant forms of two or

more subtypes. Within group M are subtypes A-D, F-H, J, and K, where each subtype exhibits a

unique set of naturally occurring polymorphisms.

Genetic diversity can reach 15 to 20% within subtypes and 25 to 30% between subtypes.

The primary geographic prevalence of each of these subgroups is given in Table 1-2.

Considering the natural frequency of mutations within HIV-1, new genetic subtypes and CRFs









will most certainly continue to develop as virus recombination and viral polymorphisms continue

to occur.

HIV-1 Viral Life Cycle

The life cycle of HIV-1 is similar to many other retroviruses, in that it is characterized by a

series of well-defined steps, as shown in the cartoon representation of the viral life cycle in

Figure 1-6. The HIV virus primarily infects cells that have CD4 cell-surface receptors, including

monocytes, macrophages, dendritic cells, T-helper cells, regulatory T-cells, natural killer (NK)

cells, hematopoietic stromal cells, and microglial cells. The gpl20 surface glycoprotein

recognizes and binds to the CD4 receptors, a conformational change in the viral protein gp41

takes place, and fusion of the virus to the host cell membrane occurs. T-helper cells are a type of

lymphocyte that plays an imperative role in the immune response. These cells exhibit no

cytotoxic or phagocytic activity, but rather they are involved in activating and directing other

cells involved in the immune response.

A mature HIV-1 virion requires a CD4 cell surface receptor for binding and fusion events

to occur. After the fusion event occurs, an uncoating event is triggered in which the RNA viral

genome and mature viral proteins are released into the cytoplasm of the host. The viral RNA is

then reverse transcribed resulting in a DNA intermediate which forms an integration complex of

viral DNA and viral proteins and is imported into the cell nucleus. At this point, the proviral

DNA is integrated into the host cell chromosome and transcribed and translated via host cell

machinery. The viral polyproteins subsequently accumulate on the outer membrane of the host

cell, triggering a budding event in which non-infectious, immature viral particles are released

from the host cell. In a final step, viral maturation takes place as HIV-1 protease cleaves the

viral polyproteins gag and gag-pol into their respective protein components.









-ALM LAHIV


CD4-Receptor 0
S0 Cell membrane
-._ .-- I __.___


Reverse Tr ansiptase
Synthesizes RNA y
into DNA
VVAAAvV


IntegraSe AW\
Integrates viral
DNA into the
cell genome A

/ C



/




Viral RNA leaves
the nucleus

Translation

Prolease
Cuts up
the protein
F


r -


N


Integration



hoc
ranscription







Virus protein


Virus RNA


Double-
stranded
DNA

Cell
SNucleus









Cyloplasm



Virus
RNA


ecostru
Reconstruction 4


** -p "


* *


-o-

New virus


Figure 1-6. Cartoon representation of the HIV-1 life cycle; steps include receptor recognition
and binding, fusion, uncoating and reverse transcription, nuclear import and
integration, transcription and translation, protein assembly and budding, and
maturation. Figure adapted from NIAIDS.


P-









The HIV-1 Viral Genome

The HIV genome consists of one strand of RNA that encodes three major genes, including

gag, pol (or gag-pol), and env, as well several other regulatory genes (tat, rev, nef vif vpr, and

vpu). The long terminal repeat (LTR) serves as a promoter of transcription. A cartoon

representation of the HIV-1 viral genome is given as Figure 1-7.

+-rev-+o |
< nef -
gag vif env
LTJR LTR
pol vpr L -tat-


Figure 1-7. Cartoon representation of the HIV-1 viral genome. Figure modified from NIAIDS.

The gag gene encodes for four proteins which are important in building the core of the

virus; these proteins include the capsid protein p24, the matrix protein p17, the nucleocapsid

protein p9, and protein p6. The pol gene encodes for four proteins that are imperative for the life

cycle of the virus, and include reverse transcriptase, HIV-1 protease, RNAse H, and Integrase.

Reverse transcriptase is required to copy the viral RNA into DNA once inside a host cell. HIV-1

protease functions in regulating post-translational processing of viral polyproteins, which is

required for viral maturation. RNAse H breaks down the viral genome following infection.

Integrase is involved in integrating the reverse-transcribed viral DNA into the host's DNA

genome. The env gene encodes for only one protein, called gpl60, which is cleaved to become

gpl20 and gp40 after new viral particles bud off from the host cell.

Accessory genes encoded by HIV-1 include tat, rev, nef vif vpr, and vpu. tat encodes for a

regulatory protein that increases the rate of transcription of HIV. rev encodes for a regulatory

protein which leads to production of other viral proteins. nefencodes for a regulatory protein

that accelerates endocytosis of CD4 from the surface of infected cells. The vif vpr, and vpu









genes encode proteins that appear to play a role in generating infectivity and pathologic effects.

Vif also called p23, increases viral infectivity by antagonizing APOBECC3G, a protein that

prevents spread of the infection by causing G to A hypermutation (Malim and Emerman 2008).

Vpr, also referred to as p15 has several functions including a role in the pre-integration complex

(Malim and Emerman 2008). Vpu, also referred to as p16 is an integral membrane protein that

helps bind CD4 receptors (Malim and Emerman 2008).

Structure and Function of HIV-1 Protease


A B






Flaps H
C D


region
Activesit
pocket








Figure 1-8. Structure of Subtype B HIV-1PR (PDB ID 2BPX); A) ribbon diagram, and B) space
filling model of Subtype B HIV-1PR (top view), and C) ribbon diagram, and D) space
filling model of Subtype B HIV-1PR (side view). Structures rendered with Chimera
(Pettersen, Goddard et al. 2004).

Human immunodeficiency virus type 1 (HIV-1) protease (HIV-1PR) (EC 3.4.23.16) is a

member of the aspartic protease family (A02.001) (Barrett, Rawlings et al. 1998). The enzyme

is a homodimer of two 99 amino acid monomers and is essential for the life cycle and maturation

of the retrovirus HIV-1 because it functions in regulating post-translational processing of the

viral polyproteins gag and gag-pol into an array of individual functional proteins that assemble









into mature and infectious HIV particles. Inhibition of HIV-1PR prevents viral maturation and

lengthens the life span, reduces viral load, and increases CD4 cell counts in HIV sufferers; as

such, this enzyme is a primary target of AIDS antiviral therapy (Ashorn, McQuade et al. 1990).

As of January 2010, more than 315 structures of HIV-1 Protease, both apo and in complex

with various substrates and inhibitors, were deposited in the Protein Data Bank (PDB), the first

of which was reported in 1989 (Miller, Schneider et al. 1989). A ribbon diagram of the side and

top view of HIV-1 protease, as well as a space filling model emphasizing the inaccessibility of

the active site to a peptide substrate when the flaps are in the closed conformation, are given in

Figure 1-8.

The nomenclature for the substrate amino acid cleavage positions is P4-P3-P2-P1/P1'-P2'-

P3'-P4', where the slash represents the scissile bond. Table 1-3 provides a listing of the

polyprotein processing sites of HIV-1 Protease. HIV-1PR has been crystallized with non-

hydrolyzable CA-p2 bound and is shown in Figure 1-9.

Table 1-3. HIV-1PR polyprotein processing sites.
Location HXB2 Consensus Cut Site Sequence
P5-P4-P3-P2-P1/P1'-P2'-P3 '-P4'-P5'
gag polyprotein
MA/CA VSQNY / PIVQN
CA/p2 KARVL / AEAMS
p2/NC TSAIM / MQRGN
NC/p 1 ERQAN / FLGKI
Pl/p6gag RPGNF / LQSRP
pol polyprotein
NC/TFP ERQAN / FLREN
TFP/p6gag-pol EDLAF / LQGKA
P6Po1/PR TSFSF / PQITC
PR/RTp51 CTLNF / PISPI
RT/RTp66 GAETF / YVDGA
RTp66/INT IRKVL / FLDGI
Nef polyproteins
Nef AACAW / LEAQE





















Figure 1-9. HIV-1PR (silver ribbon) bound to the non-hydrolyzable substrate mimic CA-p2
(colored ball and stick). Structures rendered with VMD (Humphrey 1996); A) side
and B) top views.

The dissertation work describes experiments with a non-hydrolyzable substrate mimic of

the CA-p2 cleavage site of the sequence Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala-Met-Ser; the

substrate mimic has the sequence H-Arg-Val-Leu-r-Phe-Glu-Ala-Nle-NH2 (r = reduced).

Alanine at position P1' has been replaced by phenylalanine and methionine at position P4' has

been replaced by the methionine analog Norleucine (Nle), shown in Figure 1-10A; methionine is

shown in Figure 1-10B for comparison. Norleucine is a non-natural amino acid isomeric to

leucine and isoleucine.

A 0 B 0

H3C OH H3C "SOH
NH2 NH2

Figure 1-10. Structures of A) norleucine and B) methionine.

Accessibility of substrate to the active site is mediated by two P-hairpins termed the flaps,

which must undergo a large conformational change from an open to a closed conformation

during substrate binding and catalysis, as demonstrated by nuclear magnetic resonance (NMR),

isothermal titration calorimetry (ITC), and molecular dynamic (MD) simulations. NMR has

been used to elucidate the structure of HIV-1PR in solution, both of the monomer (Ishima,









Ghirlando et al. 2001; Ishima, Torchia et al. 2003; Louis, Ishima et al. 2003) and the dimer

(Ishima, Freedberg et al. 1999; Freedberg, Ishima et al. 2002). Torchia et al. were able to

provide evidence of millisecond-microsecond timescale flap motion in unliganded protease and

nanosecond to subnanosecond flap motion in ligand-bound protease. Additionally, they were

able to demonstrate via nuclear Overhauser effect (NOE) that the flap region undergoes an

unordered-to-ordered transition upon inhibitor binding (Figure 1-11).


1.0 4
0.9
0.8 T



> 0.5 T
O
0.4- I
3 0.3
S *Liganded
S0.2 oUnliganded
0 10 20 30 40 50 60 70 80 90 100
Residue Number

Figure 1-11. HIV-1PR NOE values in black refer to the liganded form of HIV-1PR and NOE
values in grey refer to the unliganded HIV-1PR.

Isothermal Titration Calorimetry (ITC) has been used to examine many aspects of HIV-1

Protease, including stability and ligand binding. ITC is a technique that is used to determine the

thermodynamic parameters of a system, including binding affinity and stoichiometry of a

reaction, as well as changes in enthalpy and entropy. Freire et al. used the technique to

determine that the flap region is the least stable part of the protein, indicating the presence of a

major conformational change in that region (Todd, Semo et al. 1998; Todd and Freire 1999;

Todd, Luque et al. 2000; Velazquez-Campoy, Todd et al. 2000).









Molecular dynamics simulations have provided some of the most direct evidence of the

necessary conformational changes within the flap region to facilitate ligand binding and

catalysis. Molecular dynamics (MD) simulations performed by Simmerling et al. have indicated

three distinct conformations of the flaps in HIV-1 protease, namely the closed, semi-open, and

wide-open conformations, as seen in Figure 1-12 (Hornak, Okur et al. 2006).

A B C







Y- ts^-/C /








Figure 1-12. Flap conformations of HIV-1 protease captured by molecular dynamics
simulations; namely A) closed in presence of inhibitor, B) semi-open, and C) wide-
open. Structures rendered with Chimera (Pettersen, Goddard et al. 2004).

The closed conformation is found primarily in the presence of an inhibitor, and the semi-

open and wide-open conformations are seen in the absence of inhibitor. This was a landmark

study because prior to the work of Simmerling et al., no all-atom MD simulations were able to

capture flap reclosing, and the results were in agreement with previous NMR and X-Ray

crystallographic evaluations of the flap ensembles. These MD simulations have an advantage

over NMR and X-Ray in that X-Ray tends to capture a single conformation and NMR reports on

the average conformation.









Conformational Sampling and the Conformational Ensemble of the HIV-1 Protease Flaps

As noted above, the protease samples several distinct flap conformations. The sum of each

of these conformations can be described as the conformational ensemble of the protease. The

conformational sampling of the protease changes when inhibitors or substrate are added

(Blackburn, Veloro et al. 2009), polymorphisms are incorporated into the protease (Kear,

Blackburn et al. 2009), or the conditions of the protein solution are modified [Blackburn,

unpublished]. The double electron-electron resonance (DEER) methodology described in later

chapters allows us to examine the individual conformational populations that comprise the

conformational ensemble, rather than evaluating the average of the ensemble (as by NMR), or by

capturing a single conformation of the protease (as by X-Ray crystallography).

Hydrophobic Sliding Mechanism

As previously described, it is known that HIV-1PR must undergo a dramatic

conformational change for substrates or inhibitors to gain access to the active site pocket. A

mechanism, called the hydrophobic sliding mechanism, has been proposed by Schiffer et al. that

explains how this conformational change takes place, in addition to providing an explanation as

to how secondary mutations (those mutations outside the active site) in the protease confer drug

resistance (Foulkes-Murzycki, Scott et al. 2007). Analysis of molecular dynamics simulations

have suggested that nineteen core hydrophobic residues facilitate the conformational changes by

sliding past one another, exchanging van der Waal contacts with little to no energy penalties.

This type of motion would preserve hydrogen bonding that is likely imperative for maintaining

structural integrity. Seven of the nineteen core hydrophobic residues described above are

isoleucines which have numerous rotameric states that may facilitate the sliding mechanism.

Additionally, this mechanism offers insight into the effect of mutations in these regions because









changes in the hydrophobic residues would likely affect the conformational sampling and

flexibility of the protease (Foulkes-Murzycki, Scott et al. 2007).

HIV-1 Protease Construct Terminology

Numerous different HIV-1 protease constructs, including Subtypes B, C, F, CRFO1_A/E,

and patient isolates V6 and MDR769, will be discussed throughout this dissertation. For that

reason, abbreviations will be used to quickly identify which construct is being discussed. Each

of these abbreviations is given in Table 1-4, and a description of the construct is also included.

Before proceeding, a brief introduction to the abbreviations and nomenclature used throughout

this dissertation is necessary. Amino acid substitution code (e.g., D25N) is given by amino acid

residue to be substituted out, followed by the residue number, followed by the amino acid to be

substituted in (e.g., D25N the aspartic acid residue at position number 25 was mutated to an

asparagine residue).

A subscript "s" refers to stabilization against autoproteolysis. It is well known that HIV-1

protease undergoes autoproteolysis under the high concentrations required for biophysical

studies. In Subtype B, the cleavage sites have been identified as occurring between positions 6

and 7, 33 and 34, and 63 and 64 (Mildner, Rothrock et al. 1994); thus, the stabilizing mutations

Q7K, L33I and L63I were introduced to avoid self-proteolysis. The subscript "i" refers to

inactive protease in which a D25N mutation was engineered into the sequence. This mutation

has been shown by X-Ray and NMR not to perturb the structure of the protease. In addition, all

constructs possess mutations C67A and C95A to facilitate site-directed spin-labeling and to

avoid non-specific disulfide bonding.









Table 1-4. HIV-1PR construct abbreviations and descriptions.
Construct abbreviation Construct Details
A/Es CRF01 A/E; K55C, C67A, C95A, Q7K, L33I, L63I
A/Esi CRF01 A/E; K55C, C67A, C95A, Q7K, L33I, L63I, D25N
Bs Subtype B; K55C, C67A, C95A, Q7K, L33I, L63I
Bsi Subtype B; K55C, C67A, C95A, Q7K, L33I, L63I, D25N
Cs Subtype C; K55C, C67A, C95A, Q7K, L33I, L63I
Csi Subtype C; K55C, C67A, C95A, Q7K, L33I, L63I, D25N
Fs Subtype F; K55C, C67A, C95A, Q7K, L33I, L63I
Fsi Subtype F; K55C, C67A, C95A, Q7K, L33I, L63I, D25N
V6 Patient isolate V6; K55C, C67A, C95A
V6i Patient isolate V6; K55C, C67A, C95A, D25N
MDR769 Patient isolate MDR769; K55C, C67A, C95A
MDR769i Patient isolate MDR769: K55C, C67A, C95A, D25N

HIV-1 Subtype B Protease

Subtype B is the dominant HIV subtype in the United States and Europe. For this reason, a

substantial proportion of HIV-1PR research, and most drug development, clinical trials, and

observational studies have been performed on Subtype B virus. However, only about 10% of

HIV worldwide is Subtype B (2009). Top and side views of a ribbon diagram as well as a space

filling model of the crystal structure (PDB ID 2BPX) of Subtype B HIV-1PR were shown in

Figure 1-8.

The Los Alamos HIV database contains four primary sequences of the Subtype B viral

genome, namely HXB2 (Wong-Staal 1985), BK132 (Hierholzer, Montano et al. 2002), 671

(Geels, Comelissen et al. 2003), and 1058 (Bemardin, Kong et al. 2005). HXB2 (Accession

Number K03455) is the primary reference, and is a specific clone from the French isolate LAI.

The HXB2 sequence was utilized for all Subtype B work throughout the dissertation (with the

exception of several necessary mutations described in detail later). BK132 (Accession Number

AY173951) was isolated in Thailand in 1990. 671 (Accession Number AY423387) was isolated

in the Netherlands in 2000, and 1058 (Accession Number AY331295) was isolated from the

United States in 1998.









HIV-1 Subtype F Protease

The subtype F protease is generally differentiated from the LAI consensus sequence of

Subtype B by the following polymorphisms: I15V, E35D, S37N, R41K, and R57K. None of

these polymorphisms are primary, i.e., occur in the substrate binding pocket, but rather, are

located on the hinge region and on the outer wall of the protease. Two crystal structures of

Subtype F protease have been reported in the Protein Data Bank (PDB ID 2P3C and 2P3D).

2P3C, shown in Figure 1-13, is a structural complex between the protease and the TL-3 inhibitor.

TL-3 is a protease inhibitor developed for the feline immunodeficiency virus (FIV), but has been

shown to have broad-based activity in that it functions to inhibit replication of the human,

simian, and feline immunodeficiency viruses (Buhler, Lin et al. 2001).

A B










Figure 1-13. Crystal structure of HIV-1PR subtype F (PDB ID 2P3C); A) side and B) top views.
Structure was rendered with VMD (Humphrey 1996).

There are eight primary sequences for the Subtype F viral genome in the Los Alamos HIV

database. These include 93BR020-1 (Gao, Robertson et al. 1998), VI850 (Laukkanen, Carr et al.

2000), FIN9363 (Laukkanen, Carr et al. 2000), MP411 (Triques, Bourgeois et al. 2000), MP255

(Triques, Bourgeois et al. 2000), MP257 (Triques, Bourgeois et al. 2000), 0016BBY (Kijak,

Sanders-Buell et al. 2004), and CM53657 (Carr, Torimiro et al. 2001). The 93BR020-1

(Accession number AF005494) was isolated in Brazil in 1993; VI850 (Accession number

AF077336) was isolated in 1993 in Belgium; FIN9363 (Accession number AF075703) was









isolated in Finland in 1993; MP411 was isolated in France in 1996; MP2555 and MP257 were

isolated in Cameroon in 1995; AY371158 was isolated in Cameroon in 2002, and AF377956 was

isolated in Cameroon in 1997.

HIV-1 Subtype C Protease

Subtype C is the most prevalent subtype of HIV worldwide, accounting for approximately

48% of all cases. This subtype is found in Sub-Saharan Africa, as well as Asia and India.

Subtype C generally differs from Subtype B by the following polymorphisms: T12S, I15V, L19I,

M36I, S37A, H69K, N88D, L89M, and I93L. Three crystal structures of Subtype C have been

reported in the literature, one in the apo form, one in complex with NFV (shown in Figure 1-14),

and one in complex with IDV.


A B










Figure 1-14. Crystal structure of Subtype C (PDB ID 2R5Q) in complex with NFV (not shown
in figure). Structure was rendered with VMD (Humphrey 1996).

There are many full-length genomes reported in the HIV database, some of which are

ETH2220 (Accession number U46016) isolated from Ethiopia in 1986 (Salminen, Johansson et

al. 1996), 92BR025.8 (Accession number U52953) isolated from Brazil in 1992 (Gao, Robertson

et al. 1996), IN21068 (Accession number AF067155) isolated from India in 1995 (Lole,

Bollinger et al. 1999), and SK164B1 (Accession number AY772699) isolated from South Africa

in 2004 (Kiepiela, Leslie et al. 2004).









HIV-1 Protease Recombinant Form CRF01 A/E

Most commonly, CRF01_A/E differs from Subtype B protease by the following

polymorphisms: I13V, E35D, M36I, S37N, R41K, H69K, and L89M. The primary sequence of

CRF01_A/E in the Los Alamos HIV database is CM240 (Accession number U54771) and was

isolated in Thailand in 1990. CRF01_AE one of many reported circulating recombinant forms.

This particular CRF is becoming highly prevalent in Asia, but originated from Central Africa.

Circulating recombinant forms, as the name implies, are recombinant forms of the virus.

CRF01_A/E is a putative subtype A/E recombinant. Figure 1-15 shows a diagram of what

regions of the genome recombined from what subtype. The CRF01_A/E protease gene

recombined entirely from Subtype A making the protease sequences the same.

4-rev 4
ner
gag vit | env
LTR 4 tLTR
pol |vpr| L- tat-|




A E ] U (Unclassified)

Figure 1-15. Graphical description of the circulating recombinant form CRF01_A/E, portions of
DNA from several subtypes recombined to give the CRF01_A/E sequence. Portions
in red are from Subtype A, those in yellow are from a parent subtype E, and those in
white are from an unclassified parent source.

Interestingly, no full-length genome has been found for a pure subtype E. However,

because CRF01_A/E is often referred to as Subtype E, and the E designation is the common

name for the env region of the genome, the current name will not be changed so as to avoid

confusion. In the future, regions of recombinants for which there is no full-length parental strain

will be considered unclassified (U), thus a recombinant form of Subtype A and an unclassified

region would be called CRF01_A/U.









Therapeutic Approaches to HIV-1 Infection and Inhibition of HIV-1 Protease


,% ceptor 6
4 0 Cell membrane


L Fusion inhibitors


Reverse Transcaptase
Synthesizes RNA
inot DNA


Nucleoside and non- nucleoside
reverse transcri tas inhibitors
Integrase
Integrates viral
DNA into the
cell genome / N


Nuceus


y Integration
T Integrase inhibitors
_. I


[K


Transcription


Viral RNA leaves
the nucleus

Translation

Protease
Cuts up
the protein
F


0*



4


Virus protein


'/

/

Cytoplasm



Virus
RNA


e sruion
Reconstruction 4


i,; --', ""o ; _....
** 'e *
-',c-C Protease inhibitors


New virus q '-? f-o
HIV-1 Protease e--a
Viral maturation,
cleaves gag and gag-pol


Figure 1-16. Points of inhibition within the HIV-1 viral life cycle.


CD4-Re


Virus RNA

Double-
stranded
DNA

Cpll


r









There are several points within the HIV-1 viral life cycle which may be targeted for

inhibition in an attempt to treat HIV-1 infection, including the fusion, reverse transcription,

integration, and maturation steps, as illustrated in Figure 1-16. There is still no cure for HIV or

AIDS, but rather just drugs that simply function to suppress the virus. As of January 2010, there

were 31 antiretroviral drugs (ARVs) approved by the U.S. Food and Drug Administration (FDA)

to treat HIV infection, grouped into several classes based upon point of inhibition within the viral

life cycle, given in Table 1-5. These include reverse transcriptase inhibitors of two classes,

nucleoside (NRTIs) and non-nucleoside (NNRTIs), fusion inhibitors, entry inhibitors, and

integrase inhibitors. Patients with HIV/AIDS undergo a treatment known as Highly Active

Antiretroviral Therapy, or HAART, which is a combined treatment of one or more inhibitors of

the HIV viral life cycle.

Table 1-5. FDA-approved drugs for the treatment of HIV/AIDS.
PIs NNRTIs NRTIs Els IIs
Saquinavir Delavirdine Zidovudine Enfuvirtide Raltegravir
Ritonavir Efavirenz Emtricitabine Celsentri
Indinavir Nevirapine Lamivudine
Nelfinavir Stavudine
Amprenavir Abacavir
Lopinavir Didanosine
Atazanavir Tenofovir
Tipranavir
Fosamprenavir
Darunavir

Protease inhibitors target the active site of the HIV-1PR with the objective of binding to

the active site tightly enough to prevent binding by the substrate via a mechanism called

competitive inhibition. HIV-1 protease inhibitors were first developed in 1989 by researchers at

Hoffmann- La Roche Inc., Abbott Laboratories and Merck & Co., Inc. HIV-1 protease inhibitors

are used in the treatment of patients with HIV and AIDS, and can lower viral load in these

patients. They function by specifically binding to the active site by mimicking the tetrahedral










intermediate of the substrate. Currently there are ten FDA-approved HIV-1 protease inhibitors

used in the treatment of HIV-1 infection, including Ritonavir (with trade name Norvir), Indinavir

(Crixivan), Lopinavir (Kaletra and Aluvia), Darunavir (Prezista), Saquinavir (Invirase and

Fortovase), Tipranavir (Aptivus), Atazanavir (Reyataz), Amprenavir (Agenerase),

Fosamprenavir (Lexiva and Telzir), and Nelfinavir (Viracept). Details about the protease

inhibitors, including structural information, are given in Table 1-6.

Table 1-6. FDA-approved protease inhibitors for HIV-1 HAART treatment.
Inhibitor (Market Name) Abbreviation Structure Formula Mol. wt. Solubility

Amprenavir (Agenerase) APV C,,H,,NOS 505.63 DMSO
H CH2OH
SC EtOH

/' C1 .1"N MetC12
OH

Atazanavir (Reyataz) ATV C38H52N607 802.9 4-5mg/mL
SH20O
DMSO
EtOH
0 OH 0




Darunavir (Prezista) DRV NH. C27H37N30OS 593.73 0.15mg/mL










Indinavir (Crixivan) IDV C36H47N504 711.88 HO20
e'7 CH30H











Table 1-6. Continued.
Inhibitor (Market Name)
Ritonavir (Norvir)


Abbreviation
RTV


Structure Formula Mol. wt.
C37H48N605S2 720.96


Saquinavir (Invirase and Fortovase) SQV





-0Tip



Tipranavir (Aptivus) TPV


Lopinavir (Kaletra and Aluvia) LPV


Nelfinivir (Viracept)


C38H5oN605 675.10 DMSO
CH3OH


C31H33F3N20sS


602.7


EtOAc


C37H48N405 628.81 DMSO
CH3OH
EtOH
CH2Cl2
MetCl


C32H45N304S


567.78 4.5mg/mL
H20


Solubility
DMSO
Chloroform
Toluene









In December 1995, Saquinavir was the first HIV-1 protease inhibitor (and sixth

antiretroviral) to gain FDA approval. The drug was manufactured by Hoffman-La Roche and

was given the brand name Invirase. In 1997 the drug was reapproved under the brand name

Fortovase after being reformulated for higher bioavailability. Finally, in 2006 Fortovase was

discontinued and Saquinavir is now given in the form of Invirase coupled with Ritonavir. Unlike

most HIV protease inhibitors, Saquinavir inhibits both the protease from HIV-1 and HIV-2

(Collier, Coombs et al. 1996; Noble and Faulds 1996; Vella and Floridia 1998).

Ritonavir, manufactured by Abbott Labs and given the brand name Norvir, was next to

receive FDA approval in March 1996. Ritonavir is now most commonly used as a booster of

other protease inhibitors, as it also inhibits cytochrome P450-3A4, an enzyme found in the liver

which is known to break down protease inhibitors. Thus, when low doses are given in tandem

other HIV protease inhibitors, bioavailability of those PIs is increased substantially (Zeldin and

Petruschke 2004; Ortiz, Dejesus et al. 2008). Indinavir, manufactured by Merck under the name

Crixivan, also received FDA approval in March 1996. When first introduced into the market,

Indinavir was much more effective in the treatment of HIV/AIDS than any other retroviral

known at that time (2008; Wolters Kluwer Health 2008; Hou 2009).

Nelfinavir mesylate was next to be approved by the FDA for treatment of HIV in March

1997, making it the fourth HIV protease inhibitor and the twelfth antiretroviral. Nelfinavir,

marketed under the name Viracept, was originally manufactured by Agouron Pharmaceuticals,

which is now a subsidiary of Pfizer. Like Saquinavir, Nelfinavir also inhibits proteases from

both HIV-1 and HIV-2 (Pai and Nahata 1999; Bardsley-Elliot 2000; Zhang, Wu et al. 2001;

Gills, Lopiccolo et al. 2007).









The fifth HIV protease inhibitor, Amprenavir, gained FDA approval in April 1999. The

drug was manufactured by GlaxoSmithKline under the brand name Agenerase. Amprenavir is

no longer being manufactured. However, the pro-drug of Amprenavir, called Fosamprenavir,

also manufactured by GlaxoSmithKline, obtained FDA approval in October 2003 under the

name Lexiva. By taking the pro-drug of Amprenavir rather than Amprenavir itself, the body is

forced to metabolize Fosamprenavir, releasing Amprenavir and thereby increasing the amount of

time that the drug is available to the body (Bulgheroni, Citterio et al. 2004).

Lopinavir was the next HIV protease inhibitor to receive FDA approval which was granted

in September 2000. Lopinavir is marketed and manufactured by Abbott labs as both Kaletra and

Aluvia. The drug is given with a sub-therapeutic dose of Ritonavir to inhibit CP450-3A4

(Bulgheroni, Citterio et al. 2004; Ortiz, Dejesus et al. 2008).

Atazanavir, manufactured by Bristol Myers under the name Reyataz received its FDA

approval for the treatment of HIV/AIDS in June 2003. In October 2006 the FDA approved a

second formulation designed to lower the number of pills required by the therapy. Atazanavir is

also used in combination with Ritonavir to boost bioavailability (Clemente, Coman et al. 2006).

Tipranavir disodium is manufactured by Boehringer-Ingelheim under the name Aptivus

and was approved for treatment of HIV/AIDS by the FDA in June 2005. Aptivus is a member of

the 4-hydroxy-5,6-dihydro-2-pyrone sulfonamide class, with activity as a non-peptide protease

inhibitor (Cheonis 2004). It is administered with Ritonavir for improved bioavailability.

Notably, Tipranavir has been shown to inhibit proteases that have gained drug resistance to other

protease inhibitors, and it has been suggested that resistance to Tipranavir requires numerous

drug-pressure induced mutations. Unfortunately, however, the use of Tipranavir is associated









with an increase in more severe side effects when compared to other protease inhibitors (Larder,

Hertogs et al. 2000; Bulgheroni, Citterio et al. 2004; Cheonis 2004; Kandula 2005).

The most recent HIV protease inhibitor to gain FDA approval is called Darunavir, which

was approved for use in June 2006. The drug, marketed as Prezista, was developed and

manufactured by Tibotec. Darunavir is a second-generation protease inhibitor developed to

overcome drug resistance problems associated with other protease inhibitors. It has been shown

to be effective on proteases from numerous strains of HIV-1, as well as proteases from

previously PI-exposed patients who harbor multiple drug-resistant proteases. However, the drug

costs approximately $9000 for a one-year supply (Ortiz, Dejesus et al. 2008).

The FDA approved protease inhibitors have shown great promise in the treatment of

HIV/AIDS by substantially decreasing mature viral load. Each of these inhibitors were designed

with respect to the LAI sequence of Subtype B, and most clinical trials were carried out using

Subtype B. As discussed above, however, the high mutation rate ofHIV-1 Protease, as with any

retrovirus, can render the clinical efficacy of certain inhibitors with certain subtypes (Rose, Craik

et al. 1998; Wlodawer and Vondrasek 1998; Velazquez-Campoy, Vega et al. 2002; Clemente,

Coman et al. 2006; Coman, Robbins et al. 2007; Sanches, Krauchenco et al. 2007;

Bandaranayake, Prabu-Jeyabalan et al. 2008; Coman, Robbins et al. 2008). For this reason, it is

imperative to understand how other non-B subtypes compare to Subtype B in structure,

flexibility, dynamics, and kinetics.

Drug Resistance in Protease Inhibitor-Exposed Patient Isolates of HIV-1 Protease

Drug resistance is a major problem concerning the efficacy of HIV protease inhibitors.

Because of the high error rate in the production of the DNA intermediate, a high number of

natural polymorphisms can become incorporated into the viral genome resulting in numerous










variants even within a single patient. Patients that have been exposed to protease inhibitors often

develop further drug-induced polymorphisms.

100 100
S90 A 90 B
S80- ace sie aps 80-
S70- 70-
760 60-
p,50 60 50
S40 40
C. 30 30
o 20 a 20-

04 04
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
Residue Residue

Figure 1-17. Regions of amino acid conservation in A) protease inhibitor naive and B) protease
inhibitor exposed HIV-1PR.

A comparison of HIV-1PR sequences from the Stanford HIV-1 Database of both protease

inhibitor naive and exposed patients shows that there are several regions of conservation in the

naive protease that lose conservation after prolonged exposure to protease inhibitors. These

regions include the flap region, the active site, and the dimerization region (Figure 1-17). The

active site is required for binding and catalysis of substrate and the flaps are necessary for

regulation of accessibility of substrate to the active site. The dimerization domain is required to

stabilize the dimer, as the monomer has essentially no activity.

Many patterns of drug-induced mutations that render resistance have accumulated, and are

recorded in Stanford's HIV-1 Database. The mutations occurring in the HIV-1 Protease are

classified as either primary or secondary mutations. Primary mutations are found in or around the

active site and provide the protease with some level of drug resistance. Secondary mutations are

found outside the active site, often on the periphery of the protease. Secondary mutations,









though they are not found in the active site, provide some level of drug resistance, often in

combination with primary mutations or other secondary mutations.

Multi-drug Resistant Patient Isolate MDR769

The multi-drug resistant patient isolate called MDR769 was isolated from a patient

previously treated with Indinavir (IDV), nelfinavir (NFV), Saquinavir (SQV), and Amprenavir

(APV). The protease most often differs from Subtype B by the following polymorphisms: L10I,

M36V, M46L, I54V, I62V A71V, V82A, I84V, and L90M, and each of these polymorphisms

has been reported to confer drug resistance to protease inhibitors (Logsdon 2004). One crystal

structure has been reported in the PDB (1TW7), and is shown in Figure 1-18.


A B










Figure 1-18. Crystal structure of HIV-1PR multi-drug resistant patient isolate MDR769 (PDB
ID 1TW7); A) side and B) side views. Structure rendered with VMD (Humphrey
1996).

The 1.8 A resolution crystal structure shows an expanded active site cavity and flaps that

stay open wider with respect to Subtype B protease. Polymorphisms at positions 82 and 84

exchanged V and I residues for smaller A and V residues, respectively, thus lending more space

to the active site cavity. An MDR769 crystal structure of the protease in complex with

Lopinavir has shown different binding interactions between the inhibitor and the protease, when

compared with Subtype B. Additionally, surface plasmon resonance (SPR) measurements point

to higher koff rates between inhibitor and protease (Logsdon 2004).









Drug Resistant Patient Isolate V6

The drug-resistant patient isolate called V6 was isolated from a pediatric patient previously

treated with Ritonavir (RTV). V6 generally differs from Subtype B by the following

polymorphisms: K20R, V32I, M36I, A71V, V82A, L90M. V32I and V82A are primary

mutations associated with Ritonavir resistance (found in or around the active site), while the rest

are non-active site polymorphisms. The I84V polymorphisms has been reported to have a 10-

fold decrease in catalytic efficiency when compared to protease inhibitor naive Subtype B

(Clemente, Moose et al. 2004).

Introduction to Prorenin

Hypertension and Its Impact on Society

Hypertension is a medical term that describes the condition of having high blood

pressure. Blood pressure readings are given as systolic pressure/diastolic pressure, both in units

of millimeters of mercury (mmHg), where systolic pressure is the pressure that is created when

the heart beats and diastolic pressure is the pressure inside the blood vessels when the heart is at

rest. There are several classifications of hypertension, as shown in Table 1-7 (Chobanian 2003).

The American Heart Association reported that in 2008 the percentage of non-institutionalized

adults over the age of 20 living with some degree of hypertension was approximately 32%, and

approximately 24,000 deaths were attributed to hypertension in the United States alone

(Clemente, Moose et al. 2004). The National Heart, Lung, and Blood Institute (NHLBI)

estimated in 2002 that hypertension cost the United States approximately 47 billion dollars

(Clemente, Hemrajani et al. 2003). Although a person with hypertension can often experience

little or no symptoms, possible symptoms can include headaches, blurred vision, nose bleeds,

and dizziness.









Table 1-7. Classifications of hypertension and respective systolic and diastolic pressure ranges
(Chobanian 2003).
Classification Systolic Pressure (mmHg) Diastolic Pressure (mmHg)
Normal 90 119 60 79
Pre-hypertension 120 139 80 89
Stage I Hypertension 140 -159 90 99
Stage II Hypertension >160 > 100

Hypertension can be caused by a variety of factors, both genetic and lifestyle-induced.

Approximately 30% of hypertension cases can be contributed to genetic factors, although the

genes that contribute to hypertension have yet to be identified. Abnormalities in the arteries or in

the adrenal hormone glands are known to cause hypertension. Additional factors such as obesity,

high salt intake, smoking, pregnancy can cause an elevation in blood pressure leading to

hypertension (Clemente, Moose et al. 2004). There exist many different types of FDA-approved

treatments for hypertension, including alpha blockers, beta-blockers, angiotensin-converting

enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), diuretics, renin inhibitors, and

vasodilators. Renin inhibitors are a relatively new class of protease inhibitors that function on

the juxtaglomerular cells to inhibit the production of renin.

Renin, Prorenin, and the Renin-Angiotensin System

The renin-angiotensin system (RAS) is considered one of the key modulators of blood

volume, blood pressure, and cardiac and vascular function. As such, many hypertension drugs

function by regulating blood pressure at various points in the RAS (Danser 2003). Renin, also

known as angiotensinogenase, is an aspartic protease that plays a vital role in blood pressure

regulation by catalyzing the first and rate-limiting step in the activation pathway of its substrate

angiotensinogen. It cleaves angiotensinogen to form the decapeptide angiotensin I, which is then

converted into angiotensin II by angiotensin I converting enzyme. A schematic of this pathway

is shown in Figure 1-19.










Prorenin Decrease renal perfusion
-Angiten- gen- Decreased NaCI to distal tubule
Angiotensinogen _.. .
3- Renin I Increased sympathetic stimulation
Angiotensin I Bradykinin
-(- Angiotensin Converting Enzyme 2 4
Angiotensin II Inactive Degredation
Products
S1 = adrenergic antagonists
2 = ACE inhibitors
3 = Renin inhibitors
Angiotensin II Receptor 4 = AH Receptor antagonists


Figure 1-19. Renin-angiotensin system and the sites of inhibition within the RAS; 1) adrenergic
antagonists; 2) ACE inhibitors; 3) renin inhibitors; and 4) angiotensin II (AII)
receptor antagonists.

This significant cascade has an effect on aldosterone release, vasoconstriction, electrolyte

imbalance, congestive heart failure, and an increase in blood pressure leading to hypertension

(Danser 2003; Morris 2003; Marathias, Agroyannis et al. 2004; Schweda and Kurtz 2004;

Berecek, Reaves et al. 2005). Because renin is involved in the control of blood pressure through

the formation of the vasoactive peptide angiotensin II, the proteins renin, and perhaps prorenin,

represent potential therapeutic targets in the regulation and control of hypertension (Danser

2003; Morris 2003).

Biosynthesis and Intercellular Processing

Human renin in synthesized via two separate pathways, the first is a constitutive pathway

for prorenin secretion and the second is a regulated pathway for secretion of mature renin. Renin

is synthesized in the granular cells of the juxtaglomerular apparatus in the kidney and secreted in

response to several factors, including a decrease in arterial blood pressure or a decrease in

sodium chloride (NaC1) levels. Prorenin is an inactive zymogen of renin, meaning that it has a

pro-segment on its N-terminal end that functions as the activation modulator. Prorenin circulates

through the plasma until it reaches the secretary granules, where the pro-segment is cleaved and

active renin is released (Danser 2003). Interestingly, prorenin is found in much higher









concentrations in the bloodstream than renin, representing a likely enzymatic control mechanism

and it is unclear if other biological or physiological roles of prorenin exist.

Table 1-8. Pro-peptide sequences of several aspartic protease zymogens.
Aspartic Protease Pro-Peptide Amino Acid Sequence
Human Prorenin LPTDTTTFKRIFLKRIMPSIRESLKERGVDMARLGPEWSQPMKR
Mouse Prorenin FSLPTGTTFERIPLKKMPSVREILEERGVDMTRLSAEWKVFTKR
Rat Prorenin SFSLPTDTASFGRILLKKMPSVREILEERGVDMTRISAEWGERKK
Bovine Prochymosin AEITRIPLYKGKSLRKALKEHGLLEDFLQKQQYGISSKYSGF
Human Procathepsin D LVRIPLHKFTSIRETMSEVGGSVEDLIAKGPVSKYSQAVPAVTE
Human Pepsinogen C AWKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRFGDL
Human Pepsinogen A IMYKVPLIRKKSLRRTLSERGLLKDFLLKKHNLNPARKYFPQWEALPTL
Porcine Pepsinogen A LVKVPLVRKKSLRONLIKDGKLKDFLKTHKHNPASKYFPEAAAL

In vitro, conversion of prorenin into active renin appears to be a two-step process involving

the generation of an intermediary form of activated prorenin, where the active site is exposed but

the pro-segment has not yet been proteolytically cleaved from the enzyme. The first step is

thought to be an acid-induced activation that occurs as the result of a conformational change in

the pro-segment. In the inactive form, (based upon structural homology to pepsinogen) the pro-

segment is expected to be folded over the active site and in tertiary contact with other regions of

the protein. At low pH, it is believed that a series of residues become protonated, thus breaking

three salt bridges that are implicated in holding the pro-segment over the active site and resulting

in an unstructured conformation of the pro-segment that is no longer in contact with the active

site. This conformer is referred to as the acid-activated form of prorenin. This first step has been

shown via enzymatic assays to be reversible via a pH switch back to neutral conditions.

The second step in the activation of prorenin to renin is the proteolytic removal of the pro-

segment resulting in active renin (Danser 2003). The three amino acids thought to partake in

salt-bridge formation are shown to be conserved in numerous aspartic protease zymogens (Table

1-8), leading to the hypothesis that perhaps most aspartic protease zymogens function in a

similar fashion (Derkx, Schalekamp et al. 1987).









Structure and Function of Prorenin

Pepsinogen, the inactive zymogen of pepsin, is a well-studied member of the aspartic

protease family. Though no crystal structure exists for prorenin, the crystal structures for pepsin

and renin are very similar, also suggesting that the mechanisms of activation are similar. The

crystal structure of pepsinogen illustrates how the pro-segment physically blocks the active site

of the enzyme.


Figure 1-20. Crystal structures of aspartic proteases A) renin, B) pepsin, C) an overlay of the
renin (orange) and pepsin (purple) for comparison of structural similarity, and D) an
overlay of renin (orange) and pepsinogen (blue, with green pro-segment) for
comparison of structural similarity and prediction for positioning of renin pro-
segment; crystal structure of pepsinogen shows the pro-segment of pepsinogen
(green) physically blocking the active site of the protease.

In the case of pepsinogen, it is generally accepted, though there is no direct structural

evidence, that acid liberates the pro-segment from the active site of the enzyme, mature pepsin is

then generated through an autocatalytic process where the pro-segment is cleaved at residue 66









(in preprorenin numbering), leaving a 340 amino acid renin of approximately 37 kDa. Although

the pro-segment of prorenin is not autocatalytically removed like that of pepsinogen, prorenin is

thought to behave in a similar manner. Figure 1-20 demonstrates the high level of similarity

between the crystal structures of renin and pepsin. Renin is shown Figure 1-20A, pepsin is

shown in Figure 1-20B, and an overlay of the two is shown in Figure 1-20C. Shown in Figure 1-

20D is an overlay of renin and pepsinogen, the inactive zymogen of pepsin. This provides a

model for what the crystal structure of prorenin might look like. Understanding the structure and

conformational changes that take place in the activation of prorenin could lead to potentially new

avenues for rational drug design in controlling hypertension.

Difficulties in Expression and Purification of Prorenin

Currently, very little structural data on prorenin is available in the literature. This is likely

related to the fact that current methods for recombinant bacterial expression of aspartic proteases

from Escherichia coli (E. coli) have been plagued by difficulty. Most aspartic proteases have

multiple disulfide bonds; hence these proteins, when cloned using the E. coli system, are usually

expressed as inclusion bodies. Inclusion body proteins require that they be denatured and then

refolded in order to obtain properly folded, functional protein. Refolding, however, does not

ensure that the protein will be refolded correctly and active (Nishimori, Kawaguchi et al. 1982;

Imai, Cho et al. 1986; Kaytes, Theriault et al. 1986; Masuda, Nakano et al. 1986; Lin, Wong et al.

1989; Yamauchi, Nagahama et al. 1990; Chen, Koelsch et al. 1991).

Prorenin is known to be notoriously difficult to refold, thus protein is generally obtained

from renal isolation or via expression and secretion from insect cells; both techniques are costly

and time-consuming. Thus, part of my Ph.D. research focused on developing a new method for

expression and purification of prorenin, and the results of that study are reported in Chapter 5 of

this dissertation. Prorenin was successfully cloned into a bacterial expression vector, over-









expressed in E. coli BL21(DE3) and Origami(DE3) cells as a fusion construct with thioredoxin,

and purified in small yield. A CD spectrum was obtained that suggests that the protein was

properly folded, and SDS-PAGE gel electrophoresis confirmed that the size of the purified

protein corresponded with the prorenin-thioredoxin fusion construct. An activity assay

confirmed that the fusion construct possessed some level of catalytic activity. Numerous CYS

mutants were generated for spin-labeling and EPR studies of the motion of the pro-segment.

However, to date, expression levels remain low. An addition problem that remains involves

stability of the zymogen following removal of the thioredoxin fusion tag.

Scope of the Dissertation

This first chapter of the dissertation, entitled Introduction to Aspartic Proteases, served as

an introduction to the biological aspects of the research included in this dissertation. As the title

of the work implies, the research in this dissertation focuses on the aspartic proteases HIV-1

protease and prorenin. HIV was introduced as the causative agent of AIDS. HIV and AIDS

have become a world epidemic, and a large proportion of medical research focuses on the virus.

HAART treatment and other therapeutic approaches to treatment are described, followed by a

detailed description of the function and structure of HIV-1 protease. Conformational ensembles

and conformational sampling is described with respect to flap motion and dynamics. A summary

of previous NMR, ITC, MD, and X-ray data suggesting that the flaps of HIV-1 protease must

undergo a large conformational change to facilitate binding and catalysis of substrate is given.

Natural and drug-pressure selected polymorphisms are introduced and a description of groups,

subtypes, circulating recombinant forms, and drug-resistant patient isolates are discussed. Next,

a discussion of the aspartic protease renin and its zymogen prorenin is discussed. Renin is an

integral part of the renin-angiotensin system (RAS), which is a modulator of blood volume,

blood pressure, and cardiac and vascular function; therefore, many hypertension drugs function









by regulating blood pressure at various points in the RAS. The structure of renin and prorenin

are discussed and compared to pepsinogen, a well-studied zymogen of the aspartic protease

pepsin. The problems associated with recombinant bacterial expression of aspartic proteases

with pro-segments due to the necessity of protein refolding from inclusion bodies are discussed,

and were related to the fact that very little structural data on prorenin is available in the literature.

Current methods used to obtain prorenin for biophysical studies are described, and most

commonly include expression and purification from human embryonic kidney (HEK) or Chinese

hamster ovary (CHO) cells, the baculovirus (BEV) system, and isolation from human kidney

cells. Project goals, including developing a novel method for recombinant bacterial expression

and purification without the need for refolding, are described.

Chapter 2, entitled Backgroundfor Techniques and Methodologies, begins by providing a

summary of the types of techniques and methodologies performed during the course of this

work. The first major section of chapter two serves to discuss the theory of recombinant protein

expression from E. coli and subsequent purification of the target protein. Characteristics of

proteins that can be exploited for the purposes of purification, including size, charge, and binding

affinity are detailed, along with the methods that can be utilized based upon each particular

characteristic. Circular dichroism spectroscopy was utilized to ensure proper secondary structure

of each individual recombinant protein, thus the theory and instrumentation of the technique are

examined. A large proportion of the work presented in this dissertation deals with site-directed

spin-labeling in conjunction with electron paramagnetic resonance spectroscopy. This chapter

details the theory of site-directed spin-labeling, as well as the procedures used in the spin-

labeling reaction and different types and characteristics of spin labels. Next, the principles of

continuous wave and pulsed EPR are discussed, including a condensed mathematical









background, discussion of protein requirements, and detailed discussion of data analysis.

Detailed data and error analyses have been developed for the DEER data presented in this

dissertation, and those aspects are discussed in great detail.

Chapter 3 is entitled Continuous-Wave Electron Paramagnetic Resonance Studies ofHIV-

1 Protease. The first part of this chapter gives results of CW EPR studies of subtype F and

CRF01_A/E with each of 9 FDA approved protease inhibitors and the non-hydrolyzable

substrate mimic CA-p2. As described in detail, the CW EPR line shapes do not report on the

true motions of the protease flaps as previously determined by other methodologies, thus

providing a reason to expand these studies to pulsed EPR (discussed in Chapter 4). The second

part of Chapter 3 summarizes the results of a study that utilized site-directed spin-labeling and

electron paramagnetic resonance spectroscopy to monitor the autoproteolysis of active HIV-1

Protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy.

Chapter 4 is entitled Pulsed Electron Paramagnetic Resonance Studies ofHIV-1 Protease.

The first part of this chapter details the results of study designed to compare the apo proteases

from each of the six constructs, namely Subtypes B, C, F, CRF01_A/E, and Multi-Drug

Resistant Patient Isolates V6 and MDR769. Dipolar modulated echo data was used to extract

information regarding the conformational ensembles of the protease flaps. Interestingly, the

distance profiles for each of these constructs differ substantially from one to the next. Because

the structures of these proteases are not extremely different, these results are of great importance.

This work summarizes a publication in the Journal of the American Chemical Society entitled

"Subtype polymorphisms among HIV-1 protease variants confers altered flap conformations and

flexibility, by Kear et al.(Kear, Blackburn et al. 2009) The next part of this chapter examines the









results from DEER studies of CRF01_A/E with each of 9 FDA approved protease inhibitors and

the non-hydrolyzable substrate mimic CA-p2.

Chapter 5 is entitled Soluble Expression andPurification of Multiply-Disulfide Bonded

Proreninfrom Escherichia coli. The work described in this chapter details the setup and study

of an innovative system for expression and purification of prorenin. Thioredoxin fusion

methodology is utilized in an attempt to circumvent inclusion body formation in the production

of soluble, properly folded protein. Also included are the results of cysteine mutagenesis in

creating several DNA constructs for potential electron paramagnetic resonance studies to

examine the conformational change of the pro-segment associated with activation of the

zymogen to the active protease.

Chapter 6, entitled Conclusions andFuture Directions, provides a brief summary of the

conclusions drawn from the data presented within this dissertation. Following these conclusions

is a summary of several future studies that must be done to provide a thorough interpretation of

the data discussed herein. Our early spectroscopic results on HIV-1PR are both exciting and

promising, but there is a multitude of work still to be done.









CHAPTER 2
BACKGROUND FOR TECHNIQUES AND METHODOLOGIES

Introduction

Several techniques were utilized herein, including protein expression and purification,

circular dichroism (CD), site-directed spin-labeling, continuous-wave (CW) electron

paramagnetic resonance spectroscopy (EPR), and the pulsed technique Double Electron-Electron

Resonance (DEER), also known as pulsed electron double resonance (PELDOR). General

overviews to these methods are given in the following sections. Additionally, each research

chapter has a detailed materials and methods section for all experiments contained within.

Setting up a Protein Expression System for Escherichia coli

The first step in the characterization or study of a protein is to either isolate the protein

from natural sources or to over-express the target protein from a recombinant source, such as

Escherichia coli (E. coli). Recombinant expression involves incorporation of the DNA (or gene)

encoding the protein of choice into a double-stranded DNA vector which may then be

transformed into a bacterial cell for transcription and translation by cellular machinery. After

over-expression, proteins can be separated from unwanted cellular components and other non-

target proteins based upon numerous physical characteristics including size, charge or isoelectric

point, and binding affinity to various solid phases. Numerous considerations must be made when

setting up a bacterial expression system for a target protein, including how to obtain the DNA,

what type of bacterial cells to use, and what expression vector to use. These are all questions

that must be carefully deliberated prior to setting up a protein expression system, and each of

these considerations will be discussed in detail in the following pages.

The first consideration when setting up a protein expression system in E. coli regards the

DNA sequence to be used as a template for the target protein. If your laboratory does not have









access to a DNA synthesizer, numerous companies such as DNA2.0 (https://www.dna20.com)

for example, are available to synthesize the gene of choice at a fair cost. Synthetic genes have

the added advantage that they can be optimized to enhance expression focusing on characteristics

required by the expression system and host. Synthetic genes can be optimized for codon bias

and are free of unwanted regulatory elements such as pause or stop loops, which may hinder

protein expression from a non-native host.

If the target protein is not a natural E. coli protein, codon optimization may need to be

considered. Because there are 64 possible codons and only 20 naturally occurring amino acids,

most amino acids are encoded for by numerous codons, but most organisms preferentially

synthesize transfer RNAs (tRNAs). It is therefore imperative to compare the codon usage of E.

coli with that of the organism from which the target protein is derived, and synthesize the gene

accordingly. Without codon optimization, bacterial expression levels of eukaryotic proteins may

be very poor. Table 2-1 details differential codon usage in Homo sapiens and E. coli cells.

Table 2-1. Differential codon usage in Homo sapiens and E. coli cells.
Codon Amino acid H. sapiens Codon Usage E. coli codon usage
TTT Phenylalanine 0.43 0.57
TTC Phenylalanine 0.57 0.43
TTA Leucine 0.06 0.15
TTG Leucine 0.12 0.12
CTT Leucine 0.12 0.12
CTC Leucine 0.20 0.10
CTA Leucine 0.07 0.05
CTG Leucine 0.43 0.46
ATT Isoleucine 0.35 0.58
ATC Isoleucine 0.52 0.35
ATA Isoleucine 0.14 0.07
ATG Methionine 1.00 1.00
GTT Valine 0.17 0.25
GTC Valine 0.25 0.18
GTA Valine 0.10 0.17
GTG Valine 0.48 0.40
TCT Serine 0.18 0.11
TCC Serine 0.23 0.11









. Continued.


Table 2-1
Codon
TCA
TCG
CCT
CCC
CCA
CCG
ACT
ACC
ACA
ACG
GCT
GCC
GCA
GCG
TAT
TAC
TAA
TAG
CAT
CAC
CAA
CAG
AAT
AAC
AAA
AAG
GAT
GAC
GAA
GAG
TGT
TGC
TGA
TGG
CGT
CGC
CGA
CGG
AGT
AGC
AGA
AGG
GGT
GGC


Amino acid
Serine
Serine
Proline
Proline
Proline
Proline
Threonine
Threonine
Threonine
Threonine
Alanine
Alanine
Alanine
Alanine
Tyrosine
Tyrosine
**stop**
**stop**
Histidine
Histidine
Glutamine
Glutamine
Asparagine
Asparagine
Lysine
Lysine
Aspartate
Aspartate
Glutamate
Glutamate
Cysteine
Cysteine
**stop**
Tryptophan
Arginine
Arginine
Arginine
Arginine
Serine
Serine
Arginine
Arginine
Glycine
Glvcine


H. sapiens Codon Usage
0.15
0.06
0.29
0.33
0.27
0.11
0.23
0.38
0.27
0.12
0.28
0.40
0.22
0.10
0.42
0.58
0.22
0.17
0.41
0.59
0.27
0.73
0.44
0.56
0.40
0.60
0.44
0.56
0.41
0.59
0.42
0.58
0.61
1.00
0.09
0.19
0.10
0.19
0.14
0.25
0.21
0.22
0.18
0.33


E. coli codon usage
0.15
0.16
0.17
0.13
0.14
0.55
0.16
0.47
0.13
0.24
0.11
0.31
0.21
0.38
0.53
0.47
0.64
0.00
0.55
0.45
0.30
0.70
0.47
0.53
0.73
0.27
0.65
0.35
0.70
0.30
0.42
0.58
0.36
1.00
0.36
0.44
0.07
0.07
0.14
0.33
0.02
0.03
0.29
0.46









Table 2-1. Continued.
Codon Amino acid H. sapiens codon usage E. coli codon usage
GGA Glycine 0.26 0.13
GGG Glycine 0.23 0.12

For the purposes of sub-cloning the gene of choice into an expression vector, the gene

must be flanked by two different restriction sites, which are typically palindromic. Restriction

sites are sequences in the DNA that are recognized by restriction enzymes, which are

endonucleases found in bacteria. An example of a common restriction enzyme is BamHI, which

recognizes the restriction site G'GATCC, where the apostrophe denotes the site of cleavage.

BamHI cleaves after the first G on each strand of a double-stranded DNA sequence containing

the restriction site, leaving an overhang on each strand (GATCC). Overhangs created by

restriction sites are single-stranded unpaired regions which can be ligated to DNA sequences

containing complementary overhangs. Over 600 different restriction enzymes are commercially

available (Roberts, Vincze et al. 2007).

Restriction sites should be chosen based upon the sites available in the expression vector.

It is important, however, to ensure that the chosen restriction sites are not found naturally in the

gene. An expression vector is generally a double-stranded, circular DNA plasmid, which is a

non-chromosomal piece of DNA which can replicate independently from the chromosome.

Plasmids used as tools for gene expression are often referred to as vectors. For this purpose, the

gene of interest is inserted into the vector by a process called ligation; and the modified vector is

subsequently inserted, or transformed, into a host cell.

Expression vectors must possess four necessary components, the first of which is called the

multiple cloning site (MCS), or polylinker region. The MCS contains numerous restriction sites

for insertion of the gene of interest into the expression vector. The second necessary component

of an expression vector is an antibiotic resistance gene which is used for selection of transformed









host cells. The bla gene, which encodes TEM1-p-lactamase is one of the most common

ampicillin resistance (AmpR) markers used in molecular biology. The third essential component

of an expression vector is the origin of replication, or site within the vector at which replication

is initiated. Structure of the origin of replication varies, but all origins of replication have high

Adenine-Thymine (AT) content. A protein complex called the pre-replication complex binds the

origin of replication, unwinds the DNA, and begins the replication complex. A common

example of the origin of replication in expression vectors is called ori. Large plasmids may

require more than one origin of replication in order for timely replication to occur (Baker and

Wickner 1992). The final necessary component of an expression vector is the promoter, or the

region of the gene in which RNA transcription begins. Certain promoters are constitutive,

meaning that they function all the time. It is advantageous, however, for promoters on

expression vectors to be inducible, not constitutive. A common type of inducible promoter is the

T7 promoter, which is known as a strong promoter. An example of an expression vector map

with each of the four required features is shown in Figure 2-1.

A myriad of commercial vectors are available, each with particular purposes and

characteristics. Certain vectors can be used to tag a protein for ease of purification or for

identification via Western Blotting. Common tags for this purpose are the 6His-tag and '1His-

tags, in which 6 or 10 successive His residues are attached to either the N-terminal or C-terminal

end of the protein to provide a means for purification via nickel affinity chromatography. The

vectors used for the research in this dissertation are called pET vectors, which is named for

plasmidd for expression by the T7 RNA polymerase". There exist many varieties of pET

vectors, each with different characteristics; those chosen for use herein are called pET32a(+) and

pET23b. Specific vectors will be described in more detail in future chapters.










Multiple
Cloning Site


Origin
Replical


Antibiotic
Resistance

Figure 2-1. Example vector map highlighting necessary features of plasmid, including inducible
promoter, origin of replication, antibiotic resistance gene, and the multiple cloning
site.

Another important consideration when setting up a protein expression system for E. coi

is the choice of E. coi strain. The prototype expression strain is called BL21(DE3), with

genotype F ompT gal dcm lon hsdSB(rB mB-) X(DE3 [lacI lacUV5-T7 gene 1 indl sam7 nin5]).

BL21(DE3) contains the T7 expression cassette, which is a vector carrying a promoter sequence,

an open reading frame (ORF) corresponding to the T7 RNA polymerase, the LacI gene, and a

polyadenylation site. The lacI gene encodes a repressor that binds the promoter when lactose (or

often a lactose analog or derivative) is not present and transcription occurs only at basal levels.

Isopropyl-P-D-1-thiogalactopyranoside (IPTG) is a common lactose analog (Figure 2-2) used in

molecular biology. When added, IPTG binds to the repressor, changing its shape and preventing

it from binding to the promoter such that T7 RNA polymerase transcription can proceed. IPTG

has the advantage over lactose that it cannot be metabolized by E. coi and thus its concentration,

and concurrent rate of expression, remains constant. Standard E. coi cells do not produce T7

RNA polymerase; thus plasmids controlled by a T7 promoter have repressed expression until

induction with IPTG.























Figure 2-2. Structures of A) lactose and B) the lactose analog IPTG.

Other strains of E. coli cells discussed within the scope of this dissertation are the

OrigamiB(DE3) (taxon identifier 469008) and BL21(DE3)pLysS strains. BL21(DE3)pLysS

cells are based upon the BL21(DE3) strain, but also contains the pLysS plasmid, which carries

the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target

genes but does not interfere with levels of expression post-induction with IPTG. This type of

cell is necessary in order to produce proteins that are toxic to the E. coli cell (such as active HIV-

1 Protease). BL21(DE3)pLysS cells are of the genotype BL21(DE3)pLysS F-, ompT, hsdSB

(rB-, mB-), dcm, gal, ,(DE3), pLysS, Cmr. Finally, Origami B (DE3) cells, of the genotype F-

ompT hsdSB(rB- mB-) gal dcm lacY1 aphC (DE3) gor522 Tnl0 trxB (KanR, TetR), are E. coli

strain K-12 derivatives that have mutations in both the thioredoxin reductase (trxB) and

glutathione reductase (gor) genes. Mutations in these genes function to enhance disulfide bond

formation in the cytoplasm.

Inclusion Body Isolation and Protein Refolding

Eukaryotic cells utilize a process called secretion to synthesize proteins and release them to

the external environment. Proteins are synthesized on ribosomes on the rough endoplasmic

reticulum (ER) (Figure 2-3), which has a pH of approximately 7. As the proteins are translated,









they are translocated to the endoplasmic reticulum (ER) lumen for post-translational

modifications such as glycosylation, and chaperone proteins assist in proper protein folding.

Properly folded proteins are packaged in vesicles and trafficked to the Golgi apparatus (pH 6.5)

for further post-translational modifications, then the proteins are packaged into secretary vesicles

(pH 5.0 6.0) which fuse with the cell membrane thus releasing the properly folded, mature

proteins into the exterior environment (Anderson 2006).




SPlasma membranes


R.'.



Capsul8 g lrl l lqgellum
Nucleoid (circularDNA)



Figure 2-3. Internal structures of A) prokaryotic and B) eukaryotic cells demonstrating the
drastic differences between the two and the lack of compartmentalization in the
prokaryotic cell as opposed to the high level of structure within the eukaryotic cell; 1)
nucleolus, 2) nucleus, 3) ribosome, 4) vesicle, 5) rough endoplasmic reticulum (ER),
6) Golgi apparatus, 7) cytoskeleton, 8) smooth ER, 9) mitochondria, 10) vacuole, 11)
cytoplasm, 12) lysosome, 13) centrioles. Figures obtained from Wikipedia and were
free for copy and redistribution.

Conversely, prokaryotic cells have a reducing, non-compartmentalized internal

environment. As a result, over-expression of non-bacterial proteins in a prokaryotic system often

results in misfolded and aggregated protein. Oftentimes when this occurs, the proteins

accumulate in what are called inclusion bodies. Inclusion bodies are isolated as an insoluble

component following cell lysis and washed with a series of wash buffers designed to further

purify the inclusion bodies away from non-protein cellular components and certain non-target

proteins. The inclusion bodies are then solubilized by a denaturing solution such as urea, then









further purified and subsequently refolded. Though sometimes protein denaturation is

irreversible, protein refolding can sometimes be carried out by slow, drop wise addition to a

solution that promotes folding. This solution is often a buffer, a weak detergent, or a dilute acid

or base, and varies from protein to protein.

Common Methods of Protein Separation

Introduction

Protein purification is defined as the separation of a target protein from all non-target

proteins and other cellular components. Proteins possess many characteristics which can be

exploited for the purposes of purification, including size, charge, and affinity to various ligands

and solid-supports. Described in the following sections are some of these techniques. For a

more comprehensive discussion, readers are referred to many great review articles and textbooks

available for reference (Scopes 1994; Rosenberg 2005). Several steps, chromatographic or

otherwise, often need to be applied, in turn, to successfully purify the protein of interest. The

ideal purification scheme varies from protein to protein, as the physical characteristics of each

protein differ. Various purification schemes, including buffer compositions and ionic strengths,

protein concentrations, and type and order of chromatographic steps are often evaluated before

an ideal scheme can be determined.

Separation Based Upon Size

Proteins can be separated based upon size using several techniques. Size exclusion

chromatography requires a long, narrow column tightly packed with a porous resin. As the

protein sample is introduced onto the column and moves down, smaller proteins will move

through the pores in the resin, while larger proteins bypass the pores and move between the resin

beads, meaning the pathlength of larger proteins is smaller than that of smaller proteins, thus the

elution time is smaller. Hence, proteins will elute from the column with decreasing size.









A second protein separation method based upon size is SDS-PAGE (sodium dodecyl

sulfate polyacrylamide gel electrophoresis). This method is most often used to determine the

level of purity of a sample, but can be used to isolate a particular protein. A protein mixture is

exposed to sodium dodecyl sulfate (SDS), which is a detergent that applies a negative charge to

each protein in proportion to the protein's mass. Additionally, the SDS (Figure 2-4) most often

causes denaturation of the secondary and tertiary structures of the proteins (with the exception of

disulfide bonds); however, depending on the inherent structure of the protein, boiling of the

sample can also be required to ensure that the proteins will be separated based solely upon size

with little to no influence by structure or charge. An electric field is applied to the SDS-PAGE

gel, causing the negatively charged proteins to move through the polyacrylamide gel matrix with

a speed dependent upon the size of the protein as a result of the amount of resistance

encountered, resulting in separation based upon size.

0
O S // Na+


Figure 2-4. Structure of the detergent sodium dodecyl sulfate.

Separation Based Upon Charge or Isoelectric Point

Ion exchange chromatography can be carried out by via two different techniques, namely

anion exchange or cation exchange. Anion exchange chromatography involves binding a

negatively charged protein to a positively charged stationary phase within a column.

Conversely, cation exchange chromatography involves binding a positively charged protein to a

negatively charged stationary phase within a column. The rest of this section will provide details

on anion exchange chromatography. To ensure that the protein of interest carries a net negative

charge, the mobile phase must be at a pH higher than the isoelectric point (pI) of the protein of










interest and have a low ionic strength. Typical resin used for anion exchange chromatography is

called Q-resin, which is bound to a quaternary amine that carries a positive charge as shown in

Figure 2-5. As the protein sample is applied to the anion exchange column, proteins with a net

negative charge will bind via an ionic bond and neutral or positively charged proteins will come

out in the flow-through and wash. Often, a gradient of increasing ionic strength can then be

applied to remove the proteins bound within the column. Ion exchange chromatography is

generally a quite effective method of protein purification and thus is often used as a primary

purification step.

CH3
Q- +
-- N --CH
(I)

CH3


Figure 2-5. Anion exchange Q-resin bound to a positively charged quaternary amine.

Separation Based Upon Binding Affinity

Affinity chromatography is an effective method of protein purification that separates

proteins based upon specific interactions or affinities to various compounds retained via a solid

phase. A common example is immobilized metal ion affinity chromatography (IMAC), where

the stationary phase contains a resin with a chiral chelating group such as nitrilotriacetic acid

(NTA) or iminodiacetic acid (IDA) that is subsequently charged with a metal such as nickel,

copper, or cobalt (Figure 2-6).

A B

o H

SHO B OH
O n -iOH

Figure 2-6. Structures of A) nitrilotriacetic acid and B) iminodiacetic acid.









Proteins that were expressed with an N-terminal or C-terminal His-tag have an affinity for

these metals and will be retained within the column and proteins that have no natural affinity for

the chelated metal ions will be removed with the flow-through or wash. Finally, proteins bound

within the column can be released using a pH gradient, or a competitive binder or metal ion

ligand, such as imidazole, which can be applied to the column over an increasing gradient of

concentration.

Circular Dichroism Spectroscopy

Circular dichroism (CD) spectroscopy is a technique used to observe differences in

absorption of left and right-handed circular polarized light by optically active materials as a

function of wavelength. Circularly polarized light is, by definition, chiral, and thus it interacts

differently with chiral molecules. Circularly polarized light is defined by an angle of 0 = 450

(Figure 2-7), where ER and EL are the magnitudes of the electric field vectors of right and left-

circularly polarized light, respectively. As a result, after passage through the sample, elliptical

polarized light is measured to provide wavelength-dependent differential absorption.

ER+EL



E,-EL




Figure 2-7. Elliptical polarized light (purple) is composed of unequal contributions of right
(blue) and left (red) circular polarized light. Figure adapted from Wikipedia.

When being used to identify secondary structural elements in proteins, CD spectroscopy

is carried out in the far UV regions of approximately 180 260 nm where the chromophore is the

peptide bond. Left and right-handed circularly polarized light will be absorbed to different

extents by the chiral amino acids, thus a CD signal will arise when the peptide bond is in a










structured environment such as an a-helix or 3-sheet. CD spectroscopy in the far UV will yield

different spectra for different types of secondary structure in peptides and proteins (Figure 2-8);

thus, analysis of CD spectra can yield useful information regarding structure of a protein. Often,

the raw data reported by the CD instrument is in the units of millidegrees (0). The units in which

CD results are typically reported are mean residue ellipticity [0] (deg cm-2 dmol- residue-'), as

shown in Equation 2-1,

[Ox100 xlMr
[0] = (2-1)
CxlxNA

where [0] is the mean residue ellipticity, 0 is the ellipticity, Mr is the protein molecular weight, c

is the protein concentration (in mg/mL), / is the cuvette path length and NA is the number of

amino acids in the protein.

90

70
S a-helix
E 50

30
30 Sheet

S10 Random Coil

-10

-30

-50
190 210 230 250
Wavelength (nm)

Figure 2-8. Sample circular dichroism spectra for a-helix, 3-sheet, and random coil.

To accurately measure the secondary structural ensemble of a protein sample, several

factors must be considered in sample preparation. The protein sample needs to be as pure as

possible since non-target protein, particulate matter, or misfolded protein will contribute to the

CD signal. Ideal protein concentrations are typically in the range of 0.5 mg/mL. Buffers,









detergents, or other additives should not absorb in the region of interest (260 180 nm). Buffers

should typically be at or below 5 mM, or as low as possible while maintaining the stability of the

protein.

CD spectroscopy only requires small amounts of protein, is non-destructive to the sample,

and does not require difficult or extensive data processing. In addition, even small changes in

overall secondary structure can be monitored very accurately. The drawback, however, is that

even though secondary structure can be determined, it cannot be determined what regions of the

peptide or protein contains what elements of the secondary structure, thus CD cannot be used to

definitively determine if a protein is properly folded and should only be used as evidence of the

contrary. CD gives far less specific structural information than other techniques such as NMR or

X-ray crystallography. With respect to proteins, CD provides little information on membrane

proteins, as lipids and other membrane structures cause light scattering and are thus unreliable.

Site-Directed Spin-Labeling

Introduction

Site-directed spin-labeling (SDSL), first introduced in the mid 1960's, is a technique

utilized to incorporate a paramagnetic tag into a protein or other biological macromolecule at a

specific site, often an engineered cysteine residue, to facilitate electron paramagnetic resonance

(EPR) studies (EPR will be discussed in detail in the following section) (Griffith and McConnell

1965; Stone, Buckman et al. 1965). SDSL, in conjunction with EPR, is a well-suited technique

for studying the conformations and conformational changes of proteins.

A unique, reactive CYS residue is incorporated into a protein using a technique called site-

directed mutagenesis, in which the DNA is manipulated such that a codon for CYS is positioned

at a chosen point within the sequence. Upon protein expression, the modified DNA sequence

results in a CYS-substituted protein construct which can then be modified with a spin label. Spin









labels are incorporated into a protein via a thiol-based chemistry reaction, of which a general

reaction scheme for incorporation of (1-oxyl-2,2,5,5-tetramethyl-A3-pyrroline-3-

methyl)methanethiosulfonate (MTSL) is shown in Figure 2-9. The spin-labeled side chain can

easily be accommodated by most sites within proteins (especially when that site is aqueous

exposed), and has been shown to be no more perturbing than most other single amino acid

substitutions. The resulting modified CYS residue is often referred to as R1.

r ----------- i
J-SS02CH3 j-S1--s'-C,-C.
+ Protein -SH
6 I-- --

Figure 2-9. Site directed spin-labeling scheme showing the reaction of MTSL with a free thiol
group of an engineered CYS residue.

Conformational changes can be easily monitored with conventional EPR spectrometers, as

the EPR nitroxide spectral line shape is sensitive to local secondary structural elements, local

dynamics, and conformational changes. The EPR spectra of R1 are very sensitive to changes in

secondary structure, which provides an accurate detection of conformational changes in proteins

(Hubbell and Altenbach 1994; McHaourab, Lietzow et al. 1996; Hubbell, Gross et al. 1998;

McHaourab, Kalai et al. 1999; Hubbell, Cafiso et al. 2000; Columbus, Kalai et al. 2001;

Columbus and Hubbell 2002; Fanucci and Cafiso 2006). Three types of motion can be detected

using the SDSL/EPR technique: the intrinsic motion of the spin label, the backbone flexibility in

the region of the R1 label, and the overall tumbling of the molecule in solution. Experimental

parameters such as temperature, viscosity, or modified spin label side chains, can be adjusted

such that individual modes of motion can be studied more directly (Hubbell and Altenbach 1994;

McHaourab, Lietzow et al. 1996; Hubbell, Gross et al. 1998; McHaourab, Kalai et al. 1999;

Hubbell, Cafiso et al. 2000; Columbus, Kalai et al. 2001; Columbus and Hubbell 2002; Fanucci

and Cafiso 2006).









Choice of Spin Label

Dr. Luis Galiano, while a graduate student, examined the affect of the four different spin

labels, namely (1-Oxyl-2,2,5,5-Tetramethyl-A3-Pyrroline-3-Methyl) Methane-thiosulfonate

(MTSL), 4-Maleimido-TEMPO (MSL), 3-(2-Iodoacetamido)-PROXYL (IAP), and 4-(2-

lodoacetamido)-TEMPO (IASL), the structures of which are shown in Figure 2-10, on the CW

EPR nitroxide spectral line shape of HIV-1PR Subtype Bsi.

A B

r----------- I------------ 0--



6I I
C D
-------------- --------------- H
H H
O-N" N-C-T s-- 0C
00
I I



Figure 2-10. Structures of A) MTSL, B) MSL, C) IASL, and D) IAP.

From the structures of each of the spin labels, it is clear that each one contains very

different structure, with respect to size and mobility of head group, rotable bonds, and degrees of

freedom. Each of these spin labels are nitroxide-based where the nitroxide head-group is either a

five or six-membered ring with four methyl groups to help defer collision-induced reactions with

the radical. Each of the spin labels have a flexible linker, or group of atoms that link the

nitroxide to the a-C of the CYS residue, of between four and six bonds. MSL clearly has the

bulkiest head group, leading to its more restricted, anisotropic EPR line shape. On the other

extreme, IASL has the most degrees of freedom with respect to rotable bonds, leading to its

highly mobile, isotropic line shape. Additionally, however, one must consider reversibility of

attachment to the protein backbone. MTSL, for example, makes a S-S bond with the free thiol of









a CYS residue. Under certain conditions, the disulfide bond linking the spin label to the protein

may be reduced, liberating free spin label that could complicate EPR line shape analysis. On the

other hand, MSL makes a non-reducible C-S bond with a free thiol of a CYS residue. The

changes inferred upon the line shapes are due to the differences in inherent spin label mobility.

That data was reported in his 2008 dissertation and is shown in Figure 2-11 as derivative spectra

(Galiano 2008). All things considered, two different spin labels were ultimately chosen for use

in this work, namely MTSL and MSL.

A B









C D











Figure 2-11. Effect of choice of spin label on the derivative EPR line shape of HIV-1PR
Subtype Bsi with A) MTSL, B) MSL, C) IASL, and D) IAP. All spectra were
collected at X-band frequency with 100 Gauss sweep width.

Spin Label Conformations and the Z4/Z5 Model for MTSL

The intrinsic motions and conformations of the spin label are of important consideration,

as they contribute to the mobility of the EPR nitroxide spectral line shape. Here, we will talk

specifically about the X4/X5 model for the MTSL label, which was developed in the laboratory of









Dr. Wayne Hubbell at UCLA and is based upon the spin label positioned in a solvent exposed

site on an a-helix in T4 Lysozyme (Langen, Oh et al. 2000).

As discussed previously, MTSL has a linker region with five theoretically rotable bonds.

However, Hubbell et al. showed that the hydrogen atom on the a carbon can interact with the

sulfur at the 6 position, thus restricting the motion of the first three theoretically rotable bonds

and limiting rotational degrees of freedom to the fourth and fifth rotable bonds, labeled X4 and

X5 in the Hubbell model. Of course, this should only be taken as a model since each individual

protein has the potential to alter the intrinsic mobility of the label. Figure 2-12 graphically

illustrates the theory behind the X4/X5 model.

H3C H2
X5 xCX4 X3 S X2
H3Cx5 Cy S CH2
\ /
CH3 N
0 CH3 H
0

Figure 2-12. Graphical representation of the X4/X5 model for the MTSL label, which was
developed in the laboratory of Wayne Hubbell at UCLA and is based upon the spin
label positioned in a solvent exposed site on an a helix in T4 Lysozyme.

Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

Introduction

Electron paramagnetic resonance (EPR) spectroscopy, also known as electron spin resonance

(ESR) spectroscopy, is the study of absorption of microwave radiation by a paramagnetic species

in the presence of an external magnetic field. The unpaired electron has a net dipole due to a

magnetic moment that arises predominantly from spin angular momentum of quantum number m

= 12. In the simplest case of the free electron in solution, the electron's magnetic moment is

degenerate, meaning that the energy of the two spin states (ms = + /2 and ms = /2) is equal.









However, when a magnetic field is applied, the electron can align itself either parallel or

antiparallel to the field in what is known as the Zeeman Effect. The Zeeman Equation (2-2)

describes the difference between the energy levels E, and Ep (AE), where g is the spectroscopic

g-factor (equal to approximately 2 for most samples), 3e is the Bohr magneton, and B is the

strength of the applied magnetic field. The Bohr magneton (9.274x10-24 JT-1), is a

proportionality constant defined in Equation 2-3, where e is the electric charge, h is the Plank's

constant divided by 27n (1.054 X 10-34 J S), me is the mass of the electron (9.109 X 10-31 kg)

(Weil, Bolton et al. 1972; Poole 1983). Energy is absorbed, i.e., resonance occurs, when the

applied energy is equal to the difference in energy levels E, and Ep; this is achieved by

maintaining a constant frequency and sweeping the magnetic field. The energy diagram

describing this graphically is given as Figure 2-13A, while Figure 2-13B shows a typical

derivative absorption spectrum for a free electron in solution (Weil, Bolton et al. 1972; Poole

1983).

AE = hv = gpeB (2-2)

1e= eh / 2me (2-3)

When EPR is performed in conjunction with SDSL, the free electron (ms = */2) on the

nitroxide spin label couples with the nuclear spin from nitrogen (mi = 1) via the hyperfine

interaction. As such, both levels E, and Ep split into three Hyperfine energy levels (based upon

the 21+ 1 splitting rule), providing the system three allowed energy transitions. The energy

diagram and corresponding derivative absorption spectrum is given in Figure 2-14 (Weil, Bolton

et al. 1972; Poole 1983).










A B

E. + VgJ'BO



| < --- g"AB I




AB = h B0=
R
Bo=0 Bo
Bo
Resonance field Br

Figure 2-13. A) Energy level diagram for a free electron in an applied magnetic field, and B)
corresponding derivative of absorption spectrum.




A B
1 1

--- (0,0)

= --1 (1,1) (-1,-1)

E AhE= hv= g /Bj





B-2 1
Bo=O -



Bo Bo


Figure 2-14. A) Energy diagram for a system with a free electron (ms = 12) undergoing hyperfine
interaction with the nucleus of nitrogen (m = 1), and B) representative derivative
EPR spectrum for a nitroxide spin label.

All EPR work described in this dissertation was collected at X-band, for which resonance

occurs at a magnetic field of approximately 3480 Gauss and 9.75 GHz. A list of fields for

resonance at varying microwave frequencies commonly available in EPR is given in Table 2-2.










Table 2-2. Common microwave bands and frequencies used in CW EPR.
Band Frequency (GHz) Bresonance (Gauss)
S 3.0 1070
X 9.75 3480
Q 34.0 12000
W 94.0 34000

Nitroxide Spectral Line Shapes

The EPR spectral line shape is highly sensitive to motion in the environment of the spin

label and thus changes dramatically with changes in correlation time, as shown in Figure 2-15

(Hubbell, Gross et al. 1998; Hubbell, Cafiso et al. 2000; Fanucci and Cafiso 2006). For sites

capable of undergoing fast isotropic motion with a low rotational correlation time, the spectra

show sharp, narrow peaks (top). As motion becomes more restricted and correlation time

increases, resonance peaks become increasingly less sharp, and significant line broadening

occurs, as can be seen in the intermediate, slow (middle), and rigid spectra (bottom).



Fast
(T = 0.5 ns)


S i Intermediate
(T = 3 ns)


Slow
(T =10 ns)


Rigid
(T > 100 ns)



Figure 2-15. Dependence of EPR spectral line shape on motion.

Correlation time is defined by the level of a convolution of mobility, and can be broken

down into three modes of motion. The first mode is rotational correlation time (zR), the rate at

which the protein is tumbling in solution. This rate is modulated by the bulkiness of the protein,

as well as experimental factors such as temperature, viscosity, and the presence of solutes. The









second mode is defined as intrinsic spin label mobility, or internal correlation time (Ti), which

can is defined by torsional oscillations within the spin label, as well as bulkiness of the spin label

head group and the degrees of freedom separating the nitroxide moiety and the a-carbon of the

CYS residue to which the nitroxide moiety is attached, as discussed in previous sections. The

intrinsic spin label mobility can be experimentally determined to a certain extent by repeating

experiments with nitroxide spin labels of varying size and flexibility. The third and final class of

mobility as seen by the EPR spectral line shape is defined by local dynamics and backbone

fluctuation (TB). This type of mobility is modulated by changes in secondary and tertiary

structure, and well as conformational changes within the protein (McHaourab, Lietzow et al.

1996; Columbus, Kalai et al. 2001; Columbus and Hubbell 2002).

Protein Requirements for CW EPR

The CW EPR methodology requires nanomole quantities of spin-labeled protein. Typical

sample sizes are 3-10 microliters of 50-200 [iM protein for a loop gap resonator used at X-band

frequencies. These values are true for all work reported within.

CW EPR Data Analysis

CW EPR line shapes can be analyzed both qualitatively and quantitatively to provide

information regarding the motional ensemble of the spin labeled protein. The overall breadth

and width of EPR spectral features are narrowed by molecular motions, and as such, the line

shape can be analyzed in terms of empirical parameters. Most commonly analyzed are the peak

to peak width of the central resonance line, called AHpp, the second moment (

), and the

normalized resonance line intensities (ILF, ICF, IHF). These parameters have been shown to

correlate with protein secondary structure and can be used to characterize protein conformation

and dynamics.









AHpp is defined as the distance in Gauss between the minimum and maximum of the

central resonance line of the EPR spectrum. An EPR spectrum resulting from fast, isotropic

motion of a highly mobile spin label with short correlation time contains sharp, narrow

resonance lines. As motion becomes more restricted and/or anisotropic and the correlation time

increases, the EPR line shape becomes increasingly broadened. Thus, the value of AHpp

increases as the overall motion of the system decreases and/or anisotropy increases. For all work

presented in this dissertation, additional 20 Gauss scans were collected for calculation of AHpp;

this allows for a more precise calculation since all spectra are collected over 1024 points.

Another empirical parameter that is frequently analyzed is called scaled mobility. Scaled

mobility can be calculated by normalizing AHpp using spectral line widths of the most mobile

and immobile proteins in order to compare AHpp from data collected on different instruments,

and is given in Equation 2-4, where 6i, 6m, 6exp are AHpp from the most immobile, most mobile,

and experimental EPR spectra, respectively (Hubbell, Cafiso et al. 2000).

s-5 -d1'
M = e--
(2-4)

Resonance line peak intensities can be compared (when EPR spectra are area normalized

with respect to the number of spins) as a way to compare mobility of two or more spin-labeled

samples. Most commonly, either the high field resonance intensity (IHF), or the low field/center

field resonance intensity ratio (ILF/ICF) are used. As motion becomes more restricted and

anisotropic, the EPR line shape becomes increasingly broad and the intensity of each of the high

(-1), center (0), and low (+1) field resonances decrease. As motion in the system increases, the

high field resonance intensity and the ratio of the low field/center field resonance will increase.

Calculation of second moment is more complex than previously described spectral parameters.









H -H "
H") = A ( -H)
(2-5)

A precise baseline correction is necessary, and asymmetric spectra require a correction factor to

account for the asymmetry. The nth moment is given by Equation 2-5, where Ho is the center

field, Hj Hj_. is the step size, Hj is the field value for any point j, and yj is the intensity at point

j. Figure 2-16 shows a graphical representation of the determination of these parameters.

A AH B C
Bo

I
I-- r ,-



1HF LF CF






Figure 2-16. Pictorial representation of the determination of common spectral parameters A)
AHpp, B) ILF, ICF, and IHF, and C) second moment (

).

Pulsed Electron Paramagnetic Resonance Spectroscopy

Introduction

Pulsed EPR was pioneered by W. B. Mims in the 1960s and has since become a valuable

tool used in a wide variety of applications. The specific pulsed technique utilized in this research

is called pulsed electron double resonance (PELDOR), or double electron-electron resonance

(DEER). DEER experiments use two different microwave frequencies to measure the strength of

the coupling between two electron spins; in our case, to measure the distance between two

nitroxide spin labels. This technique can be described by a simplified Hamiltonian, given in

Equation 2-6, comprised of three sets of terms: the Zeeman terms for the spin subsets A and B,

and the dipolar coupling term that relates the two, where Q~ASz is the Zeeman term for the spin









subset A, BSzB is the Zeeman term for the spin subset B, and o0,eS'AB is proportional to the

dipolar coupling between spins.

HB = Q AS + BSZ + co eSZAz (2-6)

All pulsed EPR work reported in this dissertation made use of the common 4-pulse

DEER sequence at X-band frequencies. The pulse sequence is shown in Figure 2-17. The

"observer sequence" is comprised of a Hahn echo sequence followed by a refocusing 1800 pulse

(7). This channel is applied at a frequency o (1) which corresponds to the low field resonance

that lays approximately 26 Gauss or 72 VMHz below the central resonance, shown in Figure 2-18.

Hahn Echo Sequence Refocusing Pulse
Tr/2 TT TT

(1 1 I t I 2 2



Pump Pulse
CO (2) : t

Figure 2-17. The 4-pulse DEER sequence. The "observer sequence", applied at a frequency co()
is comprised of a Hahn echo sequence followed by a refocusing t pulse and is applied
at the low field resonance approximately 26 Gauss or 72 VMHz below the central
resonance The 7n "pump pulse" is applied at a frequency o(2) at the location of the
center resonance.

In the experiment, the Hahn Echo sequence is used to produce an echo for the spins in

resonance with the microwave frequency co (1), and consists of an initial 900 pulse (n /2) that flips

the magnetization in the XY plane. After a delay of time Ti, a 7n is applied to invert the

magnetization. After a second delay of time TI, this sequence will give rise to an echo lined up

along the Y axis. At time T2 after the first echo, a 7n pulse is applied to form a refocused echo.

Table 3 describes the standard 4-pulse table used for DEER experiments described within.

















observe

pump
3400 3450 3500
Field (Gauss)

Figure 2-18. Absorption spectra for a nitroxide spin-label with positions of the low field
transition marked as the "observe" frequency and the center field transition marked as
the "pump" frequency. The observer sequence is applied at the low field resonance
approximately 26 Gauss or 72 MHz below the central resonance, where the "pump
pulse" is applied.

Table 2-3. Standard pulse table used for DEER experiments.
+x Pulse Acquisition Trigger
Position 200 600 Position
Pulse Length 16 32 Pulse Length 16
Pos. Display 8 Pos. Display

The "pump" frequency 0(2) is applied to the central resonance located at approximately

3460 Gauss in the X-band EPR spectrum (corresponding to B spins). At the "pump" frequency

(corresponding to A spins), a 7t pulse is applied to invert the population of a separate set of

spins. This 7t pulse is then incremented in time and the intensity of the refocused echo is

monitored. The set of spins affected by the probe frequency experiences a different magnetic

environment before and after the pump 7t pulse. The change in the magnetic environment

experienced by the spins at co (1) affects how the spins realign after the refocusing 7t pulse. The

overall effect is a modulation in the intensity of the refocused echo as a function of the strength

of the coupling between spins. The strength of the coupling is proportional to the inverse cube

of the distance between the spins (proportional to 1/r3). Note, applying the "pump" pulse (o (1))

to the central resonance line results in an increased signal: noise ratio (SNR) because it is the









most populated region of the spectrum and the "observe" frequency (co (2)) is applied to the low

field transition because it is the next most populated region of the spectrum. Theoretically, the

technique is useful to extract distances in the range of 1.5 8 nm (15 80 A), but in practice this

distance is closer to 1.5 5 nm (15-50 A). DEER experiments were configured with the

parameters listed in Table 2-4.

Table 2-4. Standard pulsed EPR parameters used in this study.
Parameter Value
Shot repetition time 4000
Sweep width 160-200 G
Number of scans variable
Shots/point 100
Center Field -3460 G
Low Field -3432 G
Frequency -9.6 GHz
Pulsed Attenuation 0.1
Video Bandwidth 25 MHz5
Modulation Amplitude -1 G
Time Constant 0.082 0.164 sec
Receiver Phase 100

Figure 2-19 shows typical dipolar modulated echo data from a system containing two

separate nitroxide spin labels. The raw dipolar modulated echo curve, designated V(t) (Equation

2-7), is shown as the solid black line. The background, designated B(t), is plotted as a solid

green line. Given the concentration of protein used for DEER experiments, A spins will come in

contact with B spins from separate proteins causing a random distribution of intra-protein spin-

spin interactions, giving rise to a background signal in the form of an exponential decay. The

background corrected dipolar evolution data, designated F(t), is plotted as a solid blue line. It is

quite imperative to select the correct background for subtraction; though the most probable

distance of the distance distribution profile is not likely to change from improper background

subtraction, the high level of detail obtainable from DEER distance measurements can be lost.

V(t) = F(t)B(t) (2-7)









After background subtraction of the dipolar evolution curve, the dipole-dipole interaction can be

written as the function V(t), as shown in Equation 2-8, where K(t) is the kernel function given in

Equation 2-9.


V(t) = K(r, t)P(r)dr
) in (2-8)

1 [ r2ti ]l
K(r,t)= cos (1 3cosO)--t dO
t] (2-9)


Raw Dipolar Modulation
Background
Background Subtracted Echo
Background Subtracted Echo Fit

Co






0.0 0.5 1.0 1.5 2.0 2.5 3.0
t (PJS)

Figure 2-19. Sample dipolar evolution curve showing locations of raw dipolar modulation,
background subtraction, and background-subtracted echo curve with fit.

Phase Memory Time, Tm

For DEER experiments, Tm limits the length of the dipolar evolution curve, which can

affect data analysis particularly for longer distances. T2 is the transverse relaxation time and

describes how quickly the magnetization in the x-y plane dissipates; Tm is a broader term that

encompasses more of the processes that affect the refocusing of spins into an echo, including but

not limited to, local spin concentrations and dipolar interactions with nuclear spins. The use of

glycerol and deuterated materials are common and effective methods of extending the Tm of

samples with solvent-exposed labeling sites. Additionally, glycerol functions as a glassing agent









and cryoprotectant. Glycerol is the most common glassing agent/cryoprotectant used in pulsed

EPR studies; others include polyethylene glycol (PEG), sucrose, and ethylene glycol. A glassing

agent is an additive that increases the glass transition temperature of the sample which helps

avoid protein aggregation during the freezing process. The protein samples examined in this

work were placed in a deuterated buffer with 30% d8-glycerol. Tm is also highly dependent on

experimental temperature; all DEER experiments here were done at 65 K, where Tm is

substantially extended. To measure Tm, a simple echo decay experiment is performed using a 2-

pulse Hahn echo sequence given as 7t/2-Tz-t-z, with typical parameters given in Table 2-5.

Table 2-5. Typical parameters used for the echo decay experiment for determination of Tm.
Parameter Value (ns)
7c/2 16
71 32
T 200
AT 8














0 5000 10000 15000
Time (ns)

Figure 2-20. Typical results of an echo decay experiment using a 2-pulse Hahn echo sequence
(7t/2-T-7t-z) for determination of Tm (black). The data is fit to an exponential decay
function of the form in Equation 2-8 (red).

Spacing between 7t/2 and 27 pulses are separated by an increasing T, (position displacement,

AT). Echo intensity is measured as a function of Figure 2-20 is an example of typical echo









decay data (black), which is fit to an exponential decay function (red) of the form in Equation 2-

10, where Tm is the rate of decay, A is initial intensity, B is the time offset, C is a variable that

describes the exponential function stretching, and D is the value of y as t approaches infinity.

Modulations seen in the data are the result of proton modulation via the electron spin envelope

echo modulation (ESEEM) effect.

yt)A + D (2-10)


Protein Requirements for Pulsed EPR Experiments

The pulsed EPR methodology with the ELEXSYS E580 with MD4 or MD5 dielectric

resonator requires nanomole quantities of spin-labeled protein. Typical sample sizes are 100 [iL

of 150 300 [iM protein. These values are accurate for all work reported within, but may vary

from instrument to instrument

Analysis of DEER Data

DEER data must be analyzed very carefully and extensively to provide the most accurate

and useful information regarding distances between two spin labels in a protein system, and there

are numerous aspects that are important to consider in data analysis steps. For a comprehensive

review on DEER data collection and analysis, the reader is referred to a number of excellent

sources by Gunnar Jeschke, Jack Freed, and others (Jeschke 2002; Jeschke 2002; Jeschke 2004;

Chiang 2005; Jeschke, Chechik et al. 2006; Jeschke and Polyhach 2007). The dipolar evolution

curve is the result of the coupling between the A spins and the B spins; however, converting this

information into a distance profile is complex due to the ill-posedness of the problem (meaning

that there is not a single unique answer). There are several methods used to analyze DEER data,

the most common are the direct Fourier transform, Monte Carlo, and Tikhonov regularization.









Direct Fourier transform

The simplest method used to analyze DEER data is direct Fourier transform of the time-

domain data to give a frequency spectrum where the Pake pattern splitting is proportional to l/r3

(where r is the interspin distance). However, this method does not provide a detailed analysis or

distribution profile. Instead, this method only reports on a most probable distance between spins.

A graphical representation of this analysis method is shown in Figure 2-21.

A B C
H ~1/r3



I \ I I



0 1 2 3 -10 -5 0 5 10 20 25 30 35 40 45 50 55
T (Ls) Frequency (MHz) Distance (A)

Figure 2-21. Pictorial representation of the direct Fourier transform method of analysis, A) time
domain spectrum is converted to a B) frequency domain spectrum, where the
singularities in the Pake pattern are proportional to 1/r3, where r is C) the most
probable distance between two spins.

Curve fitting and Monte Carlo analysis

In order to gain more information than the direct Fourier transform method provides, i.e., a

distance distribution profile, a more complex analysis method is required. A common technique,

referred to as curve fitting, can be employed to solve the inverse problem of generating a

distance profile based on known information about the system, and optimize it until the

theoretical and experimental dipolar evolution curves match. In Monte Carlo (MC) analysis, the

scheme for which is shown in Figure 2-22, the distance profile has a pre-determined form (e.g.,

Gaussian) (Fajer 2006). The MC analysis has the advantage of being a fairly easy and expansive









approach to finding a suitable answer; however, it has the severe limitations that it is both

dependent on an original model and a pre-determined form.



x ) a -(x -b)/(2c2)
f (x)=ae A





0 1 2 3 20 30 40 50
t (s) Distance (A)

Figure 2-22. Pictorial representation of the Monte Carlo analysis.

Tikhonov regularization

A third approach, and the one used for all DEER analysis described in this dissertation, is

called Tikhonov regularization (TKR) (Tikhonov 1943; Hansen 1998; Chiang, Borbat et al.

2005). TKR uses the function in Equation 2-11 to find the best answer to the ill-posed problem

by balancing the quality of fit with the smoothness of the solution by varying the regularization

parameter X (often referred to as a). The first term represents the quality of the fit and the

second term represents the smoothness of the solution; P is probability of the spin-spin distance,

K and L are operators, S is the experimental data vector. Following the TKR process, the log of

rq() is plotted against the log of p(X) to produce an L-curve which is analyzed in order to

determine the optimal regularization parameter. Each individual point on the plot is given by

Equation 2-12, and rq() and p(X) are given in Equations 2-13 and 2-14.

D[P] =1 KP-S 112 +i2 II LP 2 (2-11)


G,(P) = S(t) -D(t) 1 2 2 l P(r) 12
ar (2.12)

p(A) =1 S(t)- D(t) 12 (2.13)

























0 1 2 3





1 (PS)




0.0 0.5 1.0 1.5











T (PS)
0.0 0.5 1.0 1.5
1 Ps)









0.0 0.5 1.0 1.5
T (PS)


S1under-
-12- smoothed

S16 optimal
S over-
-20 smoothed

-24
-4.2 -4.0 -3.8 -3.6


F Distance (A)









20 30 40 50
H Distance (A)



& _/L


20 30 40
Distance (A)


Figure 2-23. Selection of regularization parameter in TKR DEER analysis method; A) raw and
background subtracted dipolar evolution curve and B) typical L-curve showing C)
undersmoothed X and D) resulting distance profile, E) optimal X and F)
corresponding distance profile, and G) oversmoothed X with H) corresponding
distance profile.


(A)1 P(r) 2
r2


(2.14)










When the regularization parameter X is small, G(P) is dominated by the quality of the fit

with experimental data, and as the regularization parameter is increased, G(P) is driven by a

larger contribution from smoothness of the fit, as illustrated in Figure 2-23. A quality L-curve

contains an "elbow," where the slope of the line changes dramatically, and this point corresponds

to the optimal regularization parameter. By selecting this value of X, the optimal distance

distribution profile can be determined.

Zero-time selection

A B
Zero time = 4 ns





S0.00.5



I \ II I I I I
0.0 0.5 1.0 1.5 2.0 2.5 3.0 20 30 40 50
,C (s) Distance (A)


Figure 2-24. Example of zero-time selection for dipolar modulated echo data; A) selection of
zero-time, and B) results of incorrect zero-time selection.

In order to obtain the most accurate distance profile, it is imperative that the correct zero-

time be determined. DEER data is collected with a small amount of negative time, as shown in

Figure 2-24A. If the incorrect zero-time is selected, the distance distribution will be shifted

slightly towards either smaller or greater distances, as demonstrated in Figure 2-24B.

In order to do this, truncated dipolar modulated echo curve in the region of -300 300 ns is

plotted in Origin8.0 and fit to a GaussAmp function of the form shown in Equation 2-15, where

yo is the y-offset, w is the width, A is the amplitude, and x, is the center of the Gaussian-shaped

function, and thus designated as the zero time.









(X--X,)2
y=yo+Ae 2w
(2-15)


Self-consistent analysis

As previously described, the typical dipolar modulated echo curve is affected by the

dipolar interactions between spin labels on different proteins. To accurately analyze the DEER

data with respect to the lowly populated states in a conformational ensemble, it is absolutely

imperative to ensure that the appropriate level of background is subtracted. In efforts to obtain

information about the shape and location of minor populations within the distance profile, and to

ensure that the correct background subtraction was selected, our research group developed a

technique termed self-consistent analysis that facilitates this need, the scheme for which is shown

in Figure 2-25.

In short, the self-consistent analysis consists of an initial determination and subtraction of

background via an approximate Pake transformation using DeerAnalysis2008 software,

following by TKR analysis, resulting in a distance distribution profile. The distance distribution

profile is then regenerated, using the DeerSim program, with a series of Gaussian-shaped curves

representing individual flap conformations. The sum of the Gaussian-shaped curves is used to

generate a theoretical dipolar modulated echo curve, which is free of any contributions from

background. The theoretical and experimental dipolar modulations are compared. If the

theoretical and experimental dipolar modulations are not exactly the same, then the background

subtraction is incorrect, a new background subtraction is selected, and this process is

repeateduntil both the distance profile and the dipolar modulated echo curve are accurately

reproduced.









Experimental Dipolar Modulation


6 1 2 3

I t (ps)
Distance Distribution Profile


Distance (A)

Regenerated Distance
Distribution Profile


>,

0)
-c
0
WU


Compare Dipolar Modulations
Experimental dipolar modulation
Theoretical dipolar modulation


r (ps)


Generated Dipolar Modulation


0 1 2 3
T(|Js)


/


20 25 30 35 40 45 50 55
Distance (A)

Figure 2-25. Self-consistent analysis scheme.

Interpretation of distance distribution profiles

There are several ways to describe and/or interpret the distance distribution profiles for a

biological system. One way is a simple, semi-quantitative description in which the profile is

characterized with respect to most probable distance, average distance, and/or full width at half









max (FWHM), as shown in Figure 2-26. Here, the most probable distance is defined as the most

intense point in the distance profile, and the full width at half max is indicative of flexibility or

range of motion within the system. A sharp, narrow profile can be related to low flexibility or

small range of motion, whereas a broad profile suggests high flexibility and range of motion.

However, a more thorough, quantitative description can be made using a procedure developed in

our lab called Gaussian reconstruction, wherein individual sub-populations are identified and

used to regenerate the distance distribution profiles.



Most probable
distance
SFull
S Width at
Half Max
(FWHM)



20 30 40 50

Distance (A)

Figure 2-26. Semi-quantitative analysis of distance distribution profiles, including most
probable distance and full width and half max.

Gaussian reconstruction process

The Gaussian reconstruction process facilitates a highly quantitative interpretation of the

distance profiles of a biological system. Here, the distance profile is regenerated using a series

of Gaussian-shaped sub-populations, where the sum of the sub-populations defines the

conformational ensemble of the system. An example of this type of analysis is given in Figure 2-

27. Each of the sub-populations can be analyzed for most probable distance, FWHM, and the

relative percentage of the conformational ensemble. Figure 2-27A shows an example of a

distance distribution profile generated using DeerAnalysis2008 software (red), and a regenerated

distance distribution profile (blue dashed) created using the sum of the Gaussian shaped sub-









populations shown in Figure 2-27B. The two small populations centered at approximately 15

and 22 A were suppressed due to a negligible effect on the dipolar modulation; this process will

be discussed in more detail in the following section.

A B

-TKR Tucked
Gaussian Reconstruction Closed
-Semi-open
-Wide-open

Illi





20 30 40 50 20 30 40 50
Distance (A) Distance (A)

Figure 2-27. Quantitative description of distance distribution profiles using Gaussian
reconstruction procedure; A) distance distribution profile generated using
DeerAnalysis2008 software (red) and regenerated distance distribution profile (blue
dashed), and B) series of Gaussian shaped sub-populations used to regenerate the
distance distribution profile.

Error analysis by population suppression and validation

When DEER data analysis is performed using our Gaussian reconstruction process, a

method of error analysis called population suppression is performed. Population suppression is

utilized in order to determine if each of the sub-populations is necessary to properly regenerate

the experimental dipolar modulated echo data. The principle behind this analysis technique is to

individually remove, or suppress, each of the sub-populations one at a time, and also linearly,

then regenerate a theoretical dipolar modulated echo curve that is then overlaid with the

experimental background subtracted dipolar modulated echo data. If the theoretical curve

overlays with the experimental curve within the noise of the signal, the suppressed population

can be labeled as either questionable or unnecessary. If, however, the theoretical echo data no










longer overlays well with the experimental data, the suppressed population is known to

contribute to the overall experimental outcome. An example of the population suppression error

analysis technique is given in Figure 2-28.


30 40
Distance (A)


Distance (A)


0 1 2 3
T(MS)


0 1 2 3 0 1 2 3


Figure 2-28. Example of population suppression error analysis, A) distance profile (red) overlaid
with regenerated distance profile (blue dashed), B) series of Gaussian shaped sub-
populations used to regenerate the distance distribution profile, C-E) theoretical
dipolar modulation with various sub-populations suppressed (blue) overlaid with
experimental dipolar modulation. In each case, it can be seen that when the given
population is suppressed the theoretical echo curve no longer overlays with the
experimental echo curve data, therefore each of the sub-populations was validated as
being a real contributor to the experimental data.


-Tucked
- -Tucked


-TKR
\ bosed


-TKR
\ -- Closed and Tucked









CHAPTER 3
CONTINUOUS WAVE ELECTRON PARAMAGNETIC RESONANCE STUDIES OF HIV-1
PROTEASE

Introduction

As described in previous chapters, human immunodeficiency virus type 1 (HIV-1) protease

(HIV-1PR) is the viral enzyme responsible for virus maturation. Several different HIV-1PR

constructs will be discussed in chapter 3; namely A/Es, A/Esi, Fs, Fsi, Bs, and Bsi. Refer to

Chapter 1 for more detailed discussion on construct nomenclature and amino acid substitution

code. In summary, a subscript "s" refers to stabilization against autoproteolysis (Q7K, L33I,

L63I), and the subscript "i" refers to inactive protease (D25N). Amino acid substitution code

(e.g., D25N) is given by amino acid residue to be substituted out, followed by the residue

number, followed by the amino acid to be substituted in (e.g., D25N the aspartic acid residue at

position number 25 was mutated to an asparagine residue).

Unpublished work performed by graduate student A. Mike Veloro in our lab on the drug

resistant patient isolates V6 and MDR769 (without the three stabilizing mutations Q7K, L33I,

and L63I) has resulted in substantial autoproteolysis of the active proteases during the course of

the purification. Thus, most of our structural work to date has been focused on protease

constructs that have incorporated the Q7K, L33I, and L63I substitutions. Figure 3-1 shows a

16.5% tris-tricine SDS-PAGE gel of self-cleavage products for Subtype B, here called PMPR

(pentamutated protease), with stabilizing mutations Q7K, L33I and L63I (PMPR), and V6 and

MDR769 (MDR) with no stabilizing mutations. Aliquots were taken from freshly prepared stock

solutions immediately after purification. As seen in the SDS-PAGE gel, stabilized Subtype B

has enhanced stabilization from self-cleavage, while both V6 and MDR769 undergo rapid

degradation during the course of the purification. The self-cleavage products of V6 and MDR769

are not known.









kDa

PMPR V6 MDR MPR V6 MDR
MTSL MTSI. MTSL MSL MSI. MSL



I-,
liY PR

products



Figure 3-1. 16.5% tris-tricine SDS-PAGE gel of self-cleavage products for Subtype B with
stabilizing mutations Q7K, L33I and L63I (PMPR), and V6 and MDR769 (MDR)
with no stabilizing mutations. Aliquots taken from freshly prepared stock solutions
immediately after purification.

Because of the rapid degradation of the protease during the course of the purification, each

of the Subtype B, F, and CRF01_A/E constructs examined were stabilized against

autoproteolysis by incorporating amino acid substitutions Q7K, L33I and L63I. These sites have

been reported to slow the rate of autoproteolysis more than 100-fold. Edman degradation

sequencing was used to identify three primary sites of proteolytic cleavage for Subtype B

protease, and include the peptide bonds located between amino acid residues at positions L5-W6,

L33-E34 and L63-I64. In an attempt to slow the rate of autoproteolysis by rendering the primary

site of cleavage less labile, Rose et al. engineered a Q7K substitution into an HIV-1PR construct,

which reduced autoproteolysis in the protein more than 100-fold (Rose, Craik et al. 1998).

Subsequently, Mildner et al. introduced the additional substitutions L33I and L63I to produce a

triply substituted construct which was shown to retain the specificity and kinetic properties of the

wild-type enzyme but was highly stabilized against autoproteolysis (Mildner, Rothrock et al.

1994). When studying inactive protease, these mutations are not necessary. However, because

much of the research in our lab deals with active protease, where purification practically requires

the stabilizing mutations, all constructs examined (with the exception of the drug-resistant









constructs discussed in later chapters due to the location of certain drug-induced polymorphisms)

have incorporated Q7K/L33I/L63I mutations to allow for comparison between active and

inactive protease. Figure 3-2 shows a crystal structure of HIV-1PR highlighting the D25 active

sites residues, the K55 site chosen for modification as a reporter site, and stabilizing mutations at

positions Q7, L33, and L63.


K55 --

L331 11
D25'
L631

Q7K


Figure 3-2. Ribbon diagram of HIV-1PR structure, PDB ID 2BPX. Sites of K55 reporter site,
D25 active site residues, and sites of Q7K, L33I and L63I stabilizing substitutions are
labeled. Structure rendered in VMD (Humphrey 1996).

Within this chapter are results from continuous wave electron paramagnetic resonance

(CW EPR) studies on spin-labeled HIV-1PR constructs. The materials and methods section

contains detailed information about all aspects of the experiments described within chapter 3,

including cloning of the HIV-1PR gene, site-directed mutagenesis, expression and purification of

the protein and details of constructs examined, spin-labeling, circular dichroism, sample

preparation and storage, CW EPR and analysis of EPR results, as well mass spectrometry

experiments and analysis.

The next section of the chapter gives the results of a study on the effect of inhibitors on

the CW EPR nitroxide spectral line shape of HIV-1PR Subtype Fsi and CRF01_A/Esi. The

normalized spectra of the apo constructs were compared to those of the protease in the presence

of nine different FDA-approved protease inhibitors used in the treatment of HIV, and a non-

hydrolyzable substrate mimic CA-p2. Because the EPR nitroxide spectral line shape is sensitive









to motion, it is feasible that upon substrate binding and flap closure the line shape would appear

more emotionally restricted.

The final section of the chapter gives results from a study regarding autoproteolysis in the

active Subtype Fs and CRF01_A/Es constructs. The EPR nitroxide spectral line shape is highly

sensitive to mobility in the environment of the spin label, thus changes dramatically with

changes in correlation time. Contributions to correlation time come from three modes of motion,

including global protein tumbling (TR), intrinsic spin label mobility (zi), and backbone

fluctuations (TB). Autoproteolysis affects the rate of global protein tumbling by decreasing

rotational correlation time of the spin labeled protein. As total correlation time decreases, the

derivative EPR spectra decrease in breadth and resonance line shapes become sharper and

increasingly narrow. Here, we monitored the development of a sharp component in the high

field that is proportional to the amount of degraded protein in the sample, and the intensity of the

high field line (IHF) was quantitatively analyzed.

Dr. Luis Galiano performed a similar experiment with Subtype Bs in which autoproteolysis

was monitored for approximately 50 days using CW EPR (Galiano 2008). Minimal cleavage

occurred in the protease sample, as evidenced by the small change in the high field intensity of

the EPR line shape (Figure 3-3). Subtype B, because it is the most prevalent subtype in the

United States and Europe, is the most studied protease subtype. It is important, however, not to

neglect the structural and dynamic changes that subtype polymorphisms can incur on the

protease. Thus, this study was performed in part to compare the rates and locations of the

autoproteolysis of Subtype Fs and CRF01_A/Es to Subtype Bs. In addition, storage conditions

and affect of the inhibitor were examined. Subtype F and CRF01_A/E protease samples were








prepared and stored at 37 C, 25 C, or 4 C, and in the presence of a protease inhibitor or non-

hydrolyzable substrate mimic.

-t=0
t = 47 High-field
t=47
sharp component

tI

20 Gauss





Figure 3-3. Overlay of day 1 (black) and day 47 (grey) area normalized X-band 100 Gauss CW
EPR spectra for subtype B HIV-1 protease labeled with MSL at position 55 and
stored at 25 C.
Also performed by Galiano were experiments to determine the effect of salt concentration

on the CW EPR spectral line shape of HIV-1PR (Galiano 2008). While developing the

purification scheme, it was observed that HIV-1 PR was very sensitive to salt concentration. To

study the effect of salt concentration on the EPR spectrum of HIV-1PR, 100 Gauss X-band CW

EPR spectra were collected for 100 tM HIV-1PR, labeled with MTSL at the K55C and K55C'

positions, in 2 mM NaOAc with increasing concentrations of salt (0, 50, 500, and 2500 mM),

and results are shown in Figure 3-4.

It is known that the enzymatic activity of HIV-1PR is dependent upon ionic strength, with

greater activity observed in high salt concentrations near 2 M (Szeltner and Polgar 1996).

However, the EPR spectral line shapes from 100 tM Subtype B HIV-1PR labeled with MTSL

show significant broadening when the salt concentration is increased above 50 mM NaC1. These

changes in spectral line shapes can be indicative of protein aggregation which was confirmed









upon inspection of the capillary tube which revealed precipitated protein above 500 mM NaC1.

From these findings, buffers for all EPR experiments are prepared at low ionic strength with a

maximum of 50 mM NaCl (Galiano 2008).





A

B

C
c --- ------1 -

20 Gauss


Figure 3-4. Area normalized 100G X-band CW EPR spectra of 100 pM HIV-1PR as a function
of salt concentration; A) 2 mM NaOAc + 0 mM NaC1, B) 2 mM NaOAc + 50 mM
NaC1, C) 2 mM NaOAc + 500 mM NaC1, and D) 2 mM NaOAc + 2500 mM NaC1.

Materials and Methods

Materials

The chemicals, reagents, and supplies were obtained from Fisher Scientific (Pittsburg,

Pennsylvania) and used as received, with a few noted exceptions. pET23 DNA was purchased

from Novagen (Gibbstown, New Jersey). AG 501-X8 (D) resin, 20-50 mesh was purchased

from BioRad (Hercules, California), HiTrap Q HP Anion Exchange column, HiPrep 16/60

Sephacryl S-200 high resolution size exclusion column was purchased from GE Biosciences

(formerly Amersham, Pittsburg, Pennsylvania). HIV-1 Protease DNA was synthesized and

subsequently purchased from DNA2.0 (Menlo Park, California). 4-maleimido-2,2,6,6-

tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO, MSL) was purchased from Sigma-Aldrich

(St. Louis, MO). (1-oxyl-2,2,5,5-tetramethyl-A3-pyrroline-3-methyl) methanethiosulfonate spin

label (MTSL) was purchased from Toronto Research Chemicals, Inc. (North York, Ontario,









Canada). The QuikChange site-directed mutagenesis kit was purchased from Stratagene (La

Jolla, California). 0.60 I.D. x 0.84 O.D. capillary tubes (Cat # CV6084) were purchased from

Fiber Optic Center (New Bedford, Massachusetts). BL21*(DE3) pLysS E. coli cells were

purchased from Invitrogen (Carlsbad, California). Ritonavir, Indinavir, Tipranavir, Darunavir,

Amprenavir, Atazanavir, Nelfinivir, Saquinavir, and Lopinavir were generously received from

the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Bethesda,

Maryland) (NIH). The non-hydrolyzable substrate mimic, CA-p2 (H-Arg-Val-Leu-r-Phe-Glu-

Ala-Nle-NH2 (R-V-L-r-F-E-A-Nle-NH2, r = reduced) was synthesized and purchased from the

University of Florida Protein Chemistry Core Facility (Gainesville, Florida).

Methods

Cloning of HIV-1 protease

The Escherichia coli (E. coli) codon and expression optimized genes for HIV-1 protease

CRF01_A/Esi K55C and Subtype Fsi K45C (sequences are given in Tables 3-1 and 3-2,

respectively) were purchased from DNA 2.0 and received in pJ201:24237 and pJ201:24236

vectors (Figures 3-5A and 3-5B). Plasmids containing the CRF01_A/Esi K55C and Subtype Fsi

K45C were cleaved using the restriction enzymes Ndel and BamHI and subsequently ligated into

the pET23a vector (Figure 3-5C) that had been previously digested using the same enzymes.

Standard restriction digestion and ligation procedures were utilized. The ligated vector was then

transformed into XL1 blue cells, then isolated and purified using the Qiagen mini-prep kit and

checked by sequencing. These constructs will now be referred to as pET23aFsiK45C and

pET23_A/EsiK55C.
































Xho I(1E8
CI.,,

NgoA IV(3525). ,


BpU10 1(44)
sBbs (-)
Bag IBag B
Eco47 I11(42)

BsH k'1 i
pET-23a(+)
S(3666bp) Pvu llp7s

S t t hl 11 33
Bsl107 1(115.)
Bg(I4-i) C' Sa p1 1272)
Ahdl221^) BspLUli 138)




Figure 3-5. Vector maps of A) pJ201:24237 with HIV-1PR CRF01_A/E insert (red), B)
pJ201:24236 with HIV-1PR subtype F insert (red) (vector maps in A) and B) were
created by DNA 2.0 and received with DNA constructs), and (C) pET23 vector
(vector map adapted from Novagen).

Site-directed mutagenesis of HIV-1 protease constructs


To obtain each of the constructs described within this chapter, including protease


constructs pET23a_FsiK55C, pET23_FsK55C, and pET23_ A/EsK55C, several rounds of site-


directed mutagenesis were performed. Primer sequences, melting temperatures (Tm), and


molecular weights are given in Tables 3-1 and 3-2 for Subtype F and CRF01_A/E, respectively.


Thermal cycling parameters are given in Table 3-3, and the DNA and amino acid sequences of


all constructs discussed within this chapter are given in Tables 3-4 3-8.









Table 3-1. PCR primers utilized to introduce mutations to HIV-1PR CRF01 A/E.
Mutation Primer (5' 3') Tm (C) m.w. %GC
C55K
Forward TGGCGGCATCGGCGGCTTTATCAAA 72.0C 12,611.2 56.0%
Reverse CAATCTCGATAATGATCTGGTCGTA 71.60C 12,440.4 40.0%

N25D
Forward GTGGTCAACTGAAAGAAGCGCTGC 70.90C 14,962.7 54.1%
Reverse TCAATGACCGTATCATCCGCACCGGT 70. 1C 14,310.3 53.8%

T74C
Forward GGTAAGAAGGCAATTGGCTGCGTCTT 69.40C 12,094.8 50.0%
Reverse CCATTCTTCCGTTAACCGACGCAGAA 69. 10C 11,881.7 50.0%


Table 3-2. PCR primers utilized to introduce mutations to HIV-1PR subtype F.
Mutation Primer (5' 3') Tm (oC) m.w. %GC
C45K
Forward GACATGAATCTGCCGGGTAAGTGG 68.10C 13,718.9 54.2%
Reverse TTTGATAAGACCAATACCGCCAATC 67.80C 14,901.7 40.0%

K55C
Forward ATGATTGGCGGTATTGGTGGTTTCAT 67.20C 13,319.7 42.3%
Reverse ATGATAATCTGATCGTATTGCTTGAC 67.5C 15,008.8 34.6%

N25D
Forward CCAATTGAAGGAGGCCCTGCTGGAT 71.60C 11,769.6 56.0%
Reverse AATCACGGTATCGTCCGCACCGGTA 72.30C 12,202.9 56.0%

T74C
Forward GGCCACAAAGCGATCGGTTGTGTTC 70.30C 11,710.6 56.0%
Reverse CCGGTGTTTCGCTAGCCAACACAAG 69.80C 11,648.6 56.0%

Table 3-3. Thermal cycling parameters for HIV-1 protease site-directed mutagenesis reactions.
Segment Cycles Temperature Time
1 1 95 oC 30 seconds
2 18 95 C 30 seconds
55 oC 1 minute
68 OC 6 minutes

Table 3-4. E. coli codon-optimized HIV-1PR subtype Fsj K45C DNA and amino acid sequences.
1 ccg cag att acc ctg tgg aag cgt ccg ctg
P Q I T L W K R P L
11 gtc acg atc aaa gtt ggc ggc caa ttg aag
V T I K V G G Q L K
21 gag gcc ctg ctg aac acc ggt gcg gac gat
E A L L N T G A D D










Table 3-4. Continued.


31 acc
T
41 aag
K
51 ggt
G
61 cag
Q


gtg
V
tgg
W
ggt
G
att
I


att
I
aaa
K
ttc
F
atc
I


gag
E
ccg
P
atc
I
atc
I


gac
D
tgc
C
aaa
K
gaa
E


atg
M
atg
M
gtc
V
atc
I


aat
N
att
I
aag
K
gct
A


ctg
L
ggc
G
caa
Q
ggc
G


ccg
P
ggt
G
tac
Y
cac
H


ggt
G
att
I
gat
D
aaa
K


71 gcg atc ggt act gtt ctg gtt ggc cca acc
A I G T V L V G P T
81 ccg gtg aat atc att ggt cgc aac ttg ctg
P V N I I G R N L L
91 acg cag att ggt gca acg ctg aac ttc
T Q I G A T L N F


Table 3-5.
1 ccg
P
11 gtc
V


E. coli codon-optimized HIV-1PR
cag att acc ctg tgg aag
Q I T L W K
acg atc aaa gtt ggc ggc
T I K V G G


subtype F,
cgt ccg
R P
caa ttg
Q L


21 gag gcc ctg ctg aac acc ggt gcg
E A L L N T G A


gac
D


K55C DNA and amino acid sequences.
ctg
L
aag
K
gat
D


31 acc gtg att gag gac atg aat ctg ccg ggt
T V I E D M N L P G


41 aag
K
51 ggt
G
61 cag
Q
71 gcg
A


tgg
W
ggt
G
att
I
atc
I


aaa
K
ttc
F
atc
I
ggt
G


ccg
P
atc
I
atc
I
act
T


tgc
K
aaa
C
gaa
E
gtt
V


atg
M
gtc
V
atc
I
ctg
L


att
I
aag
K
gct
A
gtt
V


ggc
G
caa
Q
ggc
G
ggc
G


ggt
G
tac
Y
cac
H
cca
P


att
I
gat
D
aaa
K
acc
T


81 ccg gtg aat atc att ggt cgc aac ttg ctg
P V N I I G R N L L
91 acg cag att ggt gca acg ctg aac ttc
T Q I G A T L N F


Table 3-6. E. coli codon-optimized HIV-1PR
1 ccg cag att acc ctg tgg aag
P Q I T L W K
11 gtc acg atc aaa gtt ggc ggc
V T I K V G G
21 gag gcc ctg ctg aac acc ggt
E A L L D T G


subtype FsK55C DNA and amino acid sequences.
cgt ccg ctg
R P L
caa ttg aag
Q L K
gcg gac gat
A D D


31 acc gtg att gag gac atg aat ctg ccg ggt
T V I E D M N L P G










Table3-6. Continued.
41 aag tgg aaa ccg tgc atg att ggc ggt att
K W K P K M I G G I
51 ggt ggt ttc atc aaa gtc aag caa tac gat
G G F I C V K Q Y D
61 cag att atc atc gaa atc gct ggc cac aaa
Q I I I E I A G H K
71 gcg atc ggt act gtt ctg gtt ggc cca acc
A I G T V L V G P T
81 ccg gtg aat atc att ggt cgc aac ttg ctg
P V N I I G R N L L
91 acg cag att ggt gca acg ctg aac ttc
T Q I G A T L N F

Table 3-7. E. coli codon-optimized HIV-1PR A/Es K55C DNA and amino acid sequences.
1 ccg cag atc acg ctg tgg aaa cgt cca ctg
P Q I T L W K R P L
11 gtt acc gtt aag att ggt ggt caa ctg aaa
V T V K I G G Q L K
21 gaa gcg ctg ctg aac acc ggt gcg gat gat
E A L L N T G A D D
31 acg gtc att gag gac atc aat ctg ccg ggt
T V I E D I N L P G
41 aag tgg aaa ccg aaa atg att ggc ggc atc
K W K P K M I G G I
51 ggc ggc ttt atc tgc gtg cgc caa tac gac
G G F I C V R Q Y D
61 cag atc att atc gag att gct ggt aag aag
Q I I I E I A G K K
71 gca att ggc acc gtc ttg gtt ggt ccg acc
A I G T V L V G P T
81 ccg gtg aat atc atc ggt cgt aac atg ctg
P V N I I G R N M L
91 act cag att ggt gcc acg ctg aac ttc
T Q I G A T L N F

Table 3-8. E. coli codon-optimized HIV-1PR A/Es K55C DNA and amino acid sequences.
1 ccg cag atc acg ctg tgg aaa cgt cca ctg
P Q I T L W K R P L
11 gtt acc gtt aag att ggt ggt caa ctg aaa
V T V K I G G Q L K
21 gaa gcg ctg ctg aac acc ggt gcg gat gat
E A L L N T G A D D
31 acg gtc att gag gac atc aat ctg ccg ggt
T V I E D I N L P G
41 aag tgg aaa ccg aaa atg att ggc ggc atc
K W K P K M I G G I









Table 3-8. Continued.
51 ggc ggc ttt atc tgc gtg cgc caa tac gac
G G F I C V R Q Y D
61 cag atc att atc gag att gct ggt aag aag
Q I I I E I A G K K
71 gca att ggc acc gtc ttg gtt ggt ccg acc
A I G T V L V G P T
81 ccg gtg aat atc atc ggt cgt aac atg ctg
P V N I I G R N M L
91 act cag att ggt gcc acg ctg aac ttc
T Q I G A T L N F
T Q I G A T L N F

Expression of HIV-1 protease constructs

Modified pET23a vectors (pET23a FsiK45C and pET23_A/EsiK55C, pET23a FsiK55C,

pET23_FsK55C, and pET23_ A/EsK55C were transformed separately into E. coli strain

BL21*(DE3)pLysS via standard heat-shock methodology. The transformed cells were

inoculated in 5 mL sterile Luria-Bertani (LB) media (Table 3-9) and grown at 37 C with

shaking at 250 rpm to an optical density (OD600) of approximately 0.60, then transferred to 1 L

sterile LB media and grown to an OD600 of approximately 1.0 with shaking at approximately 200

RPM. Cells were then induced using 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG).

The culture was then incubated with shaking at 250 rpm for 5-6 hours at 370C. Cells were

harvested by centrifugation for 15 minutes at 8500 g using a Sorvall RC6 floor-model centrifuge

with SLA-3000 rotor at 4 OC and supernatant was discarded.

Table 3-9. Luria-Bertani media (1L).
Component Amount
Yeast extract 5 g
NaC 10 g
Tryptone 10 g
Ampicillin 1 mL (100 iM) (opt.)
Sterile HO0 Volume to 1 L

Details of protease constructs

Several different HIV-1PR constructs were designed and prepared; namely A/Es

(CRF01 A/E; K55C, C67A, C95A, Q7K, L33I, L63I), A/Esi (CRF01 A/E; K55C, C67A, C95A,









Q7K, L33I, L63I, D25N), Fs (Subtype F; K55C, C67A, C95A, Q7K, L33I, L63I), Fsi (Subtype F;

K55C, C67A, C95A, Q7K, L33I, L63I, D25N), and Bs (Subtype B; K55C, C67A, C95A, Q7K,

L33I, L63I). The D25N amino acid substitution was incorporated into some of the constructs in

order to render inactivity on the protease. The D25N substitution has been shown by X-Ray and

NMR not to perturb the structure of the protease. All constructs contained three stabilizing

mutations that help provide protection from autocatalytic cleavage: Q7K, L33I, and L63I.

Additionally, each construct had both naturally occurring cysteine residues (C67 and C95)

substituted to alanine residues. These mutations allow for site-specific labeling and protection

against intramolecular disulfide cross-linking. Sites K55 and K55', solvent exposed sites in the

flap region, were substituted with CYS residues for site-directed spin-labeling. Active constructs

were labeled with 4-Maleimido-TEMPO (MSL) and inactive constructs were labeled with (1-

oxyl-2,2,5,5-tetramethyl-A3-pyrroline-3-methyl)methanethiosulfonate (MTSL). Structures of

the spin labels and spin-labeled side chains are given in Figure 2-10.

HIV-1 protease purification buffers

For compositions of all buffers used in HIV-1PR purification, see Table 3-10. All buffers

containing urea were made fresh prior to each round of protein purification. With heat and time,

urea degrades to give products that can carbamylate free cysteines (Figure 3-6), thus limiting

spin-labeling efficiency (Stark 1965; Stark 1965; Stark 1965; Lippincott and Apostol 1999).

Additionally, all urea buffers contain glycylglycine (diGly) to function as an ion scavenger for

urea decomposition products and were ion-exchanged using a mixed-ion bed resin (AG501-X8)

by adding 5 g resin/100 mL urea.









Table 3-10. HIV-1PR purification buffers.
Buffer (Volume)
Resuspension Buffer


*Adjust pH to 7.5 and filter using a 0.22 [m membrane.
Wash Buffer 1




*Adjust pH to 7.0 and filter using a 0.22 rm membrane.
Wash Buffer 2






*Adjust pH to 7.0 and filter using a 0.22 rm membrane.
Must be fresh! Prepared an hour before use.
Wash Buffer 3




*Adjust pH to 7.0 and filter using a 0.22 [m membrane.
Inclusion Body Resuspension Buffer (250 mL)


Component
20 mM Tris HCI
1 mM EDTA
10 iM BME added fresh
Store at 250C.
25 mM Tris-HCl
2.5 mM EDTA
0.5 MNaCl
1 mM Gly-Gly
50 [iM BME added fresh
Store at 40C.
25 mM Tris-HCl
2.5 mM EDTA
0.5 MNaCl
1 mM Gly-Gly
50 iM BME added fresh
1 M urea


25 mM Tris-HCl
1.0 mM EDTA
0.5 MNaCl
1 mM Gly-Gly
50 iM BME added fresh
Store at 40C.
25 mM Tris-HCl
2.5 mM EDTA
0.5 MNaCl
1 mM Gly-Gly
50 [iM BME added fresh


9 M urea
*Adjust pH depending on isoelectric point of construct; pH should be slightly less than pi
of protein to give a good yield.
*Filter using a 0.22 [m membrane.
*Must be fresh! Prepared an hour before use.
Spin-labeling Buffer (1 L) 10 mM Tris-HCl
Adjust pH to 6.9 and filter using a 0.22 [m membrane.
Size Exclusion Column buffer (1 L) 50 mM NaOAc
Adjust pH to 5.0, sonicate to remove air, and filter using a 0.22 tm membrane.
Abbreviations: Ethylenediaminetetraacetic acid (EDTA), P-mercaptoethanol (BME), sodium
acetate (NaOAc)










0
II Heat/Time
H2N-C-NH2 NH4+ + NCO-
Urea Ammonium Cyanate

Figure 3-6. Decomposition of urea; with heat and time, urea degrades into ammonium and
cyanate.

HIV-1 protease purification

Prior to protein purification, 250 mL of 9 M and 150 mL of 1 M urea buffers were freshly

prepared in separate containers, and 1-2 g AG 501-X8 (D) resin (20-50 mesh) was added to each

of the urea buffers. These solutions were placed on a heated (30 C) stir plate and mixed by stir

bar for approximately 2 hours to dissolve the urea, and the resin was removed by filtration. All

buffer exchange steps described in the following sections were carried out using a 5 mL HiTrap

Desalting column from GE Healthcare (packed with Sephadex G25), which is first washed

successively with 3 4 column volumes (15 20 mL) of nanopure water (nH20), 1 M NaC1,

nH20, 0.5 M NaOH, nH20, and then equilibrated in the desired buffer.

A B















Figure 3-7. A) Thermo brand 35 mL French pressure cell and B) Fisher Scientific brand tip
sonicator.

Pelleted cells from 1 L growth were resuspended in 30 mL resuspension buffer (Table 3-

10), and 23.4 tL of 3-Mercaptoethanol (BME) was added to the reaction and mixed well by









swirling. The approximate weight of the wet pellet was generally 5-6 grams, but varied from

purification to purification. In order to lyse the cells and release the cellular contents, the sample

was sonicated for 2 minutes using a Fisher brand tip sonicator (Figure 3-7) at approximately 25

watts output power. Sonication was always performed in cycles of 5 seconds on followed by 5

seconds off to avoid shearing the proteins present in the lysate. The sample was then passed 3

times through a 35 mL French pressure cell (Thermo Scientific, Waltham, Massachusetts)

operating at approximately 1200 pounds per square inch (psi).

Next, the lysed cells were centrifuged for 30 minutes at 18500 x g and 4 C using the

Eppendorf 5810R centrifuge with F34-6-38 rotor to collect cell debris and protease-containing

inclusion bodies, and the supernatant was discarded. The inclusion body-containing pellet was

resuspended, homogenized (using a 50 mL Dounce Tissue Homogenizer) and sonicated in 40

mL fresh wash buffer #1 (Table 3-10), then centrifuged for 30 minutes at 18500 x g and 4 C,

and the supernatant was discarded. This process was repeating with 40 mL wash buffer #2

(Table 3-10) and 40 mL wash buffer #3 (Table 3-10). Each of these steps functioned to isolate

and wash the inclusion bodies, removing non-target proteins and remaining cellular components.

In order to solubilize the inclusion bodies, the pellet was then resuspended, homogenized and

sonicated in 30 mL inclusion body resuspension buffer containing 9 M urea, then centrifuged at

18500 x g and 4 C for 30 minutes. The supernatant, which contained solubilized target proteins

in unfolded, monomeric form, was collected. Because purification proceeds by anion exchange

chromatography, the pH of the inclusion body resuspension buffer needs must be adjusted

according to the specific isoelectric point (pI) of the protein being purified. The theoretical pi's

of the FsiK55C, FsK55C, A/EsiK55C, and A/EsK55C constructs are 8.95, 8.95, 9.53, and 9.53,









respectively; and, the respective pH values of the buffer utilized for anion exchange

chromatography are 8.5, 8.5, 9.08, and 9.08.

A 5 mL HiTrap Q HP anion exchange column was equilibrated with inclusion body

resuspension buffer on an Akta Prime liquid chromatography system, and the supernatant

containing solubilized HIV-1PR monomers was applied to the column at a rate of 5 mL/min.

Fraction collection was started immediately and flow through was collected in 4 mL fractions,

(specific fraction numbers depend on the results of the chromatogram). Figure 3-8 shows a

typical chromatogram for the anion exchange step chromatography step.


Conductivity (mS)




Gradient (here 0%)

Fraction number UV28



Figure 3-8. Typical anion exchange chromatogram (blue: UV280, red: conductivity, green:
constant buffer composition) for purification of HIV-1PR via 5 mL anion exchange Q
column. Black square highlights peak of eluted protein. Fractions 1-8 were selected
and pooled for further purification, refolding, and spin-labeling.

Fractions containing HIV-1PR were collected and pooled into a clean 50 mL Eppendorf

tube (approximately 32 mL from 8 separate 4 mL fractions), then acidified to pH 5 (typically

with approximately 39.3 tL formic acid) and stored for approximately 12 hours at 4 OC to allow

some contaminants to precipitate, after which time the protease sample was decanted into a 50

mL polypropylene Eppendorf centrifuge tube and centrifuged for 30 minutes at 12000 rpm and 6

C to separate precipitated contaminants. 300 mL 10 mM formic acid solution was prepared in a

clean beaker and cooled on ice to approximately 0 C. HIV-1PR was refolded by doing a 10-









fold stepwise dilution on ice using a peristaltic pump for approximately 2 hours, and the pH was

subsequently adjusted to approximately 3.8 (typically by adding about 1 mL 2.5 M sodium

acetate). The solution temperature was brought to approximately 30 C and the pH was adjusted

to 5 (typically by adding about 3 mL 2.5 M sodium acetate).



Conductivity







Gradient




UV280


Figure 3-9. Typical size exclusion chromatogram of HIV-1PR on S-100 column. chromatogram
(blue: UV, red: conductivity, green: constant buffer composition). Box encircles UV
peak corresponding to HIV-1PR.

After approximately 20 minutes of wait time, the solution was moved to balanced

centrifuge tubes and centrifuged for 20 minutes at 18500 x g and 23 C to remove contaminants

that precipitated during refolding. The sample was concentrated to OD280 = 0.5 using an Amicon

100 mL equipped with a Millipore 10,000 Da MW cut-off polyethersulfone membrane. If

further purification was required, the protease sample was buffer exchanged into 50 mM NaOAc,

pH 5, and 5 mL of the concentrated protein sample was loaded on to a HiPrep 16/60 Sephacryl

S-200 equilibrated with the same buffer and run at 0.5 mL/min and 2 mL fractions containing

HIV-1PR were collected (fraction numbers vary according to chromatogram). A typical size

exclusion chromatogram is shown in Figure 3-9. The result is >95% pure HIV-1 Protease.










Aliquots were collected after each step in the purification protocol and run on a Biorad Criterion

pre-cast 16.5% tris-tricine peptide SDS-PAGE gel to illustrate the purity of the protein sample

after each step of the purification protocol, as shown in Figure 3-10. Note, though many typical

protein purifications utilize protease inhibitors to prevent cellular proteases from acting upon the

target protein, no protease inhibitors were added to the lysate at any time because the target

protein is a protease.

1 2 3 4 I5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
MW LS WS W S W ~P IB Q flow-through fractions AS AP RS F

















Figure 3-10. Purification ofHIV-1PR. Biorad Criterion pre-cast 16.5% tris-tricine SDS-PAGE
gel. Lane 1: Promega broad range protein markers (MW), 2: total cell pellet (TC), 3:
kDa

75
500

25

15







Figure 3-10. Purification of HIV-1PR. Biorad Criterion pre-cast 16.5% tris-tricine SDS-PAGE
gel. Lane 1: Promega broad range protein markers (MW), 2: total cell pellet (TC), 3:
lysed cell supernatant (LS), 4: lysed cell pellet (LP), 5: wash buffer 1 supernatant
(WS), 6: wash buffer 1 pellet (WP), 7: wash buffer 2 supernatant (WS), 8: wash
buffer 2 pellet (WP), 9: wash buffer 3 supernatant (WS), 10: wash buffer 2 pellet
(WP), 11: solubilized washed inclusion bodies (IB), 12-19: Q-column flow-through
fractions, 20: acidification supernatant (AS), 21: acidification pellet (AP), 22: post-
refolding supernatant (RS), 23: post-refolding pellet (RP), 24: size exclusion
fractions (SF), 25: empty.

Spin-labeling

When spin-labeling was needed, the purified and refolded protein sample was buffer

exchanged into 10 mM Tris HC1, pH 6.9 (further details of spin-labeling discussed in next

section) after the final purification step. Approximately 1 mg of spin label (IASL, IAP, MTSL

or MSL) was dissolved in 100 [L ethanol, and added to approximately 40 mL of HIV-1PR in 10









mM Tris HC1, pH 6.9. The spin-labeling reaction was carried out in the dark (via wrapping

reaction tube in aluminum foil) at room temperature for approximately 4-6 hours followed by 6-8

hours at 4 C for inactive (D25N) constructs, and at 4 OC for approximately 8-12 hours for active

(D25) constructs. At this time, the sample solution was centrifuged at 12000 rpm for 20 minutes

at 4 C to remove solid impurities and aggregated proteins. The sample was then buffer

exchanged into 2 mM NaOAc, pH 5, and concentrated to OD280=1.25.

Circular dichroism spectroscopy

To ensure that the spin-labeled HIV-1 PR constructs contained proper secondary structure,

circular dichroism (CD) experiments were performed. All measurements were collected on an

Aviv 400 spectrometer using Hellma CD cuvettes with 1 cm pathlength with samples in 2 mM

NaOAc buffer, pH 5.0 at approximately 30 |iM protein concentration. Protein concentration was

determined by absorption at 280 nm, using an extinction coefficient of 12490 M-1 cm-1 for each

construct (calculated using Expasy's extinction coefficient calculator). Typical parameters used

for circular dichroism experiments are summarized in Table 3-11. For each data set, 3 5 scans

were taken and averaged to give a final result. Background scans of all buffers were collected

and subtracted from the final averaged spectra.

Table 3-11. Standard parameters used for circular dichroism experiments.
Parameter Value
Experiment type Wavelength
Bandwidth 1 nm
Temperature 250 C
Wavelength Start 260 nm
Wavelength End 190 nm
Wavelength Step 0.5 nm
Averaging time 3.000 sec
Settling time 1.0 sec
Multi-scan wait 1.0 sec
Scans 3









Sample preparation for EPR data collection

The first set of experiments reported in the results section of this chapter was performed in

order to analyze the effect of inhibitors and substrate on the EPR nitroxide spectral line shapes.

Samples were prepared by adding 1 ptL of 0.08 mM inhibitor to 9 p.L of 125 pM MTSL-labeled

HIV-1PR (a final molar ratio of approximately 4:1 inhibitor:enzyme). For inhibitors not

dissolved in dimethylsulfoxide (DMSO) (i.e., indinavir and tipranavir), 1 ptL DMSO was

included in the 10 p.L sample (1 p.L inhibitor, 1 ptL DMSO, 8 ptL HIV-1PR) to make all samples

isoviscous with one another (10% v/v DMSO). Samples were loaded into capillary tubes and

allowed to equilibrate to a controlled temperature, as described above.

The next set of experiments was performed in order to analyze the autoproteolysis of

active HIV-1PR under various conditions. MSL-labeled samples were utilized here for reasons

to be discussed later. Apo (non-substrate/inhibitor bound) HIV-1PR Subtype Fs and

CRF01_A/Es were prepared at 150 pM protein concentration in 2 mM NaOAc, pH 5.0. Protein

concentration was measured by absorption at 280 nm with an extinction coefficient of 12490 M-1

cm-1 for each construct. Extinction coefficients were calculated using the program ProtParam at

the EXPASY server (http://www.expasy.ch/tools). Three identical 10 [iL samples were prepared

and loaded into capillary tubes and both ends were sealed. The tubes were then stored in three

conditions, one at 37 C, another at 25 C, and the third at 4 OC for a total of 97 days. Samples

of HIV-1PR Subtype F containing 0.08 mM Tipranavir and CRF01_A/E containing 0.08 mM

CA-p2 substrate mimic (1 [iL TPV or CA-p2 in 10 [iL total sample volume) were also prepared

in sealed capillary tubes and stored at 25 C.









CW EPR measurements

CW EPR data were collected on a modified Bruker ER200 spectrometer with an ER023 M

signal channel, an ER032 M field control unit, and a loop gap resonator (Molecular Specialties,

Milwaukee, WI). A quartz dewar (Wilmad-Labglass) surrounded the loop gap resonator for

temperature controlled experiments. Nitrogen gas was passed through a copper coil submerged

in a recirculating 25 C water bath (Thermo Scientific) containing 40% ethylene glycol. This

setup is shown in Figure 3-11.

A B











C









Figure 3-11. Temperature control set-up; A) thermocouple thermometer, B) quartz dewar
surrounding the loop gap resonator, C) copper coil submerged in a recirculating water
bath containing 40% ethylene glycol, with nitrogen passing through the line into the
D) back of the quartz dewar.

Samples were removed from storage conditions and allowed to equilibrate in the loop gas

resonator with quartz dewar for at least 15 minutes prior to sample collection. CW EPR spectra

were collected with 1 Gauss modulation amplitude and 100 Gauss sweep width. Additional

spectra were collected at 20 Gauss sweep width for more approximate calculation of AHpp. Each









spectrum contains 1024 points with a center field of approximately 3450 Gauss. Spectra were

collected and averaged from between 1 20 scans with a frequency of 9.6 9.7 GHz. A

complete listing of other typical parameters is shown in Table 3-12.

Table 3-12. Standard CW EPR parameters used in this study.
Parameter Value
Number of points 1024
Center field -3450 G
Number of scans 5-25
Sweep width 20 100 G
Acquisition time 40.63 sec
Frequency -9.6 GHz
Power 20 dB 20 mW
Receiver Gain x103 5x105
Modulation Amplitude -1 G
Time Constant 0.082 0.164 sec
Receiver Phase 100

Mass spectrometry experiments

After a total of 100 days of storage at 37 C, apo samples were removed from the sealed

capillary tubes used for EPR analysis and prepared for analysis via mass spectrometry (MS).

All sample preparation and MS experiments were performed by Dr. Laura Busenlehner at the

University of Alabama. Samples were diluted to 6 pmol/tL in 50% HPLC-grade acetonitrile in

HPLC-grade water with 0.1% formic acid. Samples were introduced to the Briker HCTultra

Discovery ion trap mass spectrometer by direct infusion at a flow rate of 2 [tL/min. The

electrospray ionization source maintained a nebulizer gas pressure of 10 psi, a dry gas flow of 5

L/min, and a drying temperature of 250 C. The positive mode mass range swept was 50-2000

m/z and data-dependent collision-induced-dissociation (CID) fragmentation of peptides was

collected. MS spectra were deconvolved for charge using DataAnalysis (Briker Daltonics) with

an abundance cutoff of 10%, a molecular weight tolerance of 0.01% and an envelope cutoff of

75%. For peptides with a monoisotopic mass of less than 3000 Da, CID spectra were collected











and the fragmentation pattern analyzed de novo using MassXpert2 (Rusconi 2009) and MS-

Product (Protein Prospector, University of California San Francisco, California).

http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct). The MSL spin label

was included as a permanent modification on cysteine 55 of mass 354.28 m/z.

Results and Discussion

Affect of Inhibitors on CW EPR Line Shapes of HIV-1PR Subtype F and CRF01_A/E

CW EPR spectra were obtained for several HIV-1PR Subtype Fsi and CRF01_A/Esi, with

nine separate FDA-approved protease inhibitors, with the non-hydrolyzable substrate mimic CA-

p2, and in the apo form (free of inhibitors and substrate). Most of the inhibitors used here are

dissolved in DMSO, which changes the viscosity of the sample and thus affects the EPR spectral

line shape. As a result, each of the samples were made isoviscous to one another by ensuring

that each one has the same percent DMSO (10% v/v).

A B

Apo (10% DMSO)
APV
Ca-P2
DRV
IDV
LPV
S NFV
SQV
RTV
ATV
S TPV









Figure 3-12. 100 Gauss CW EPR nitroxide spectral line shapes for HIV-1PR subtype Fsi
K55MTSL, collected at X-band frequency, same data given in two views: (A)
waterfall and (B) overlay.










Table 3-13. CW EPR data analysis for subtype
AHpp




HIV-1PR (2 mM NaOAc)
HIV-1PR (10% DMSO)
CA-p2
RTV
APV
ATV
DRV
IDV
LPV
NFV
SQV
TPV


1.85
1.93
2.01
1.97
2.05
2.01
2.03
1.95
2.05
1.91
2.05
1.97


188
196
195
194
193
205
187
195
192
192
189
189


Fsi K55MTSL.
low/center field high/center field
intensity (normalized) intensity (normalized)
0.64 0.18
0.60 0.17
0.58 0.16
0.58 0.16
0.56 0.15
0.56 0.16
0.56 0.16
0.62 0.18
0.59 0.17
0.58 0.16
0.59 0.16
0.59 0.16


Shown in Figure 3-12 are the spectra for Subtype Fsi K55MTSL, and Table 3-13

summarizes the spectral parameters described by those data. Shown in Figure 3-13 are the

spectra for CRF01_A/Esi K55MTSL, and Table 3-14 summarizes the spectral parameters

described by those data.


A B

-- Apo (10% DMSO)
-- APV
-- Ca-P2
-- DRV
IDV
LPV
-- NFV
SQV
-- RTV
ATV
S--TPV


Figure 3-13. 100 Gauss CW EPR nitroxide spectral line shapes for HIV-1PR CRF_01A/Esi
K55MTSL, collected at X-band frequency shown in (A) waterfall and (B) overlay.









Table 3-14. CW EPR data analysis for A/Esi K55MTSL.
AHpp

low/center field high/center field
intensity (normalized) intensity (normalized)
HIV-1PR (2 mM NaOAc) 1.93 192 0.60 0.17
HIV-1PR (10% DMSO) 2.01 196 0.64 0.17
CA-p2 1.94 189 0.59 0.16
RTV 1.97 186 0.60 0.17
APV 1.94 193 0.59 0.15
ATV 1.93 189 0.58 0.16
DRV 1.97 190 0.57 0.17
IDV 1.95 195 0.61 0.18
LPV 2.03 194 0.60 0.17
NFV 1.99 192 0.58 0.17
SQV 2.05 195 0.58 0.18
TPV 1.97 196 0.57 0.16

Data from a number of biophysical techniques, including NMR and ITC, have suggested

that the flaps of HIV-1 PR become locked down into the closed conformation in the presence of

various inhibitors and that minor backbone fluctuations are the major source of protein motion

(Todd, Semo et al. 1998; Ishima, Freedberg et al. 1999; Todd and Freire 1999; Todd, Luque et

al. 2000; Velazquez-Campoy, Todd et al. 2000; Freedberg, Ishima et al. 2002; Hornak, Okur et

al. 2006). Thus, it might be expected that changes would be reported in the CW EPR line shape

for apo and ligand-bound samples; however, this is not the case. Because the change in the EPR

nitroxide spectral line shape with and without inhibitor/substrate is so minor, it is likely that the

CW EPR is not reporting on the true motion of the flaps in these particular protein systems, but

rather the internal motion of the spin label itself. As such, pulsed EPR methodology was

employed, and results from those studies will be discussed in Chapter 4.

Monitoring the Autoproteolysis of HIV-1 Protease by SDSL EPR and Mass Spectrometry

It is well known that HIV-1PR undergoes self-proteolysis, particularly at the high

concentrations necessary for many types of spectroscopic analysis, and that the sites in which

proteolytic cleavage occurs most frequently in Subtype B are positions W6-Q7, L33-E34 and

L63-I64 (Tomasselli, Mildner et al. 1995; Szeltner and Polgar 1996). In addition, it is known









that the amino acid substitutions, Q7K, L33I and L63I reduce autoproteolysis in Subtype B

protease without affecting protein folding, activity, or kinetic properties, but rather remains

similar to the wild-type (Tomasselli, Mildner et al. 1995). In agreement with the literature, the

constructs investigated here have the amino acid substitutions Q7K, L33I and L63I in order to

slow the self-proteolysis process. Note, autoproteolysis is slowed, not completely removed.

Protease inhibitors used in treatment of HIV-1 are generally designed with respect to

Subtype B (Wlodawer and Vondrasek 1998). Subtype B is the dominant HIV-1 subtype in

Western Europe and the United States, though it is estimated that only about 12% of HIV

infection worldwide is due to that strain. Additionally, various reports show that key differences

occur in structure and dynamics of the protease among the subtypes and CRFs as a result of the

assorted sequences that define each subtype. For example, we recently showed, with site-

directed spin-labeling, that subtype polymorphisms alter the conformations and flexibility of the

apo protease flaps (Kear, Blackburn et al. 2009), demonstrating the need to understand the key

variations between subtypes that may affect the efficacy of inhibitors. In addition to these

general implications, this work was also specifically applicable to the studies performed in our

lab. Due to the autoproteolytic ability of HIV-1PR, our early EPR investigations have all been

conducted with inactive (D25N) protease constructs.(Galiano, Bonora et al. 2007; Blackburn,

Veloro et al. 2009; Galiano, Ding et al. 2009; Kear, Blackburn et al. 2009) In order to continue

investigations on active protease, we need to understand how conditions during sample

preparation and storage affect the autoproteolytic process.

CW EPR was used to monitor the autoproteolysis of HIV-1PR Subtype Fs and

CRF01_A/Es. The EPR nitroxide spectral line shape is highly sensitive to mobility in the

environment of the spin label, thus changes dramatically with variations in correlation time.










Contributions to correlation time come from three modes of motion, including global protein

tumbling (TR), intrinsic spin label mobility (Ti), and backbone fluctuations (TB). Autoproteolysis

affects the rate of global protein tumbling by decreasing rotational correlation time of the spin

labeled protein. As the protease undergoes self-cleavage, a peptide fragment containing the spin

label is generated, which gives rise to an isotropic nitroxide line shape component that is easily

discernable in the high field resonance line in the EPR spectrum. By monitoring the intensity of

this spectral component over time, the autoproteolytic stability of each construct was

characterized under various conditions. Data was collected on samples that were stored at 4 OC,

25 C, and 37 C, as well as on samples that contained either 0.08mM Tipranavir or 0.08mM of

the substrate mimic CA-p2.

Circular dichroism experiments were performed prior to carrying out the EPR experiments

to ensure that the spin-labeled HIV-1 PR constructs contained proper secondary structure.

Measurements were collected on an Aviv 400 spectrometer using Hellma CD cuvettes with 1 cm

pathlength. Samples were prepared at approximately 30 iM protein concentration in 2 mM

NaOAc, pH 5.0.


9000-

6000-

3000-

0 0

-3000

-6000 -
200 210 220 230 240
Wavelength (nm)

Figure 3-14. Circular dichroism spectra for spin labeled HIV-1PR, subtype B (black), subtype F
(red), CRF01 A/E (blue).













-----r4 oC




25 oC








37 C
20 Gauss 20 Gauss





Figure 3-15. Overlay of day 1 (black) and day 30 (grey) area normalized 100 Gauss X-Band CW
EPR spectra for (A) subtype Fs HIV-1PR stored at 37 C, 25 C and 4 C, and (B)
CRF01 A/Es HIV-1PR stored at 37 C, 25 C, and 4 C.

Figure 3-15 shows overlays of spectra collected immediately after purification and labeling

(black) and after 30 days (grey) for HIV-1PR Subtype Fs and CRF01_A/Es at all three storage

conditions (37 C, 25 C, or 4 C). LabVIEW software, which was provided by Drs. Christian

Altenbach and Wayne Hubbell (University of California Los Angeles), was used for baseline

correction and double integral area normalization, allowing for easy comparison of various

spectral parameters. Subsequently, the peak-to-peak intensity of the high field line (IHF) was

measured for each spectrum and converted to mole fraction of proteolyzed, unfolded protein (Zb)

using Equation 3-1 and then plotted with respect to time, as shown in Figure 3-16. For these

calculations, t = 100 is presumed equal to Il, and Io was calculated from an EPR spectrum









collected immediately after purification, spin-labeling, and sample preparation was complete.

The value obtained is proportional to the amount of uncleaved protease remaining in the sample.

The intensity of the high field line (IHF), as expected, increased substantially over time and with

increasing storage temperature.


b ( -t 0 (3-1)


Figures 3-17 and 3-18 give an indication of the stability to self-proteolysis of HIV-1PR at

these concentrations. The prompt addition of a substrate to the enzyme solution greatly reduced

the rate of autoproteolysis of the samples, as evidenced by the small change in IHF over the

course of the 30 days when the protease samples were stored at 25 C over the course of 30 days.

These results seem to indicate that subtype F protease, with tipranavir, undergoes negligible self-

proteolysis, and the CRF01_A/E sample underwent very little. As previously stated, most of our

early EPR investigations have all been conducted with inactive (D25N) protease constructs due

to the instability of the autoproteolytic activity of the protease (Galiano, Bonora et al. 2007;

Blackburn, Veloro et al. 2009; Galiano, Ding et al. 2009; Kear, Blackburn et al. 2009). Thus, in

order to continue investigations on active protease, experiments were performed to determine

how preparation and storage affect the autoproteolytic process. From this work, it seems likely

that SDSL EPR studies could be successfully applied to active HIV-1PR if the protein is purified

quickly and the addition of inhibitor or substrate is swift following the refolding process. This

SDSL EPR methodology has shown to be very useful in monitoring the slow autoproteolysis or

degradation of a protein sample. Fitting the plots with an exponential growth function allowed

for generating a term that is proportional to the amount of uncleaved protein in the sample. This

type of methodology has the advantage over SDS-PAGE in that it does not deplete the sample.












1.0 3 37 1C 1.0- 37 .C
25 C 1 25 C F
0. C4 0.8
S0.8- 4 0.8
C1~
: 0.6* 0.6

S 0.4- 0.4

S0.2- 0.2
o 0
0.0- 0.0-
0 10 20 30 40 50 0 10 20 30 40 50
Time (days) Time (days)


Figure 3-16. Mole fraction of degraded protein (Xb) vs. time (days) for CRF01_A/E and subtype
F protease for samples stored at 37 C, 25 C, and 4 C.

Function fitting was done in Origin 8.0 with the 2 component exponential growth

function (ExpGro2) of the form given in Equation 3-2, under the assumption that there are two

pseudo-first order processes occurring in the autoproteolytic process, a fast component likely

representative of the initial conversion of intact, properly folded protease to cleaved, unfolded

protease, and a slow component likely representative of further degradation of the protease. In

this Equation, yo is equal to the y-offset, A1 and A2 are amplitudes, and ti and t2 are growth

constants.

y = yo + A1e /t + A2et2 (3-2)

The degradation constant tl (in days) was extracted for each sample as a measure of the rate of

conversion of intact, properly folded protease to unfolded/cleaved protease. For subtype F, the

degradation constants tl are 12.1 + 1.2, 24 3 and 34 6 days, for samples stored at 37 C, 25

C, and 4 C; respectively. For CRF01_A/E, the degradation constants tl are 8.2 0.9, 38 7,

and 54 10 days, for samples stored at 4 OC, 25 C, and 37 C, respectively. The values oftl


Subtype F


CRF01 A/E









generally indicate that autoproteolysis in the Subtype F protease construct proceeded more

quickly than did the autoproteolysis of the CRF01_A/E protease construct.


A B




20 Gauss




20 Gauss


Figure 3-17. Area normalized 100 G X-Band EPR spectra of HIV-1PR subtype F in the
presence of 0.08 mM tipranavir (FDA-approved protease inhibitor) over the course of
30 days, where A) the data are stacked such that day 1 spectrum is at the bottom and
day 30 is at the top and B) overlaid, with inset showing magnified high field line.


20 Gauss


Figure 3-18. Area normalized 100 G X-Band EPR spectra of HIV-1PR CRF01_A/E in the
presence of 0.08 mM non-hydrolyzable substrate mimic CA-p2 over the course of 30
days, where A) the data are stacked such that day 1 spectrum is at the bottom and day
30 is at the top and B) overlaid, with inset showing magnified high field line.

After a total of 100 days of EPR data collection, apo samples were removed from the

sealed capillary tubes and prepared for analysis via mass spectrometry in order to identify sites

of autoproteolytic cleavage. Mass spectra are reported in Figures 3-19 3-22. The identities of

smaller peptide fragments were confirmed by MSMS (data not shown), but longer peptides could









only be identified via deconvolution (too large for MSMS). Sites of autoproteolysis in subtype

Fs and CRF01_A/Es were identified by mass spectrometry. MALDI-TOF and electrospray

(ESI) mass spectrometry of the subtype F and CRF01_A/E HIV-1PR constructs indicated that

both proteins have complete MSL spin-labeling and were of the correct masses. In order to

identify the specific sites of autoproteolysis, electrospray mass spectrometry was used to

sequence peptide fragments via collision-induced dissociation (CID), more commonly known as

tandem MS. Because of the incorporation of the MSL label at position K55C, the modification

was included in the de novo analysis. The substitution of amino acids between the subtypes

clearly influences the cleavage sites. For Subtype F, four peptides which contain the MSL label

were identified conclusively by tandem MS (peptide residues 31-63, 41-55, 53-61 and 53-63). In

contrast, only one peptide from the CRF01_A/E autoproteolysis sample was identified by MS

sequencing (peptide residues 37-58), but another was identified by charge deconvolution of the

multiply-charged isotopic envelope (peptide residues 24-71). Results indicated that the sites of

autoproteolytic cleavage differed between Subtype Fs and CRF01_A/Es, and are shown in the

maps in Figures 3-23 and 3-24. The arrows denote HIV-1PR peptide fragments identified by

mass spectrometry. Note, for CRF01_A/E, there currently exist a few gaps in the sequence, and

work is ongoing to identify these peptide fragments.

Conclusions

Affect of Inhibitors on CW EPR Line Shapes of HIV-1PR Subtype F and CRF01_A/E

It is known that there exist dramatic differences between the flap motion of free and

inhibitor-bound protease; however, the spectra collected in this experiment reported very minor

changes in the spectral line shape of protease in the apo form and with various inhibitors. Thus,

it can be concluded that the CW EPR does not report on the motion of the flaps of these protease









constructs. For this reason, the system will be studied using a pulsed technique, called double

electron-electron resonance, to be discussed in depth in the following chapter.

Monitoring the Autoproteolysis of HIV-1 Protease by SDSL EPR and Mass Spectrometry

The CW EPR methodology has proven to be very useful in monitoring the slow

autoproteolysis or degradation of a protein without depletion of the sample. By evaluating the

EPR spectral line shape with respect to IHF, plots of Xb(t), fit with a two-component exponential

growth function were generated, providing a term that is proportional to the amount of uncleaved

protein in the sample. Many experimental techniques, such as NMR, EPR and ITC, require

protein concentrations near 150 riM, thus it is important to examine the rates of autoproteolysis

at that concentration. In all cases, the normalized intensity of to IHF is observed to increase over

time, indicating protease degradation and unfolding. Additionally, the normalized intensity of IHF

increased dramatically with increasing storage temperature, which indicates a temperature-

dependent autoproteolytic process. The normalized spectral intensities IHF were converted to

mole percent of proteolysed, unfolded protease (yb) and plotted as a function of time. The

degradation constant tl (in days) was extracted for each sample as a measure of the rate of

conversion of intact, properly folded protease to unfolded/cleaved protease. These results also

indicated that with a quick purification and addition of inhibitor or substrate, autoproteolysis can

be reduced substantially, if not eliminated; thus, SDSL EPR experiments on active protease seem

feasible. The presence of an inhibitor, which was added immediately upon completion of protein

purification and prior to refolding, impeded the autoproteolytic activity of the samples, as

evidenced by almost no change in the intensity of IHF over the course of the 30 days,

demonstrating, via SDSL and CW EPR, that HIV-1PR can be stabilized against autoproteolysis

via the timely addition of inhibitor, even at the high concentrations necessary for spectroscopic









studies. Mass spectrometry revealed that in both constructs, the N-terminus is sensitive to

systematic degradation, but there are also specific cleavage sites within the proteins. The most

obvious sites of autoproteolysis for Subtype F are after L23, D30, G52, 163, and T73. The

cleavages after L23 and D30 are conserved in CRF01_A/E, but a new proteolytic site appeared

after Q61.





































Figure 3-19. Mass spectrum for HIV-1PR CRF01_A/E K55MSL, 1.5 pmol/itL in 50:50 ACN/H20 + 0.1% formic acid.










149





































Figure 3-20. Mass spectrum for HIV-1PR CRF01_A/E K55MSL 6.0 pmol/jiL in 50:50 ACN/H20 + 1% formic acid.









150














































[ l11111


+MS, 0 4-1 1mm #(12-33)


991 8


9093


6927
664 7


5867


5656


5191


4735


201 2
I I ,j 2543 1


885 4


8396


7901
811 6


10909


diii i.


12121


200 400 600 800 1000 1200 1400 1600 m/z

Figure 3-21. Mass spectrum for HIV-1PR Subtype Fs K55MSL, 1.5 pmol/jiL in 50:50 ACN/H20 + 0.1% formic acid.


I~Lk Li


301 3


4185


4403


3935

i, i,


13632


13274 i
,i l ,lI I >


15576
| 16042













+MS, 0 40 8min #(11-22)


5656


519 2


4735


4185



1732
1292 J 1 301 33394


SI n.LllrLJilII 1iMHi5 7471
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z



Figure 3-22. Mass spectrum for HIV-1PR CRF01 A/E K55MSL 6.0 pmol/jiL in 50:50 ACN/H20 + 1% formic acid.


991 9


1091 0


664 5


8854


12121


8328


13635


13275

12955


kll ld


IJ LI 1 ,1,I


15581





I 16188


18175
, I


... n










P Q I T L W K R P L V T I K V G G Q L K



E A L L D T GA D D T V I E D M N L P G



K W K P K M I G G I G G F I *C* V K Q Y D



Q I I I E I A G H KA I G T V L V G P T


P V N I I G R N L L T Q I G A T L N F



Figure 3-23. Sites of autoproteolytic cleavage in HIV-1PR subtype Fs K55MSL.

P Q I T L W K R P L V T V K I G G Q L K


E A L L D T G A D D T V I E D I N L P G




K W K P K M I G G I G G F I *C* V R Q Y D




Q I I I E I A G K KA I G T V L V G P T


P V N I I G R N M L T Q I G A T L N F



Figure 3-24. Sites of autoproteolytic cleavage in HIV-1PR CRF01 A/E K55MSL.


-h









CHAPTER 4
PULSED ELECTRON PARAMAGNETIC RESONANCE STUDIES OF HIV-1 PROTEASE

Introduction

The previous chapter summarized results from continuous wave electron paramagnetic

resonance (CW EPR) studies of spin labeled apo and inhibited HIV-1 protease (HIV-1PR)

constructs of Subtype F and CRF01_A/E. Although CW EPR has provided a means for

monitoring autoproteolysis of active protease, we find that the nitroxide line shapes are

dominated by the intrinsic motion of the spin label, and thus do not provide a means to report on

changes in flap motion and dynamics. In this chapter, results from double electron-electron

resonance (DEER) experiments are reported and discussed. DEER experiments provided a

means to determine distance profiles between two spin labeled sites in the flaps, which can be

used to describe and quantify conformational sampling of protease constructs.

Semi-open

closed
p-hairpins
aka "the flaps"






Active site


Figure 4-1. Ribbon diagrams showing crystal structures of HIV-1PR in the (red) closed (PDB ID
2PBX) and (blue) semi-open (PDB ID 1HHP) flap conformations. The active site
(residues D25 and D25') and the 0-hairpin flaps are shown. In addition, the K55C
and K55C' reporter sites are shown after modification with MTSL, showing how the
distance between the spin labels is expected to change as the flaps sample different
conformations. Figure was originally made by Luis Galiano (Ph.D. 2008) and has
been modified.

Site-directed spin-labeling (SDSL) with DEER electron paramagnetic resonance (EPR)

spectroscopy was used to measure the distance between nitroxide labels attached at positions









K55C and K55C'. Shown in Figure 4-1 are ribbon diagrams from X-ray crystal structures of

HIV-1PR with the flaps in the "closed" (red) and "semi-open" (blue) conformations (PDB ID

2BPX and IHHP, respectively). Each of these ribbon diagrams has been modified at positions

K55 and K55' with MTSL modified CYS residues. As the flaps undergo a conformational

change from "closed" to "semi-open", the distance between the labels is predicted to change

from about 33 A to about 36 A, a net change of approximately 3 A. DEER distance

measurements were successful in providing a description of the conformational ensembles of the

flap region of various subtypes of HIV-1PR (Galiano, Bonora et al. 2007; Galiano, Ding et al.

2009; Kear, Blackburn et al. 2009). Distance measurements by SDSL DEER EPR are based on

the magnitude of the magnetic dipolar coupling of the unpaired spins, which is proportional to

1/r3, where r is distance between the two spins (Pannier, Veit et al. 2000; Jeschke and Polyhach

2007).

Distance profiles from spin-labeled constructs of Subtype Bsi, Subtype Csi, Subtype Fsi,

CRF01_A/Esi, and patient isolate V6i and MDR769i without ligand were analyzed in order to

characterize the conformations and flexibility of the flap region of the protease and to identify

what effect polymorphisms have on the conformational ensemble of HIV-1PR. Additionally, in

order to monitor differences in flap conformations upon inhibitor binding between constructs,

HIV-1PR CRF01_A/Esi constructs were also analyzed upon addition of inhibitors and a non-

hydrolysable substrate mimic, CA-p2. Chapter 1 provided a more detailed discussion on

construct nomenclature and amino acid substitution code. In our naming scheme, a subscript "s"

refers to sequences that have incorporated the Q7K, L33I, and L63I substitutions that stabilize

against autoproteolysis. The subscript "i" refers to inactive protease (D25N). Amino acid

substitution code (e.g., D25N) is given by amino acid residue to be substituted out, followed by









the residue number, followed by the amino acid to be substituted in (e.g., D25N the aspartic

acid residue at position number 25 was mutated to an asparagine residue).

Previous Work

Initial work on DEER measurements of HIV-1PR was performed by former Fanucci group

member Dr. Luis Galiano (Ph.D. 2008). At that time, we did not have the capability here at the

University of Florida (UF) to collect DEER data, and Galiano's experiments were performed in

the lab of Peter Fajer at the National High Magnetic Field Lab (NHMFL) with the help of post-

doctoral research fellow Marco Bonora. This original data was collected at X-band on a Briker

EleXsys E580/E680 equipped with the ER 4118X-MD5 Dielectric Ring Resonator. Dr.

Galiano's work demonstrated the success of the DEER methodology when applied to the study

of flap conformations in Subtype Bsi HIV-1PR. These results showed clear differences in the

most probable distance between the flaps and the flexibility of the flaps in the presence and

absence of the protease inhibitor Ritonavir (Galiano, Bonora et al. 2007).

A B









0.0 0.5 1.0 1.5 2.0 20 30 40 50
Time (ps) Distance (A)

Figure 4-2. DEER results of subtype B HIV-1PR with (grey) and without (black) the FDA-
approved inhibitor Ritonavir; (A) dipolar modulated echo data and (B) resulting
distance distribution profile.

The dipolar modulated echo data for the apo construct (Figure 4-2A, black) is noticeably

different than that of the protease in the presence of protease inhibitor Ritonavir (Figure 4-2A,









grey). The resulting distance distribution profiles (Figure 4-2B) report very different flap

conformations and flexibility. Data shows that the average distance between the labeled sites on

each flap shift by about 3 A (from 36 A to 33 A) upon addition of Ritonavir, indicating that the

flaps become locked in a closed position upon inhibitor binding. Additionally, the breadth of the

distance distribution profile decreased dramatically upon addition of Ritonavir, indicating that

the range of motion or flexibility of the flaps decreased as well. These results provided a

glimpse into the structural mechanism of inhibitor resistance.

This work was noticed and expanded upon by the lab of Steve Kent at University of

Chicago. Kent and co-workers obtained both active and inactive (D25N) K55MTSL-labeled

HIV-1 protease constructs via total chemical synthesis and reported on interflap distances,

determined by DEER, in the presence of three peptidomimetic inhibitors. Each of the inhibitors,

namely MVT-101, KVS-1, and JG-365, represent different stages of the enzyme-catalyzed

peptide bond hydrolysis reaction; MVT-101 is structurally similar to an early transition state,

KVS-1 mimics the tetrahedral intermediate in the reaction, and JG-365 mimics a later transition

state (Baca and Kent 1993; Torbeev, Mandal et al. 2008). The resultant distance distribution

profiles demonstrated that the flaps adopted different catalytic properties throughout the course

of the catalytic reaction. In the early stages of the reaction, the flaps are found predominantly in

the closed conformation with little flexibility. As the reaction proceeds, data indicated that the

flaps adopt a more open conformation with increased flexibility, perhaps aiding in product

release (Torbeev, Raghuraman et al. 2008).

Further work in the Fanucci lab, performed by Luis Galiano with the assistance of Dr.

Ralph Weber, Senior Applications Scientist for Bruker's EPR division, focused on understanding

the role of secondary polymorphisms on acquired drug-resistance by the protease. Specifically,









the V6 and MDR769 drug-resistance constructs were investigated. V6 is a clinical isolate from a

pediatric patient treated with RTV and MDR769 was isolated from a patient previously treated

with IDV, NFV, SQV, and APV. Dr. Galiano was able to show that mutations that arise in

response to PI treatment alter the flap conformations of the apoenzyme, thus affecting the

conformational ensemble of the protease. In order to provide structural insight into the

experimental data, MD simulations were performed in the lab of Carlos Simmerling, and the

trends are in excellent agreement. Subtype B adapts an average conformation similar to the

semi-open conformation described by X-ray crystallographic studies. Conversely, MDR769

adopts a more open average conformation and V6 a more closed average conformation with

respect to Subtype B. These results were important because they demonstrated that drug-induced

polymorphisms, often secondary with respect to proximity to the active site, can affect flap

conformations and flexibility, likely contributing to drug resistance. These findings that were

featured in an issue of Chemical and Engineering News (Drahl 2009) will likely contribute

toward the design of inhibitors that can tolerate various point mutations while maintaining

binding affinity.

Subsequent work on Subtype B protease was performed by Mandy E. Blackburn, who

compared the distance distribution profiles of HIV-1PR in the apo form with those in the

presence of nine separate FDA-approved protease inhibitors and a substrate mimic (Figure 4-3)

(Blackburn, Veloro et al. 2009). With this work came the discovery that with sufficiently high

signal to noise ratio (SNR) in the dipolar modulated echo curve, information about the relative

population distributions of the major flap conformations can be extracted in order to describe the

energy landscape and conformational sampling of HIV-1PR (a detailed discussion of the

described analysis method can be found in Chapter 2). Inhibited HIV-1PR distance profiles were









split into two groups, those that showed a "strong/moderate" affect on flap closing (Figure 4-

3A), and those that revealed a "weak" affect (Figure 4-3B), and detailed analyses were

performed to provide distinct relative population percentages for the conformational ensemble of

the flaps upon interaction with inhibitor or substrate (Figure 4-3C). Inhibitors defined as having

strong interactions have at least 70% of the conformational ensemble in the closed flap

conformation. IDV, NFV, and ATV were placed in the "moderate/weak" affect category.

Figure 4-3C shows that the flaps, in the presence of those "moderate/weak" inhibitors, were

predominantly found in the semi-open conformation. Moderate affects were seen for ATV,

where approximately 40% of the conformational ensemble is in the closed conformation. Weak

affects were seen for IDV and NFV with less than 20% of the conformational ensemble in the

closed conformation (Blackburn, Veloro et al. 2009).

m sqv
A "Strong" Apo Ca-P2 tpv C
"Moderate" RTV SQV m rtv
LPV APV m drv
ITPV DRV m Ipv
7 cap2
apv 100
M atv
m nfv 80 g
20 30 40 50 m idv 60
B "WeaklModerate" Apo apo 0
n gNFV 0 OR
SNV Closed 20
ATV Semi-open 4 to
Wide-open Xz
o Tucked
Se, Curled
20 30 40 50
Distance (A)

Figure 4-3. Distance distribution profiles of subtype B HIV-1PR with inhibitors that have
displayed a (A) "strong" effect on flap closing and (B) those inhibitors that have
shown a "weak"/"moderate" effect on flap closing. The apo distance profile is given
in both groups for reference. Error is estimated at +2.5%.









Comparisons were made between the percentage of the closed conformer seen with DEER

and enzymatic inhibition constants, thermodynamic dissociation constants, and the number of

non-water-mediated hydrogen bonds identified in crystallographic complexes. No strong

correlation seems to exist between the relative percent closed conformation and the Ki or KD

values; however, the DEER data does seem to be in agreement with the number of non-water

mediated hydrogen bonds between the inhibitor and the protease, suggesting a correlation

regarding inhibitor effectiveness. This work was also important because it showed that with a

high signal to noise ratio (SNR) in the dipolar modulated echo curve, information can be gained

regarding the relative conformational ensembles of the flaps of HIV-1PR.

Table 4-1. Comparison of relative percentage of closed flap conformation of HIV-1PR subtype
B to published values of Ki, KD, and the number of non-water-mediated hydrogen
bonds between inhibitor and protease construct (Blackburn, Veloro et al. 2009).
Inhibitor Relative % Ki KD # non-water
(abbreviation) closed conformation (nM) (pM) mediated Hydrogen bonds
(+5%) (Blackburn, Veloro et al. 2009)
Saquinavir (SQV) 93 1.3a 280c 7d
Tipranavir (TPV) 91 0.019b 19b 6e
Ritonavir (RTV) 90 0.7a 100 7f
Darunavir (DRV) 87 0.010b 10b 6d
Lopinavir (LPV) 84 0.05a 36c 3g
Amprenavir (APV) 76 0.17a 220c 5d
Atazanavir (ATV) 41 0.48a NA 3b
Indinavir (IDV) 14 3.9a 590c 3d
Nelfinavir (NFV) 14 1.2a 670c 2d
*a. (Clemente, Moose et al. 2004), b. (Muzammil, Armstrong et al. 2007), c. (Yanchunas,
Langley et al. 2005), d. (Prabu-Jeyabalan, King et al. 2006), e. (Nalam, Peeters et al. 2007), f
(Prabu-Jeyabalan, Nalivaika et al. 2003), g. (Reddy, Ali et al. 2007)

The SDSL DEER EPR results presented in this chapter were collected in order to examine

conformational sampling in the apoenzyme and to determine how naturally evolved and drug-

induced polymorphisms alter the effect of inhibitors on the conformational ensemble and flap

flexibility of non-B subtypes and drug-resistant patient isolates from protease inhibitor exposed

patients. The dipolar modulated echo data and resulting distance distribution profiles differed









greatly among the apo protease constructs. From detailed analysis of the echo data, we reported

differences in populations of four distinct flap conformations, namely wide-open, semi-open,

closed, and tucked/curled. These results demonstrated that natural and drug-induced

polymorphisms in the amino acid sequence of various subtypes and patient isolates, whether

because of naturally occurring amino acid substitutions or drug pressure selected mutations alter

the average flap conformations and flexibility of the flaps. These results, which may indicate

how select mutations may play a role in viral fitness and drug resistance, were featured in an

issue of AIDS Weekly (30 Nov. 2009).

Materials and Methods

Materials

The chemicals, reagents, and supplies were obtained from Fisher Scientific (Pittsburg,

Pennsylvania) and used as received, with a few noted exceptions. pET23 plasmid DNA was

purchased from Novagen (Gibbstown, New Jersey), and sequence specific information is given

in the appendix. HiTrap Q HP Anion Exchange column, HiPrep 16/60 Sephacryl S-200 high

resolution size exclusion column was purchased from GE Biosciences (formerly Amersham,

Pittsburg, Pennsylvania). HIV-1PR DNA was synthesized and subsequently purchased from

DNA2.0 (Menlo Park, California). 4-maleimido-2,2,6,6-tetramethyl- 1-piperidinyloxy (4-

maleimido-TEMPO, MSL) was purchased from Sigma-Aldrich (St. Louis, MO). (1-oxyl-

2,2,5,5-tetramethyl-A3-pyrroline-3-methyl) methanethiosulfonate spin label (MTSL) was

purchased from Toronto Research Chemicals, Inc. (North York, Ontario, Canada). The

QuikChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, California).

0.60 I.D. x 0.84 O.D. capillary tubes were purchased from Fiber Optic Center (New Bedford,

Massachusetts). BL21*(DE3) pLysS E. coli cells were purchased from Invitrogen (Carlsbad,

California). NaOAc (C2D302), D20, ds-glycerol were purchased from Cambridge Isotope Labs









(Andover, MA). Ritonavir, Indinavir, Tipranavir, Darunavir, Amprenavir, Atazanavir,

Nelfinivir, Saquinavir, and Lopinavir were generously received from the AIDS Research and

Reference Reagent Program, Division of AIDS, NIAID, NIH (Bethesda, Maryland) (NIH). The

non-hydrolysable substrate mimic, CA-p2 (H-Arg-Val-Leu-r-Phe-Glu-Ala-Nle-NH2 (R-V-L-r-F-

E-A-Nle-NH2, r = reduced) was synthesized and purchased from the University of Florida

Protein Chemistry Core Facility (Gainesville, Florida).

Methods

Details of protein constructs

Six different HIV-1PR constructs will be discussed in this chapter; namely A/Esi

(CRF01 A/E; D25N, K55C, C67A, C95A, Q7K, L33I, L63I), Fsi (Subtype F; D25N, K55C,

C67A, C95A, Q7K, L33I, L63I), Bsi (Subtype B; D25N, K55C, C67A, C95A, Q7K, L33I, L63I),

Csi (Subtype C; D25N, K55C, C67A, C95A, Q7K, L33I, L63I), V6i (Patient isolate V6; D25N,

K55C, C67A, C95A), and MDR769i (multi-drug resistant patient isolate MDR769; D25N,

K55C, C67A, C95A). V6 was isolated from a pediatric patient previously treated with the

protease inhibitor Ritonavir, and MDR769 is a multi-drug resistant protease isolated from a

patient previously treated with Saquinavir, Indinavir, Amprenavir, and Nelfinivir. All constructs

were labeled with (1-Oxyl-2,2,5,5-Tetramethyl-A3-Pyrroline-3-Methyl)methanethiosulfonate

spin label (MTSL) at the K55C and K55C' sites. Structures of the spin labels and spin labeled

side chains are shown in Figure 2-10. HIV-1PR variant constructs contain the following

polymorphisms relative to the LAI consensus sequence of Subtype B:

Subtype C T12S, I15V, L19I, M36I, S37A, H69K, N88D, L89M, I93L
Subtype F I15V, E35D, S37N, R41K, R57K
CRFO1 A/E I13V, E35D, M36I, S37N, R41K, H69K, and L89M
V6 K20R, V32I, M36I, A71V, V82A, L90M
MDR769 L10I, M36V, M46L, I54V, I62V A71V, V82A, I84V, L90M










Tables 4-2 (residues 1-50) and 4-3 (residues 51-99) give details of protein sequences for

each construct. Highlighted in grey are the positions of the stabilizing mutations, of the D25N

active site mutations, of the K55C reporter site mutations, and of the CYS to ALA mutations

required to facilitate SDSL. Italicized and in yellow are the residues on each variant that differ

from those in the LAI consensus sequence of Subtype B. Figure 4-4 shows ribbon diagrams of

HIV-1PR structure highlighting the sites of the protein constructs where polymorphisms occur.

Table 4-2. HIV-1PR variant sequence alignment residues 1-50.
1 50
Subtype B: PQITLi ii -PLVTIKIGG'.i F -I..li LTGADD'i i -EE ii L.PGRWKPKMIGGI
Subtype C: PQITLi i -FlPLVSIKVGG i T, .i.I TLGADDI T-I E E ._L.PGRWKPKMIGGI
Subtype F:PQITLi ii -FPLVTIKVGG',.i F T.L.[lTADD' T-I IE i 'PGKWKPKMIGGI
CRFO1 A/E:PQITLi ii-F:PLVTVKIGGI,. i TI ..lliTGADDITT IE. -,' -I.PGKWKPKMIGGI
V6: PQITLWQRPLVTIKIGGiL.. -E 1.1.I TGADDTIFEEISLPGRWKPKMIGGI
MDR769: PQITLWQRPIVTIKIGGi. LF T-i.-I. LTADDTVLEEVNLPGRWKPKLIGGI

Table 4-3. HIV-1PR variant sequence alignment residues 51-99.
51 99
Subtype B:GG F I ,- F ,-YDQI IT E i:.Hi --IGTVLVGPTPVNI IGRNLLTQIG-T LNF
Subtype C:GGFI T- 1F ,YDQI I E I-i _- 1- I GTVLVGPTPVNIIGRNMLTQLG TLNF
Subtype F:GG I ,:' -. -QYDQI I E I H .Hi '--I GTVLVGPTPVNI IGRNLLTQIG-T LNF
CRFO1_A/E : GG I -F ,-YDQIIE IT E-. 1 T_--I GTVLVGPTPVNI I GRNMLTQI -T LNF
V6: GG F I ,: --F YDQI I- E i-' Hi IGTVLVGPTPANIIGRNLMTQIG T LNF
MDR769: GG E :-,i F QYDQV- -I --E, H i '-GTVLVGPTPANVIGRNLMTQIG- TLNF


Subtype C


Subtype F


CRF01 A/E


V6 MDR769


Figure 4-4. Ribbon diagrams of HIV-1PR (PDB ID: 2PCO) with amino acid differences relative
to the LAI consensus sequence highlighted by colored spheres. All diagrams were
rendered with VMD (Humphrey 1996).









Expression of HIV-1 protease

The over-expression of each protein construct was carried out in the pET-23a vector from

Novagen. Expression of Subtype F and CRF01_A/E constructs was described in Chapter 3, and

expression of Subtype B and C proceeded via a similar method. After induction with IPTG, the

over-expression of MDR769 and V6 constructs was carried out at 20 OC for longer periods of

time, approximately 10 hours each, due to lower expression levels.

Purification of HIV-1 protease

Purification of HIV-1 PR was carried out as described in Chapter 3, with the exception of a

change in the pH of the anion exchange buffer. The pH of this buffer is dependent upon the

isoelectric point (pI) of the protein, which was calculated using the isoelectric point calculator

program on EXPASY (http://www.expasy.ch/tools). The theoretical pi's of Subtypes B, C, and

F, CRF01 A/E, V6, and MDR769 constructs are 9.39, 9.59, 9.28, 9.53, 9.12, and 9.06,

respectively; thus the respective pHs of the anion exchange buffer were 8.85, 9.05, 8.75, 9.05,

8.65, and 8.50. Using this purification scheme, we are able to produce protein that was estimated

to be >95% pure by SDS-PAGE.

Spin-labeling

Spin-labeling was carried out as described in Chapter 3. All DEER experiments were

carried out using MTSL spin label. When spin-labeling was needed, the purified and refolded

protein sample was buffer exchanged into 10 mM Tris HC1, pH 6.9 (further details of spin-

labeling discussed in next section) after the final purification step. Approximately 1 mg of spin

label was dissolved in 100 [L ethanol, and added to approximately 40 mL of HIV-1PR in 10

mM Tris HC1, pH 6.9. The spin-labeling reaction was carried out in the dark (via wrapping

reaction tube in aluminum foil) at room temperature for approximately 4-6 hours followed by 6-8

hours at 4 C for inactive (D25N) constructs, and at 4 OC for approximately 8-12 hours for active









(D25) constructs. At this time, the sample solution was centrifuged at 12000 rpm for 20 minutes

at 4 C to remove solid impurities and aggregated proteins. The sample was then buffer

exchanged into 2 mM NaOAc, pH 5, and concentrated to OD280=1.25.

Buffer requirements

To extend the spin-memory relaxation time, Tm, which improves the signal-to-noise ratios

(SNR) of the experimental echo curves, samples were buffer exchanged into deuterated solvent

composed of 2 mM NaOAc (C2D302) in D20, pH 5.0, with 30% ds-glycerol (used as a glassing

agent). The buffer exchange process is carried out using a 5 mL HiTrap Desalting column from

GE Healthcare (packed with Sephadex G25), which is first washed successively with 3 4

column volumes (15 -20 mL) of nanopure water (nH20), 1M NaC1, nH20, 0.5 M NaOH, nH20,

and NaOAc (pH 5). The column is then equilibrated with 10 mL deuterated NaOAc (pH 5). 1

mL sample (OD280 = 2.5 to 2.7) is then injected, followed by 0.5 mL of deuterated NaOAc; all

flow-through to this point is discarded. Another 1.5 mL of deuterated NaOAc is injected while

collecting the buffer-exchanged sample in another tube. Finally, 0.5 mL of nH20 is injected

while collecting the last 0.5 mL of buffer-exchanged sample. The 2 mL of buffer exchanged

sample is then concentrated to the appropriate volume.

Circular dichroism spectroscopy

To demonstrate that HIV-1 PR constructs retained proper secondary structure after spin-

labeling, circular dichroism (CD) experiments were performed as described in Chapter 3.

Typical parameters used for circular Dichroism experiments are summarized in Table 3-10. The

results of these experiments are given in Figure 4-5. Results match published circular dichroism

results for HIV-1PR, indicating proper folding of the protease.











9000- -
Subtype C
6000- CRF01 A/E
30- Subtype F
I 3000- \ V6
l MDR769
01)
-3000

-6000
200 210 220 230 240
Wavelength (nm)

Figure 4-5. Circular dichroism spectra for spin-labeled HIV-1 PR, subtype B LAI consensus
sequence cyann), subtype C (red), CRF01_A/E (green), subtype F (blue), V6 (black),
and MDR769 (purple).

DEER experiments

Pulsed EPR data were collected on a Bruker EleXsys E580 EPR spectrometer (Billerica,

MA) equipped with the ER 4118X-MD-5 dielectric ring resonator at a temperature of 65 K

(cooled via liquid helium) and 4-pulse DEER sequence, described in Chapter 2. DEER data

collection was typically preceded by a series of preliminary experiments, also described in

Chapter 2, designed to accurately determine the center field, Tm, the do (time in ns at which the

echo begins) and pulse gate (the breadth of the echo in ns), and appropriate positions for the

observer sequence and the pump pulse. The "observer sequence" is applied at the low field

resonance approximately 26 Gauss below the "pump pulse" at central resonance.

DEER data analysis

Data analysis was carried out as described previously by Blackburn et al. (Blackburn,

Veloro et al. 2009). The raw experimental data was processed via Tikhonov Regularization

using DeerAnalysis2008 software, available at http://www.epr.ethz.ch/software/index. Distance









profiles were reconstructed with a series of Gaussian functions via an in-house Matlab based

program called DeerSim.

Population validation

Population validation was performed as discussed previously by Blackburn et al.

(Blackburn, Veloro et al. 2009). The validity of each population with a relative percentage of

10% or below was tested via population suppression using DeerSim and DeerAnalysis2008.

This concept is discussed in detail in Chapter 2.

Results and Discussion

Subtype Polymorphisms Found Among Subtypes B, C, F, CRF01_A/E and Patient Isolates
V6 and MDR769 Confer Altered Flap Conformations and Flexibility in the Apo
Protease

Introduction

As discussed in detail in previous chapters, HIV-1 is categorized into different groups,

subtypes (or clades), and circulating recombinant forms (CRFs). Groups refer to distinctive viral

lineages, subtypes are specific taxonomic groups within a lineage, and CRFs are recombinant

forms of the virus comprised of different viral strains (Kantor, Katzenstein et al. 2005). Each

subtype and CRF is defined by a unique set of naturally occurring polymorphisms. Previous

SDSL DEER results have provided detailed information about the flap conformations sampled in

Subtype B. Distinctive flap conformations have been described and are referred to as curled,

closed, semi-open, and wide-open. Each of these conformations has been detected in the

distance profile of Subtype B protease and has been modeled with molecular dynamics (MD)

simulations (Galiano, Bonora et al. 2007; Ding, Layten et al. 2008; Kear, Blackburn et al. 2009;

Torbeev, Raghuraman et al. 2009). Protease inhibitors used in treatment of HIV-1 are often

designed with respect to subtype B (Wlodawer and Vondrasek 1998), thus it is of great

importance to understand how subtype polymorphisms affect protein structure and flexibility and










thus the efficacy of inhibitors (Rose, Craik et al. 1998; Velazquez-Campoy, Vega et al. 2002;

Clemente, Coman et al. 2006; Coman, Robbins et al. 2007; Sanches, Krauchenco et al. 2007;

Bandaranayake, Prabu-Jeyabalan et al. 2008; Coman, Robbins et al. 2008).

Zero-time selection

A B C









-100 0 100 200 -100 0 100 200 300 400 500 -100 0 100 200 300 400
T (ns) T (ns) T (ns)
D E F









-100 0 100 200 300 400 500 -100 0 100 200 300 400 500 -300-200-100 0 100 200 300 400
T (ns) T (ns) (ns)
Figure 4-6. Dipolar modulated echo curves used for zero time selection of A) subtype Bsi, B)
subtype Csi, C) subtype Fsi, D) CRF01 A/Esi, E) V6i, and F) MDR769i.

The first step in DEER data analysis by TKR is to accurately determine the zero-time. As

discussed in Chapter 2, DEER data was collected with a small amount of negative time (Figure

2-17A). If the incorrect zero-time is selected, the distance distribution will be shifted towards

either smaller or greater distances (Figure 2-17B). DEER data in the form of a dipolar

modulated echo curve was truncated in the range of = -300 to 300 and plotted in Origin8.0

(Figure 4-6), then fit to a function called GaussAmp (the amplitude version of the Gaussian peak

function) of the form given in Equation 4-1, where yo is the y-offset, A is the amplitude, y is









equal to yo + A, w = the full width at half max, and xc is the center of the function; thus Xc is

equal to the zero-time. Table 4-4 reports the zero times chosen for DEER data analysis.

(x-x,)2
y =y +Ae 2w2
(4-1)

Table 4-4. Zero-times chosen for apo data analysis.
Subtype Zero-time
Bsi 110 ns
Csi 115 ns
Fsi 305 ns
CRF01 A/Esi 108 ns
V6i 108 ns
MDR769i 307 ns

Background subtracted dipolar modulated echo curves

Background subtracted echo data with overlaid TKR fits are shown for data analyzed

without and with a long-pass filter, respectively, in Figure 4-7A and 4-7B. The long-pass filter,

an optical filter that selectively attenuates shorter wavelengths, was applied using

DeerAnalysis2008 TKR analysis software. Calculated values of SNR are given in Table 4-5.

The following sections provide details for all data analysis, including:

A) Long-pass filtered and background subtracted dipolar echo curve (black) overlaid with the

TKR regenerated echo curve from DeerAnalysis (red) and with the reconstructed echo

curve from DeerSim (blue) corresponding to the sum of distance profiles comprising the

Gaussian populations shown in D.

B) L-curve utilized to choose the appropriate regularization parameter.

C) TKR distance profile (red) overlaid with summed Gaussian population profile (blue).

D) Individual populations in Gaussian reconstruction of the TKR distance profile.

E) Pake dipolar pattern resulting from Fourier transform of the background subtracted dipolar
echo curve (black) and the TKR fit echo curve from DeerAnalysis (red).









Additionally, a table is provided which summarizes values determined from both TKR

analysis Gaussian reconstruction. Population validation procedures and a Figure summarizing

the population validation process are given, and include:

A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population

profile (blue dashed). Populations marked with 'x' were discarded and populations

marked with '*' were deemed questionable.

B) Individual populations for the Gaussian reconstruction.

C-J, where applicable) Background subtracted dipolar echo curve (black) overlaid with the

TKR fit (red) and the modified echo curve for the distance profile with one or more

populations suppressed (blue).


0 1 2 3
T (JS)


0 1 2 3
T (j4S)


Figure 4-7. Background subtracted time-domain echo data (black) with fits generated by TKR
for both A) non-optically filtered and B) long-pass filtered data

Table 4-5. Values of signal:noise ratios (SNRs) for background subtracted echo data.
Construct Raw SNR Long-pass filtered SNR
Subtype B 20 28
Subtype C 20 21
Subtype F 13 20
CRF01 A/E 13 20
V6 13 23
MDR769 11 19











Data analysis and population validation process: Subtype Bsi


Background Subtracted Echo Data
TKR
-Gaussian Reconstruction








0 1 2 3
Tr(s)


-7 -6 -5
log r


20 30 40 50 20 30 40 50
Distance (A) Distance (A)


0 5
f(MHz)


Figure 4-8. Data analysis for HIV-1PR subtype Bsi apo. A) Background subtracted dipolar echo
curve (black) overlaid with the TKR regenerated echo curve (red) and reconstructed
echo curve (blue) comprising the Gaussian populations shown in D, B) L-curve, (X =
10), C) distance profile from TKR analysis (red) overlaid with the summed Gaussian
population profile (blue dashed), D) individual populations necessary for Gaussian
reconstruction process, and E) Pake dipolar pattern.


-4 -3














-TKR
- Gaussian Reconstruction









20 30 40 50
Distance (A)

TKR
--Tucked









0 1 2 3
T(ps)

TKR
---Unassigned and Wide-open


20 30 40 50
Distance (A)

- TKR


I I I I
0 1 2 3
T(ps)

TKR
--Tucked and Wide-open


S 1 2 3 0 1 2 3
0 1 2 3 0 1 2 3


- TKR


I I +I I
0 1 2 3
T(ps)

TKR
--Unassigned and Tucked


I I I
0 1 2 3
a(us)

TKR
--Unassigned and Tucked
I and Wide-open


0 1 2 3


Figure 4-9. Population validation process for HIV-1PR subtype Bsi apo. A) Distance profile
from TKR analysis (red) overlaid with the summed Gaussian population profile (blue
dashed). B) Individual populations for the Gaussian reconstruction. D-J) Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).


Table 4-6. Results of Gaussian reconstruction and population validation procedures for Bsi.
Subtype Bsi Apo R (A) FWHM (A) TKR % Final %
Unassigned 22.2 3.0 2 0
Tucked 28.5 4.0 5 5
Closed 33.3 3.9 23 24
Semi-open 36.1 5.2 65 67
Wide-open 40.4 2.8 5 4











Data analysis and population validation process: Subtype Csi


Background Subtracted Echo Data
TKR
Gaussian reconstruction








0 1 2 3


-6 -5 -4 -3
log r


30 40 50 20 30 40
Distance (A) Distance (A)


0 5
f(MHz)


Figure 4-10. Data analysis for HIV-1PR subtype Csi apo. (A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. (B)
L-curve, (X = 3.1623). (C) Distance profile from TKR analysis (red) overlaid with
the summed Gaussian population profile (blue dashed). (D) Individual populations
necessary for Gaussian reconstruction process, and (E) Pake dipolar pattern.


T

I
**
m'i'mm m.....














- Curled
- Closed
- Semi-open
- Wide-open






20 30 40 50
Distance (A)

TKR
Closed and Tucked


0 1 2 3 0 1 2 3


I I + I
0 1 2 3
C (gs)


Figure 4-11. Population validation process for HIV-1PR subtype Csi apo. A) Distance profile
from TKR analysis (red) overlaid with the summed Gaussian population profile (blue
dashed). B) Individual populations for the Gaussian reconstruction. D-J) Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).

Table 4-7. Results of Gaussian reconstruction and population validation procedures for Csi.
Subtype Csi Apo R (A) FWHM (A) TKR % Final %
Tucked 29.7 3.1 13 13
Closed 33.3 2.8 8 8
Semi-open 36.7 4.0 52 52
Wide-open 40.2 3.3 27 27


- TKR
--- Tucked


Distance (A)


- TKR
-- -Closed












Data analysis and population validation process: Subtype Fsi


-Background Subtracted Echo Data
TKR
Gaussian Reconstruction









0 1 2 3
T (us)


B
-5

-10

-15

--20

-25

-30


Distance (A)


U



--
-





-4.5 -4.0 -3.5 -3.0 -2.5
log r1
- Tucked
- Closed
- Semi-oper









20 30 40 50
Distance (A)


0 5
f(MHz)


Figure 4-12. Data analysis for HIV-1PR subtype Fsi apo. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve, (X = 100). C) Distance profile from TKR analysis (red) overlaid with the
summed Gaussian population profile (blue dashed). D) Individual populations
necessary for Gaussian reconstruction process, and E) Pake dipolar pattern.















- TKR
- -Gaussian Reconstruction






x x

20 30 40 50
Distance (A)

TKR
-- Wide-open









0 1 2 3
T ([S)

-TKR
Wide open and Unassigned


0 1 2 3
T (gs)


Tucked
Closed
Semi-oper.








20 30 40 50
E Distance (A)
TKR
-- Unassigned









0 1 2 3
H cT (S)
TKR
\- Tucked and Unassigned


0 1 2 3


- TKR
S--Tucked


0 1 2 3
T (gS)

-TKR
S- Tucked and Wide-open


0 1 2 3
T ([S)

TKR
Tucked and Wide open
and Unassigned


0 1 2 3


Figure 4-13. Population validation process for HIV-1PR subtype Fsi apo. A) Distance profile
from TKR analysis (red) overlaid with the summed Gaussian population profile (blue
dashed). B) Individual populations for the Gaussian reconstruction. D-J) Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).


Table 4-8. Distance distribution profile for apo HIV-1PR Fsi.
Subtype Fsi Apo R (A) FWHM (A) TKR % Final %
Tucked 28.8 4.8 12 12
Closed 32.1 4.9 16 17
Semi-open 35.8 6.8 68 71
Wide-open 46.5 5.4 2 0
Unassigned 53.0 5.9 2 0












Data analysis and population validation: CRFO1_A/Esi


1 2 3
T(gs)


Distance (A)


-5 0 5
f(MHz)


-10


-15


S-20


-25


-6 -5 -4 -3
log q

- Tucked
- Closed
- Semi-open








20 30 40 50
Distance (A)


Figure 4-14. Data analysis for HIV-1PR CRF01_A/Esi Apo. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve, (X = 100). C) Distance profile from TKR analysis (red) overlaid with the
summed Gaussian population profile (blue dashed). D) Individual populations
necessary for Gaussian reconstruction process, and E) Pake dipolar pattern.


Background Subtracted Echo Data
-TKR
- Gaussian Reconstruction


't
















-TKR
* Gaussian Reconstruction





x x

I I I
20 30 40 50
Distance (A)


I I I +
0 1 2 3
T(US)

-TKR
-- Tucked and Curled








0 I I2
0 1 2 3


- Tucked
- Closed
- Semi-open








20 30 40 50
Distance (A)


I I I I
0 1 2 3
T(US)

-TKR
Curled and Unassigned








0 1 2 3


-TKR
Tucked








0 1 2 3
I s)

-TKR
Tucked and Unassigned








0 1 2 3


Figure 4-15. Population validation process for HIV-1PR CRF01_A/Esi apo. A) Distance profile
from TKR analysis (red) overlaid with the summed Gaussian population profile (blue
dashed). B) Individual populations for the Gaussian reconstruction. D-J) Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).


Table 4-9. Distance distribution profile for HIV-1PR A/Esi.
CRF01_A/Esi Apo R (A) FWHM (A) TKR % Final %
Curled 21.2 3.1 1 0
Tucked 29.7 5.2 13 14
Closed 33.3 5.4 25 25
Semi-open 36.7 6.5 59 61
Wide-open 40.2 4.7 2 0











Data analysis and population validation: V6i


I II
0 1 2 3
T (gs)


-8 -7 -6 -5
log ri


30 40 50 20 30 40
Distance (A) Distance (A)


0
f(MHz)


Figure 4-16. Data analysis for HIV-1PR patient isolate V6i apo. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve, (X = 50). C) Distance profile from TKR analysis (red) overlaid with the
summed Gaussian population profile (blue dashed). D) Individual populations
necessary for Gaussian reconstruction process, and E) Pake dipolar pattern.


Background Subtracted Echo Data
- TKR
-Gaussian Reconstruction













- TKR
- Gaussian Reconstruction








20 30 40 50
Distance (A)

TKR
--Closed


Tucked
Closed
Semi-open







20 30 40 50
E Distance (A)

TKR
Only Semi-open



" ---


0 1 2 3
T (gs)


0 1 2 3 0 1 2 3
T (gs) t (Ls)


Figure 4-17. Population validation process for HIV-1PR V6i apo. A) Distance profile from TKR
analysis (red) overlaid with the summed Gaussian population profile (blue dashed).
B) Individual populations for the Gaussian reconstruction. D-J) Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).

Table 4-10. Distance distribution profile for V6i.
V6i Apo R (A) FWHM (A) TKR % Final %
Tucked 29.3 3.85 8 8
Closed 33.2 6.0 10 10
Semi-open 36.0 6.5 82 82


- TKR
--- Tucked











Data analysis and population validation: MDR769i


0 1 2 3
T (gs)


20 30 40 50
Distance (A)


-5.5 -5.0
log rI
- Curled
- Closed
- Semi-opet
-Wide-opei,
L


20 30 40
Distance (A)


0 5
f(MHz)


Figure 4-18. Data analysis for HIV-1PR patient isolate MDR769i apo. A) Background
subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve
(red) and reconstructed echo curve (blue) comprising the Gaussian populations shown
in D. B) L-curve, (X = 70). C) Distance profile from TKR analysis (red) overlaid
with the summed Gaussian population profile (blue dashed). D) Individual
populations necessary for Gaussian reconstruction process, and E) Pake dipolar
pattern.


Background Subtracted Echo Data
-TKR
- Gaussian Reconstruction




























30 40
Distance (A)


0 1 2 3
r(Vs)

TKR
20.4 A and 26.3 A










0 1 2 3
r(Vs)

TKR
26.3 A and closed




26. A an L


- Curled
- Closed
- Semi-opel.
--Wide-opei.








20 30 40 50
Distance (A)

TKR
Closed








I I I
0 1 2 3
r(Vs)

TKR
20.4 A and closed









I I I I
0 1 2 3
r (Vs)

TKR
S- 26.3 A and wide-open


TKR
-20.4 A










0 1 2 3
T(rs)
TKR
-- Wide-open








I I I +
0 1 2 3
T( -s)

TKR
20.4 A and wide-open










0 1 2 3

r(Vs)

TKR
Closed and wide-open


0 1 2 3 0 1 2 3 0 1 2 3
T (Vs) T (Us) T (Us)


Figure 4-19. Population validation process for HIV-1PR MDR769i apo. A) Distance profile

from TKR analysis (red) overlaid with the summed Gaussian population profile (blue
dashed). B) Individual populations for the Gaussian reconstruction. D-J) Background

subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed

(blue).


- TKR









Table 4-11. Distance distribution profile for MDR769i.
MDR796i Apo R (A) FWHM (A) TKR % Final %
Unassigned 20.4 3.4 7 0
Curled 26.3 3.4 7 7
Closed 33.4 3.5 7 9
Semi-open 36.4 5.4 74 79
Wide-open 44.9 3.3 5 5

Polymorphism-induced shifts in the conformational ensemble

The distance profiles for each of the apo proteases from subtypes B, C, F, CRF01_A/E,

V6, and MDR769, are shown in Figure 4-20A (Kear, Blackburn et al. 2009). For each of the

constructs, a peak centered near 33 A corresponding to the closed conformation is required for

regeneration of the TKR distance profile. The semi-open conformation, with a distance of 36 A,

was found to be the major sub-population required to regenerate each of the distance profiles.

This conformation was assigned from molecular dynamic (MD) simulations. A sub-population

centered at 25-30 A is required for adequate fitting of the distance profiles. These distances

correspond to conformations in which the flaps are tucked in towards one another or into the

active site pocket (Scott and Schiffer 2000; Heaslet, Rosenfeld et al. 2007). Distances closer to

25 A are assigned to a curled conformation, while distances closer to 30 A are assigned to a

tucked flap conformation. Finally, a population with average distances of 40-45 A is needed to

regenerate each of the distance profiles for the apo proteases. These distances correspond to a

wide-open conformation of the flaps, which has seen in MD simulations of Subtype B (Ding,

Layten et al. 2008). Figure 4-20B shows the relative percentages of each of the sub-populations

utilized in the Gaussian reconstruction of each of the TKR distance profiles, and Figures 4-21

and 4-22 show this same data, but broken into individual conformations and contracts,

respectively. Figure 4-23 shows individual overlays of the apo Subtype B distance profile with

each other apo construct's profile for comparison.


































20 30 40
Distance (A)


Figure 4-20. DEER results for HIV-1PR subtypes B, C, F, CRF01_A/E, and drug resistant
patient isolates in the apo; A) distance distribution profiles of each construct, and B)
results of population analysis.


A

100.

S80.

60"

S40.

2 20


8 C


Tucked/Curled


- r ,,,,V MDR7,69
Variant


100Semi-open
100
80- -- --

60- -

40-

20-


I? C P CPVV6 Alb)?a
SVariant69
Variant


Closed
100Inn


S80-
o
60-
-
S40-
S20-
' 20-
-


a C -P C',,,,V6 M R769
Variant


Wide Open
100

80-

60-

40-

20-
20

a C P CP01 R769
Variant


Figure 4-21. Relative percentage of A) tucked/curled, B) closed, C) semi-open, and D) wide-
open conformations of protease constructs in the apo form.



184


-- -nnn --











A B C
Subtype B Subtype C Subtype F


100
1UU -----------------

80-

60- a

40- -

20- -
0

Conformation

CRF01 A/E
i n .


100 100

80 80


S40 40 ~



10 0 1 0 ,
' 20 3 20

o0IlI1 I I I 0-L ------
Conformation Conformation
E F
V6 MDR769
loof 1-loof


80- 8 80- 80

60- -r 60- a a a 60 a a

40 4 ? 40 -

S 20- 20- 20-

0 -0 I I
Conformation Conformation Conformation

Figure 4-22. Individual plots showing relative percentage of tucked/curled, closed, semi-open
and wide-open conformations in the apo form of A) subtype B, B) subtype C, C)
subtype F, D) CRF01_A/E, E) V6, and F) MDR769 variants.

Table 4-12. Summary of distance parameters obtained from DEER distance profiles of HIV-1PR
constructs.
Construct Range (Span) Most Probable distance Average Distance
(+1 A) (+0.2 k) (0.2 A)
Bsi 24 45 (21) 35.2 35.2
Csi 25-45 (20) 36.9 36.5
Fsi 24 45(21) 35.1 34.3
CRF01_A/Esi 25 45(20) 34.8 35.2
V6i 25 45 (20) 35.8 35.2
MDR769i 22 49 (27) 36.3 35.9


The different in the overall shape and breadth of the distance profiles shown in Figures 4-


20A and 4-23 indicate that the individual polymorphisms that define various subtypes, CRFs,


and patient isolates have a drastic impact on average flap conformation and flexibility. Table 4-


12 lists values of the overall span, the most probable distance and the average distance for each


construct. The span of flap motion was calculated using the derivative of the distance profile,


and the double derivative was used to help locate the centers of the individual Gaussian peaks for










the reconstruction procedure, as shown in Figure 4-24. The most probable distance is simply the

distance corresponding to the most intense point in the distance profile (Kear, Blackburn et al.

2009).


- Subtype B
- Subtype C





I \



20 30 40 50
Distance (A)


Distance (A)


30 40 50 20 30 40
Distance (A) Distance (A)


20 30 40 50
Distance (A)


Figure 4-23. Distance distribution profile overlays of subtype B and A) subtype C, B)
CRF01 A/E, C) subtype F, D) V6 and E) MDR769.










A B

Subtype B
Subtype C
Subtype F
CRFO1 A/E
V6
MDR769

20 30 40 50 20 30 40 50
Distance (A) Distance (A)


Figure 4-24. A) Derivative spectra of distance profiles of each variant used to calculate the span
of the distance profile, and B) second derivative spectra of distance profiles of each
variant used to help identify the centers of the individual Gaussian peaks for the
reconstruction procedure.

From analysis of the relative percentages of each construct's sub-populations, the affects of

natural and drug-pressure selected polymorphisms on the average flap conformation can be

understood as shifting the conformational ensemble of the system and changing the flap

flexibility. The distance profile for Subtype C indicates a relatively large percentage of wide-

open flap conformation. It is possible that this difference could be attributed to the presence of

three unique polymorphisms located at positions 12, 19, and 93. Changes in flexibility are

inferred from the breadth of each of the sub-populations. The breadths of the closed populations

of Subtype F, CR01_A/E and V6 have broader than average breadths seen for the other apo-

constructs, indicating either an increase in flap flexibility or flap instability for the closed

conformation (Kear, Blackburn et al. 2009).

The distance profiles for V6 and MDR769 differ slightly from those in Galiano's earlier

report (Galiano, Ding et al. 2009); however, the data reported here are consistent with the

conclusion that MDR769 has a larger relative percentage of wide-open conformation than









Subtype B. The distance profile for V6 shows a greater relative percentage of V6 in the

tucked/curled conformation, however the average value for flap conformation matches within

error that of B.

Inhibitor-Induced Flap Closure in CRF01_A/E Constructs

As previously described, there are currently 9 FDA-approved protease inhibitors that are

administered to HIV-1 patients in the form of a cocktail. These PIs were designed with respect

to subtype B (Wlodawer and Vondrasek 1998); thus, however the efficacy of the inhibitors on

other subtypes, circulating recombinant forms (CRFs), and patient isolates differ dramatically. It

is thus of great importance to understand how variant-specific polymorphisms alter protein

structure and flexibility and thus efficacy of inhibitors (Rose, Craik et al. 1998; Velazquez-

Campoy, Vega et al. 2002; Clemente, Coman et al. 2006; Sanches, Krauchenco et al. 2007;

Bandaranayake, Prabu-Jeyabalan et al. 2008; Coman, Robbins et al. 2008). Additionally, it

would be very beneficial to identify which inhibitors or combinations of inhibitors effectively

close the flaps of each individual variant, thereby possibly reducing the drug-load given to each

HIV-1 patient. Apo data was reported and discussed in the previous section and will not be

repeated here. This section specifically reports on DEER data for HIV-1PR CRF01_A/E with

each of nine FDA-approved protease inhibitors and the non-hydrolyzable substrate mimic CA-

p2. The following sections provide all details for data analysis and population validation, as

described in the previous section.

CRF01_A/Esi dipolar modulation zero-time determination

The proper zero-times were determined as described previously. Figure 4-25 shows the

truncated dipolar evolution curves fit to a GaussAmp function of the form shown in Equation 4-

1. Table 4-13 reports on the zero-times chosen for CRF01_A/Esi data analysis.











Table 4-13. Zero-times chosen for CRF01 A/Esi data analysis.


Inhibitor/Substrate
RTV
IDV
LPV
TPV
SQV
DRV
NFV
ATV
APV
CA-n2


-100 0 100 -100 0 100
T (ns) T (ns)
F





0
wU


-300-200-100 0 100 200 300
r(ns)


-300-200-100 0 100 200 300
r(ns)


-300-200-100 0 100 200 300
(ns)









-300-200-100 0 100 200 300
r(ns)


-300-200-100 0 100 200 300
T (ns)









-300-200-100 0 100 200 300
(ns)


-300-200-100 0 100 200 300
S(ns)


-100 0 100


Figure 4-25. Truncated dipolar modulated echo curves used for zero-time selection of
CRF01_A/E with A) RTV, B) IDV, C) LPV, D) TPV, E), SQV, F) DRV, G) NFV,
H) ATV, I) APV, and J) CA-p2.


Zero-time
105 ns
113 ns
311 ns
309 ns
308 ns
311 ns
307 ns
311 ns
311 ns
106 ns











CRF01_A/Esi with CA-p2


0 1 2 3
T( s)
-TKR
- -Gaussian Reconstruction








20 30 40 50
Distance (A)


0
f(MHz)


-6 -5 -4
log q
Curled
Tucked
-t'-






- Closed
- Semi-open
Wide-open





20 30 40 50
Distance (A)


Figure 4-26. Data analysis for HIV-1PR CRF01 A/E with CA-p2. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


- TKR
-- Unassigned











B C
\ TKR
| Wide-open and Unassigned


0 1 2 3 0 1 2 3 0 1 2 3


T (s)


T (s)


T (s)


Figure 4-27. Population validation process for HIV-1PR CRF01_A/E with CA-p2. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) unassigned, B) wide-open and unassigned, and C) curled, tucked,
semi-open, wide-open, and unassigned suppressed.

Table 4-14. Distance distribution profile for CRF01 A/E with CA-p2.
CRF01 A/Esi CA-p2 R (A) FWHM (A) TKR % Final %
Curled 26.2 1.5 7 7
Tucked 30.5 1.8 8 8
Closed 33.1 2.5 75 77
Semi-open 36.7 1.8 5 5
Wide-open 42.4 2.0 3 3
Wide-open 48.7 2.0 2 0


- TKR
- Unassigned


- TKR
- Curled and Tucked and
Semi-open and Wide-open
and Unassigned


I I I I


I I I I

















TKR
Gaussian Reconstruction









0 1 2 3
(gs)

-TKR
- -Gaussian Reconstruction









20 30 40 50
Distance (A)


-5 0
f(MHz)


Distance (A)


Figure 4-28. Data analysis for HIV-1PR CRF01_A/E NFV. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


CRF01_A/Esi with NFV


L


j
'._
-
-
-























0 1 2 3 0 1 2 3 0 1 2 3


T(ts)


T(S )


T(S )


Figure 4-29. Population validation process for HIV-1PR CRF01_A/E NFV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) curled, B) curled and wide-open, and C) curled, tucked, and wide-
open suppressed.

Table 4-15. Distance distribution profile for CRF01 A/E NFV.
CRF01 A/Esi NFV R (A) FWHM (A) TKR % Final %
Curled 26.6 2.9 4 0
Tucked 30.6 5.1 7 8
Closed 32.2 5.0 26 30
Semi-open 36.1 4.9 60 62
Wide-open 41.0 4.7 3 0


- TKR
- Curled


- TKR
- Curled and Wide-open


-TKR
- Curled and Tucked
and Wide-open











CRF01 A/Esiwith TPV


S 1 2 3
S(ts)
-TKR
- -Gaussian Reconstruction









20 30 40 50
Distance (A)


- TKR
-Gaussian Reconstruction


-5 0 5
f(MHz)


Figure 4-30. Data analysis for HIV-1PR CRF01_A/E TPV. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


-6 -5 -4
log r
- Curled
- Tucked
- Closed
Wide-open







20 30 40 50
Distance (A)











B C
TKR
-- 17.5 A and 22.8 A and
Wide-open and 45.5 A



0 0
oC C
o 1 O
LU I / \ ^S. o rI ^


0 1 2 3 0 1 2 3 0 1 2 3


T(S )


T(gLs)


T (Ls)


Figure 4-31. Population validation process for HIV-1PR CRF01_A/E TPV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) unassigned peaks, B) wide-open and unassigned peaks, and C) tucked,
wide-open, and unassigned peaks suppressed.

Table 4-16. Distance distribution profile for CRF01 A/E with TPV.
CRF01 A/Esi TPV R (A) FWHM (A) TKR % Final %
Unassigned 17.5 1.5 1 0
Unassigned 22.8 1.5 1 0
Curled 26.1 1.7 10 10
Tucked 30.6 2.0 2 3
Closed 32.9 2.3 82 85
Wide-open 40.2 1.0 2 2
Unassigned 45.5 2.0 2 0


- TKR
-- 17.5 A and 22.8 A and
45.5 A


- TKR
-- 17.5 A and 22.8 A and
Tucked and Wide-open
and 45.5 A


I I I











CRF01 A/Esi with LPV


Background Subtracted Echo Data
-TKR
Gaussian Reconstruction








0 1 2 3
S(us)
- TKR
- Gaussian Reconstruction


20 30 40
Distance (A)


0
f (MHz)


-5

-6 -5 -4
log r


20 30 40
Distance (A)


Figure 4-32. Data analysis for HIV-1PR CRF01_A/E LPV. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.























0 1


2 3 0


C (Gis)


1 2
C (Gis)


Figure 4-33. Population validation process for HIV-1PR CRF01_A/E LPV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) wide-open, and B) semi-open and wide-open suppressed.

Table 4-17. Distance distribution profile for CRF01 A/E LPV.
CRF01 A/Esi LPV R (A) FWHM (A) TKR % Final %
Curled 26.0 2.1 11 11
Closed 32.8 3.1 80 81
Semi-open 37.4 2.7 8 8
Wide-open 43.4 2.0 1 0


-- TKR
-- Wide-open


S- TKR
-- Semi-open and Wide-open











CRF01_A/Esi with SQV


- Background Subtracted Echo Data
- TKR
- Gaussian Reconstruction







I I I I
0 1 2 3
T(@S)


Distance (A)


-10- 1



-15. .
', -5-..


-20

-6 -5 -4
D log n
Curled
Closed
Semi-open








20 30 40 50
Distance (A)


-5 0 5
f(MHz)


Figure 4-34. Data analysis for HIV-1PR CRF01_A/E SQV. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.











TKR
--Wide-open








0 1 2 3
C (Gis)


0 1 2 3
-C (Gs)


Figure 4-35. Population validation process for HIV-1PR CRF01_A/E SQV. A, B) Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) wide-open, and B) semi-open and wide-open suppressed.

Table 4-18. Distance distribution profile for CRF01 A/E SOV.
CRF01 A/Esi SOV R (A) FWHM (A) TKR % Final %
Curled 26.1 1.8 11 11
Closed 32.7 2.5 78 80
Semi-open 36.4 2.0 9 9
Wide-open 43.2 1.6 2 0


- TKR
- Semi-open and Wide-open











CRF01_A/Esi with ATV



A
Background Subtracted Echo Data
-TKR
Gaussian Reconstruction







0 1 2 3
C (gs)
-TKR
Gaussian Reconstruction








20 30 40 50
E Distance (A)


log i-
- Closed
- Semi-open
Wide-open







20 30 40 50
Distance (A)


f (MHz)


Figure 4-36. Data analysis for HIV-1PR CRF01 A/E ATV. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.



















0
C-
u-



0 1 2 3
T (s)

Figure 4-37. Population validation process for HIV-1PR CRF01_A/E ATV. A) Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).

Table 4-19. Distance distribution profile for CRF01 A/E ATV.
CRF01 A/Esi ATV R (A) FWHM (A) TKR % Final %
Closed 32.9 5.3 58 58
Semi-open 36.5 5.5 34 34
Wide-open 40.2 4.5 8 8


- TKR
- -Wide-open











CRF01_A/Esi with DRV


-Background Subtracted Echo Data
TKR
-Gaussian Reconstruction








0 1 2 3
T (gs)


Distance (A)


-6 -5 -4
log n
- Curled
-Tucked
-Closed
- Semi-open







20 30 40 50
Distance (A)


-5 0 5
f(MHz)


Figure 4-38. Data analysis for HIV-1PR CRF01_A/E DRV. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.






















0 1 2 3
T(RS)
TKR
Tucked and Semi-open
and Wide-open


C
TKR TKR
Semi-open and Wide-open Tucked and Wide-open




0
LL


0 1 2 3 0 1 2 3


T (gs)


T (gs)


6 1 2 3
T (gs)
Figure 4-39. Population validation process for HIV-1PT CRF01_A/E DRV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) wide-open, B) semi-open and wide-open, C) tucked and wide-open
suppressed.

Table 4-20. Distance distribution profile for CRF01 A/E DRV.
CRF01 A/Esi DRV R (A) FWHM (A) TKR % Final %
Curled 25.9 1.35 10 10
Tucked 28.9 1.2 6 6
Closed 33.0 2.15 78 79
Semi-open 36.3 1.2 5 5
Wide-open 42.3 1.3 1 0


- TKR












CRF01_A/Esi with APV


TKR
Background Subtracted Echo Data
Gaussian Reconstruction









0 1 2 3
S(gs)

-TKR
-Gaussian Reconstruction









20 30 40 50
Distance (A)


-5

-10

a- -15.

-20
-20


-25


D






&--


-




**.



-5
-6 -5 -4
log ir

Curled
- Closed
- Semi-open
- Wide-open







20 30 40 50
Distance (A)


-5 0 5
f(MHz)


Figure 4-40. Data analysis for HIV-1PR CRF01_A/E APV. A) Background subtracted dipolar
echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.













- TKR
- Unassigned and Wide-open


I I I I
0 1 2 3
T(4s)

-TKR
-- 22 4 and Tucked and
Wide-open and 45 35







0 1 2 3


-TKR
-- 22 4 and Curled and Tucked
and 45 35


I I +
0 1 2 3
T(4S)


-TKR
-- Unassigned and Tucked and
Wide-open


0 1 2 3


Figure 4-41. Population validation process for HIV-1PR CRF01_A/E APV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) unassigned and wide-open, B) curled, tucked, and unassigned peaks,
and C) tucked, wide-open, and unassigned, and D) tucked, wide-open, and unassigned
peaks suppressed.

Table 4-21. Distance distribution profile for CRF01 A/E APV.
CRF01 A/Esi APV R (A) FWHM (A) TKR % Final %
Unassigned 22.4 1.0 1 0
Curled 26.15 1.9 5 5
Tucked 30.55 1.4 2 0
Closed 32.85 3.0 65 68
Semi-open 36.15 3.4 21 22
Wide-open 40.7 2.3 5 5
Unassigned 45.35 1.5 1 0











CRF01_A/Esi with RTV


- Background Subtracted Echo Data
- TKR
- Gaussian Reconstruction








0 1 2 3
T (us)


Distance (A)


0
f (MHz)


-5 -4


-Curled
- Closed
- Semi-open
Wide-open







20 30 40 50
Distance (A)


Figure 4-42. Data analysis for HIV-1PR CRF01_A/Esi with RTV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


















O




0 1 2 3
T (GlS)

Figure 4-43. Population validation process for HIV-1PR CRF01_A/Esi with RTV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).

Table 4-22. Distance distribution profile for CRF01 A/Esi with RTV.
CRF01 A/Esi RTV R (A) FWHM (A) TKR % Final %
Curled 26.3 2.4 14 14
Closed 32.9 2.8 57 57
Semi-open 35.9 3.4 22 22
Wide-open 40.8 2.5 7 7


-TKR
C--Curled











CRF01_A/Esi with IDV


A


\





-6 -5 -4
log i-


-Background Subtracted Echo Data
-TKR
Gaussian Reconstruction








0 1 2 3
T (gs)


0
f(MHz)


20 30 40
Distance (A)


Figure 4-44. Data analysis for HIV-1PR CRF01_A/Esi with IDV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlaid with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


30 40
Distance (A)

















-C I

I I I I I I I
.. \ I \




0 1 2 3 0 1 2 3
z (Iis) C (his)

Figure 4-45. Population validation process for HIV-1PR CRF01_A/E. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with A)
unassigned and B) unassigned and curled suppressed.

Table 4-23. Distance distribution profile for CRF01 A/Esi with IDV.
CRF01 A/Esi IDV R (A) FWHM (A) TKR % Final %
Unassigned 16.7 5.0 6 0
Curled 27.0 5.2 11 11
Closed 33.1 6.4 2.8 30
Wide-open 36.0 7.3 55 59

A comparison of distance profiles from CRFO1_A/Esi with various inhibitors

The distance profiles, error analyses, and conformational ensemble report for CRF01_A/E

with each of nine FDA-approved inhibitors and the substrate mimic CA-p2 are shown above.

Figure 4-46 shows the distance distribution profiles for each inhibitor, and Figure 4-47 (A)

shows those that demonstrated a "strong/moderate" affect on flap closure and (B) a "weak"

affect on flap closure. Figure 4-48 shows a graph of each of the sub-populations utilized in the

Gaussian reconstruction of each of the TKR distance profiles, summarizing the differences in the

conformational ensembles of the CRF01_A/E protease, with respect to inhibitor. Indinavir,

Nelfinavir, and Atazanavir did not induce a drastic change in the relative percent closed flap

conformation with respect to apo, thus were placed in the "weak" category.


-TKR
-- Unassigned


- TKR
-- Unassigned and Curled










CRF01 A/E


20 30 40 50
Distance (A)


Figure 4-46. Overlay of distance distribution profiles for CRF01_A/Esi in the apo form and with
each of 9 FDA-approved protease inhibitors and CA-p2 substrate.


20 30 40
Distance (A)


Apo
IDV
NFV
ATV





20 30 40 50
Distance (A)


Figure 4-47. Distance distribution profiles of CRF01_A/Esi with inhibitors that exhibited an (A)
"strong/moderate" affect of flap closure and (B) a "weak" affect on flap closure.


sqv
tpv
m rtv
m dry
M Ipv
m cap2
=apv
M atv
m nfv
m idv
m apo


Closed
Semi-open ^
Wide-open I
Oo Tucked
Curled

4


Figure 4-48. Population analysis for CRF01_A/E. Error is estimated at + 2.5%.









Inhibitor-Induced Flap Closure in Subtype Fsi

This section reports on DEER data for HIV-1PR Subtype F with each of nine FDA-

approved protease inhibitors and CA-p2 substrate mimic. The following sections provide details

for data analysis and population validation, including long-pass filtered and background

subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve from

DeerAnalysis (red) and with the reconstructed echo curve from DeerSim (blue) corresponding to

the sum of distance profiles comprising the Gaussian populations needed to regenerate the

profile, the L-curve, TKR distance profile (red) overlaid with summed Gaussian population

profile (blue), individual populations in Gaussian reconstruction of the TKR distance profile,

Pake dipolar pattern, and a table which summarizes values determined from both TKR analysis

Gaussian reconstruction and all appropriate population validation figures.

Subtype Fsi dipolar modulation zero-time determination

The proper zero-times were determined as described previously. Shown in Figure 4-49

are the truncated dipolar evolution curves in the range oft = -300 to 300, each fit to a GaussAmp

function (the amplitude version of the Gaussian peak function) of the form shown in Equation 4-

1. Table 4-24 reports on the zero-times chosen for Subtype Fsi data analysis determined by the

aforementioned fitting.

Table 4-24. Zero-times chosen for Fsi data analysis.
Inhibitor/Substrate Zero-time
RTV 307 ns
IDV 308 ns
LPV 311 ns
TPV 308 ns
SQV 307 ns
DRV 107 ns
NFV 311 ns
ATV 310 ns
APV 306 ns
CA-o2 108 ns























-300-200-100 0 100 200 300
T(ns)


-300-200-100 0 100 200 300
S(ns)


-300-200-100 0 100 200 300
S(ns)


-300-200-100 0 100 200 300
T (ns) F


-300-200-100 0 100 200 300
T (ns)


-300-200-100 0 100 200 300


-300-200-100 0 100 200 300


-100 0 100
T(ns)


-300-200-100 0 100 200 300


-100 0 100 200 300 400
T(ns)


Figure 4-49. Truncated dipolar modulated echo curves used for zero time selection of
CRF01 A/E with A) RTV, B) IDV, C) LPV, D) TPV, E) SQV, F) DRV, G) NFV, H)
ATV, I) APV, and J) APV.















212











Subtype Fsi with RTV


Distance (A)

TKR
Background Subtracted Echo Data
\- Gaussian Reconstruction


-3O


10-


15 *.


20-

-7 -6 -5

D log r
Curled
Tucked
Closed
Wide-open







20 30 40 50
Distance (A)


f (MHz)


Figure 4-50. Data analysis for HIV-1PR Subtype Fsi with RTV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.





















0 1 2 3 0 1 2 3 0 1 2 3
T (UiS) T (UiS) T (UiS)

Figure 4-51. Population validation process for HIV-1PR Fsi with RTV. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with A)
unassigned and B) unassigned and curled suppressed.

Table 4-25. Distance distribution profile for Subtype Fsi with RTV.
Subtype Fsi RTV R (A) FWHM (A) TKR % Final %
Curled 26.5 2.0 17 17
Tucked 29.2 1.3 3 0
Closed 33.1 2.5 71 71
Semi-open 36.1 2.0 3 0
Wide-open 41.4 2.1 6 9


- TKR
-- -Curled


- TKR
- Tucked


- TKR
-- Wide-open











Subtype Fsi with IDV


A


20 30 40
Distance (A)


-5 0 5
f(MHz)


- TKR
- Background Subtracted Echo Data
i-Gaussian Reconstruction


Figure 4-52. Data analysis for HIV-1PR subtype Fsi with IDV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


-6 -5
log r

- Tucked
- Closed
- Semi-open








20 30 40 50
Distance (A)


0 1 2 3
T (ps)

-TKR
- Gaussian Reconstruction






















0 1 2 3
C (Gis)

Figure 4-53. Population validation process for HIV-1PR subtype Fsi with IDV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue).

Table 4-26. Distance distribution profile for subtype Fsi with IDV.
Subtype Fsi IDV R (A) FWHM (A) TKR % Final %
Unassigned 23.5 4.1 3 0
Curled 27.0 4.8 8 9
Closed 33.0 6.1 41 44
Semi-open 36.1 6.8 43 47
Unassigned 51.6 5.1 5 0


- TKR
-- Unassigned and unassigned










Subtype Fsi with LPV


-TKR
- -Gaussian Reconstruction







20 30 40 50
Distance (A)

TKR
Gaussian Reconstruction





7


-3


-10


-15 .


-20
-7 -6 -5
log Ti
D
Curled
Tucked
Closed
Wide-open
--Clse


0 1 2 3 20 30 40
T (us) Distance (A)


-5 0 5
f(MHz)

Figure 4-54. Data analysis for HIV-1PR subtype Fsi with LPV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.























C (us)

Figure 4-55. Population validation process for HIV-1PR Fsi with LPV. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with
unassigned population suppressed.

Table 4-27. Distance distribution profile for subtype Fsi with LPV.
Subtype Fsi LPV R (A) FWHM (A) TKR % Final %
Unassigned 22.9 1.3 6 0
Curled 25.8 1.9 7 8
Tucked 28.9 1.5 10 10
Closed 33.1 2.5 67 71
Wide-open 41.7 2.1 10 11


- TKR
-- Unassigned























30 40
Distance (A)


I







8 -7 -6
log i-
Closed
Wide-open







20 30 40 50
Distance (A)


-5 0 5
f(MHz)

Figure 4-56. Data analysis for HIV-1PR subtype Fsi with TPV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


Subtype Fsi with TPV


T (us)

























T (Us)

Figure 4-57. Population validation process for HIV-1PR Fsi with TPV. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with A)
unassigned and B) unassigned and curled suppressed.

Table 4-28. Distance distribution profile for subtype Fsi with TPV
Subtype Fsi TPV R (A) FWHM (A) TKR % Final %
Unassigned 18.6 1.0 2 0
Curled 25.9 1.0 2 0
Tucked 29.0 1.0 1 0
Closed 32.9 2.1 83 88
Wide-open 41.3 1.9 12 12


-TKR
-- unassigned and curled
and tucked








I I I I











Subtype Fsi with SQV


30 40
Distance (A)


- Background Subtracted Echo Data
- TKR
- Gaussian Reconstruction








0 1 2 3
T (gs)


-5 0 5
f(MHz)


I


-5^




-7 -6 -5 -4
log ir

- Curled
- Closed
Wide-open








20 30 40 50
Distance (A)


Figure 4-58. Data analysis for HIV-1PR subtype Fsi with SQV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.














- Unassigned


0
C-



II I -
0 1 2 3
T(gs)

Figure 4-59. Population validation process for HIV-1PR Fsi with SQV. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with A)
unassigned and B) unassigned and curled suppressed.

Table 4-29. Distance distribution profile for subtype Fsi with SQV.
Subtype Fsi SQV R (A) FWHM (A) TKR % Final %
Curled 26.5 1.8 13 15
Closed 33.2 3.0 74 75
Wide-open 42.0 3.0 10 10
Unassigned 50.0 2.6 3 0











Subtype Fsi with DRV

A


T (Rs)


- TKR
- Gaussian Reconstruction








20 30 40 50
Distance (A)


I







-5 -4
log n

- Curled
- Tucked
- Closed







20 30 40 50
Distance (A)


f (MHz)


Figure 4-60. Data analysis for HIV-1PR subtype Fsi with DRV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.























z (ilS)


z (ilS)


Figure 4-61. Population validation process for HIV-1PR subtype F with DRV. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) unassigned, B) semi-open and unassigned suppressed.

Table 4-30. Distance distribution profile for subtype Fsi with DRV.
Subtype Fsi DRV R (A) FWHM (A) TKR % Final %
Unassigned 21.5 1.6 6 0
Curled 26.8 1.7 15 16
Tucked 30.5 4.7 17 19
Closed 33.4 2.2 60 65
Semi-open 38.1 1.3 3 0











Subtype Fsi with NFV


A


30 40
Distance (A)


-TKR
-Background Subtracted Echo Data
Gaussian Reconstruction







0 1 2 3


-5 0 5
f (MHz)


-10-


-15-


-20.


-25


D





0L


-6 -5 -4
log 1

- Tucked
- Closed
-Semi-oper.
Wide-oper.




A
20 30 40 50
Distance (A)


Figure 4-62. Data analysis for HIV-1PR subtype Fsi with NFV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.























I I I I
0 1 2 3
T (s)
-TKR
-- Unassigned and curled
and wide-open


-TKR
-- Unassigned


0 1 2 3 0 1 2 3
r (itS) r (itS)

Figure 4-63. Population validation process for HIV-1PR Fsi with NFV. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with A)
unassigned and B) unassigned and curled suppressed.

Table 4-31. Distance distribution profile for subtype Fsi with NFV.
Subtype Fsi NFV R (A) FWHM (A) TKR % Final %
Curled 25.0 3.3 4 0
Tucked 29.3 4.3 10 10
Closed 33.1 5.2 27 29
Semi-open 36.2 5.4 50 54
Wide-open 40.0 3.1 7 7
Unassigned 59.0 5.0 2 0


-TKR
-- Unassigned and curled








+ I I
0 1 2 3
T (uTs)
-TKR
-- Unassigned and wide-open











Subtype Fsi with ATV


-TKR
- Gaussian Reconstruction









20 30 40 50
Distance (A)

Gaussian Reconstruction
Background Subtracted Echo Data
TKR








0 1 2 3


0
f(MHz)


-10-


-15.


s-20- ."


-25-

-7 -6 -5
log 1-
D
Tucked
-Closed
-Semi-open
Wide-open







20 30 40 50
Distance (A)


Figure 4-64. Data analysis for HIV-1PR subtype Fsi with ATV. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


















O
t0
IJ



0 1 2 3
T (uls)

Figure 4-65. Population validation process for HIV-1PR Fsi with ATV. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with A)
unassigned and B) unassigned and curled suppressed.

Table 4-32. Distance distribution profile for subtype Fsi with ATV.
Subtype Fsi ATV R (A) FWHM (A) TKR % Final %
Unassigned 21.4 2.6 2 0
Tucked 28.9 5.2 13 13
Closed 33.0 5.6 56 57
Semi-open 36.0 4.9 19 19
Wide-open 40.5 4.5 10 11


- TKR
-- Unassigned












Subtype Fsi with APV


A
TKR
-Gaussian Reconstruction









20 30 40 50
Distance (A)

-Gausslan Reconstruction
Background Subtracted Echo Data
-TKR








0 1 2 3
T (us)


0
f (MHz)


-7 -6 -5
log q
--Curled
Tucked
Closed
Wide-open








20 30 40 50
Distance (A)


Figure 4-66. Data analysis for HIV-1PR Fsi with APV. A) Background subtracted dipolar echo
curve (black) overlaid with the TKR regenerated echo curve (red) and reconstructed
echo curve (blue) comprising the Gaussian populations shown in D. B) L-curve. C)
Distance profile from TKR analysis (red) overlain with the summed Gaussian
population profile (blue dashed). D) Individual populations necessary for Gaussian
reconstruction process, and E) Pake dipolar pattern.


















0. 1



0 1 2 3
T (Us)

Figure 4-67. Population validation process for HIV-1PR Fsi with APV. Background subtracted
dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo
curve for the distance profile with one or more populations suppressed (blue); with A)
unassigned and B) unassigned and curled suppressed.

Table 4-33. Distance distribution profile for subtype Fsi APV.
Sutype Fsi APV R (A) FWHM (A) TKR % Final %
Unassigned 17.5 2.0 3 0
Curled 26.4 3.0 12 12
Tucked 28.9 2.8 8 8
Closed 33.5 3.5 67 70
Wide-open 40.3 2.6 10 10


- TKR
-- Unassigned











Subtype Fsi with CA-p2


- TKR
- Gaussian Reconstruction









20 30 40 50
Distance (A)


0
f (MHz)


-TKR
- Gaussian Reconstruction


Figure 4-68. Data analysis for HIV-1PR subtype Fsi with CA-p2. A) Background subtracted
dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and
reconstructed echo curve (blue) comprising the Gaussian populations shown in D. B)
L-curve. C) Distance profile from TKR analysis (red) overlain with the summed
Gaussian population profile (blue dashed). D) Individual populations necessary for
Gaussian reconstruction process, and E) Pake dipolar pattern.


a


-5


-6 -5 -4
log ri
-Curled
-Tucked
-Closed
- Semi-open
Wide-open






20 30 40 50
Distance (A)












TKR
--16A








0 1 2 3
r (Gs)

TKR
-16 Aand19.6A
and Wide-open


B C
TKR
--19.6 A




0 1 2




E (Gts) F

S- TKR
16 A andl9.6 A and
Semi-open and Wide-open


- TKR
--16A and 19.6 A








0 1 2 3
T (Gs)

TKR
16 A andl9.6 A and Tucked
and Semi-open and Wide-open


0 1 2 3 0 1 2 3 0 1 2 3


r (Gs)


r (Gs)


r (Gts)


Figure 4-69. Population validation process for HIV-1PR subtype Fsi with CA-p2. Background
subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the
modified echo curve for the distance profile with one or more populations suppressed
(blue); with A) unassigned 16 A, B) unassigned 19.6 A, C) both unassigned 16 and
19.6 A, D) both unassigned and wide-open, E) both unassigned, semi-open and wide-
open, and F) both unassigned, tucked, semi-open and wide-open suppressed.

Table 4-34. Distance distribution profile for subtype Fsi with CA-p2.
Subtype Fsi CA-p2 R (A) FWHM (A) TKR % Final %
Unassigned 16.0 1.0 1 0
Unassigned 19.6 1.2 4 0
Curled 26.1 1.3 13 14
Tucked 30.9 1.4 3 3
Closed 33.1 2.2 72 76
Semi-open 36.0 1.3 4 4
Wide-open 41.5 1.3 3 3









A comparison of distance profiles from Subtype F with various inhibitors


Stpv
m cap2 -
sqv
M Ipv 100
M rtv 80
80
apv
Sdry 60 "
Satv
idv 40 o
unfv U 20 o

Closed 0 o
Semi-open
0 Wide-open 4
o, Tucked 4
-% Curled 4,o


Figure 4-70. Population analysis for subtype Fsi. Error is estimated at + 2.5%.

Conclusions

The DEER results reported in this work show that natural and drug-pressure selected

polymorphisms within subtypes, CRFs, and patient isolates of HIV-1 protease alter the average

flap conformations and flexibility of the apo protease. Each of the apo distance profiles is

strikingly different, and the changes can be described as shifts in the conformational ensemble of

the protease, comprising of four distinctive sub-populations of HIV-1PR conformations.

Additionally, certain constructs showed enhanced flexibility or structural instability. These

important differences may play a role in viral fitness and drug-resistance (Kear, Blackburn et al.

2009). Additionally, the distance distribution profiles for CRF01_A/E protease showed a similar

trend with respect to inhibitor-induced flap closure as previously reported work by Blackburn et

al. for subtype B protease. Indinavir, Nelfinavir, and Atazanavir had a weak affect on flap









closure, while Ritonavir, Lopinavir, Tipranavir, Saquinavir, Amprenavir, Damnavir, and CA-p2

each had a strong affect on flap closure (Blackburn, Veloro et al. 2009).









CHAPTER 5
SOLUBLE EXPRESSION AND PURIFICATION OF MULTIPLY DISULFIDE BONDED
PROTEINS FROM ESCHERICHIA COLI

Introduction

Prorenin is the inactive zymogen of renin, which is an aspartic protease that plays a vital

role in blood pressure regulation by catalyzing the first and rate-limiting step in the activation

pathway of its substrate angiotensinogen. Discovered and characterized in 1898 by Robert

Tigerstedt of the Karolinska Institute of Stockholm (Tigerstedt and Bergman 1898; Phillips and

Schmidt-Ott 1999), renin is secreted via two separate pathways, a constitutive secretion of

prorenin and a regulated pathway for secretion of the mature enzyme (Pratt, Flynn et al. 1988).

Renin is a highly specific protease that hydrolyzes angiotensinogen into angiotensin I with a KM

= 1 [M in the absence of ATP6AP2 and 0.15 [M in the presence of membrane-bound

ATP6AP2. Angiotensin I is then further hydrolyzed by angiotensin converting enzyme (ACE)

into vasoactive angiotensin II (Nguyen and Sraer 2002; Fujino, Nakagawa et al. 2004). This

pathway is part of the Renin-Angiotensin System (RAS), a key modulator of blood plasma,

lymph, and interstitial fluid volume, arterial vasoconstriction, blood pressure, and cardiac and

vascular function. As such, many hypertension drugs function by regulating blood pressure at

various points in the RAS. Prorenin circulates through the plasma until it reaches the secretary

granules, where the pro-segment is cleaved and active renin is released. Curiously, prorenin is

found in much higher concentrations in the bloodstream than renin, representing a likely

enzymatic control mechanism (Danser 2003; Morris 2003; Marathias, Agroyannis et al. 2004;

Schweda and Kurtz 2004; Berecek, Reaves et al. 2005).

The primary structure of prorenin consists of 452 amino acid residues, of which 46

correspond to the pro-sequence. The enzyme functions as a monomer with aspartate active site

residues located at positions D104 and D292 (using preprorenin numbering system). Reports









indicate that human renin is glycosylated (N-linked) at the N71 and N141 positions, and that

those post-translational modifications are necessary for proper secretion from mammalian cells

(Rothwell, Kosowski et al. 1993). Additionally, three disulfide bonds are formed from six CYS

residues at positions 117-124, 283-287, and 325-362 (Imai, Miyazaki et al. 1983; Hardman

1984). A ribbon diagram, rendered in VMD, showing the crystal structure of renin is provided in

Figure 5-1. Catalytic aspartate residues are shown in the active site as purple space-filling model

residues. Disulfide bonds are shown via blue space-filling model residues.

















Figure 5-1. Crystal structure of human renin (PDB ID 2REN).

In vitro, conversion of prorenin into active renin appears to be a two-step process involving

the generation of an intermediary form of activated prorenin, where the active site is exposed but

the pro-segment has not yet been proteolytically cleaved from the enzyme. The first step is

thought to be an acid-induced activation that occurs as the result of a conformational change in

the pro-segment. In the inactive form (based upon structural homology to pepsinogen), the pro-

segment is expected to be folded over the active site and in tertiary contact with other regions of

the protein (Derkx, Schalekamp et al. 1987; Dunn 2002). At low pH, it is believed that a series

of residues become protonated, thus breaking three salt bridges that are implicated in holding the









pro-segment over the active site and resulting in an unstructured conformation of the pro-

segment that is no longer in contact with the active site. This conformer is referred to as the acid-

activated form ofprorenin. This first step has been shown via enzymatic assays to be reversible

via a pH switch back to neutral conditions. The second step in the activation of prorenin to renin

is the proteolytic removal of the pro-segment resulting in active renin (Derkx, Schalekamp et al.

1987). Physiologic activation mechanisms have not yet been identified, nor has the identity of

the activating enzyme been determined.

Currently, very little structural data on prorenin is available in the literature. This is likely

related to the fact that current methods for recombinant expression of aspartic proteases have

been plagued by difficulty. Current methods of isolating prorenin include recombinant

expression and secretion from Chinese Hamster Ovary (CHO) (Mercure, Thibault et al. 1995)

and Human Embryonic Kidney (HEK) cells, and the Baculovirus (BEV) system. In comparison

to these methods, bacterial expression offers a relatively inexpensive, quick and high yield

system; however, most aspartic proteases, including prorenin, have multiple disulfide bonds,

hence these proteins, when cloned using the E. coli system, are usually expressed as inclusion

bodies. Inclusion body proteins require that they be denatured and then refolded in order to

obtain properly folded, functional protein. Refolding, however, does not ensure that the protein

will be both properly refolded and active (Nishimori, Kawaguchi et al. 1982; Imai, Cho et al. 1986;

Kaytes, Theriault et al. 1986; Masuda, Nakano et al. 1986; Lin, Wong et al. 1989; Yamauchi,

Nagahama et al. 1990; Chen, Koelsch et al. 1991). The expression method described within this

paper overcomes these difficulties.

The bacterial system does not produce glycosylated prorenin; however, structural and

enzymatic studies have shown that the non-glycosylated form is an adequate structural and









functional model. The crystal structures for both the glycosylated and non-glycosylated renin are

quite similar, indicating that glycosylation has little effect on the structure. A previous site-

directed mutagenesis study, where both of the N-glycosylation sites were replaced by alanine,

has shown that lack of glycosylation has no effect on the specific activity of the enzyme (Hori,

Yoshino et al. 1988; Rahuel, Priestle et al. 1991).

A bacterial expression methodology aimed at producing soluble, properly folded and

functional prorenin, activatable by pH or enzymatic removal of the pro-segment, was

investigated using thioredoxin fusion construct methodology. As described in detail in Chapter

2, the cytoplasmic reducing potential of E. coli often causes accumulation of proteins with

disulfide bonds into insoluble inclusion bodies. Once in inclusion bodies, proteins require

denaturation and refolding steps that often result in improperly folded protein. Most remarkably,

fusion constructs with E. coli thioredoxin (trxA) can eliminate the formation of inclusion bodies

by aiding in proper folding of water-soluble proteins containing disulfide bonds (Vallie, DiBlasio

et al. 1993). Thioredoxin is small (11.6 kD), thermally stable, and is involved in a variety of

cellular functions, including the reduction of protein disulfides and sulfate metabolism. A ribbon

diagram showing the x-ray crystal structure of thioredoxin is provided in Figure 5-2, showing the

two native cysteine residues, C32 and C35, involved in a disulfide bond.

Under physiological conditions, thioredoxin is found under an equilibrium of both the

oxidized disulfidee form) and reduced (dithiol form) enzyme. The mechanism by which

thioredoxin fusion constructs function is thiol-disulfide exchange, a simple chemical reaction in

which a free thiolate group attacks a sulfur atom within a disulfide bond and the principle

reaction by which disulfide bonds are formed and rearranged in a protein. The original disulfide









bond is broken and its other sulfur atom is released as a free thiolate, and a new disulfide bond

forms between the attacking thiolate and the original sulfur atom.
















Figure 5-2. Ribbon diagram showing x-ray structure of oxidized thioredoxin, PDB ID 2TRX.

Materials and Methods

Materials

The chemicals, reagents, and supplies were obtained from Fisher Scientific (Pittsburg,

Pennsylvania) and used as received, with a few noted exceptions. pET32a DNA, Ni-NTA His-

bind resin, and Xarrest agarose were purchased from Novagen (Gibbstown, New Jersey).

HiTrap Chelating HP column, HiTrap Q HP anion exchange column, and HiPrep 16/60

Sephacryl S-300 high resolution size exclusion columns were purchased from GE Biosciences

(formerly Amersham, Pittsburg, Pennsylvania). Prorenin DNA was synthesized and subsequently

purchased from DNA2.0 (Menlo Park, California). The QuikChange site-directed mutagenesis

kit and PfuUltra DNA polymerase were purchased from Stratagene (La Jolla, California).

BL21(DE3) and Origami(DE3) E. coli cells were purchased from Invitrogen (Carlsbad,

California). Spin MiniPrep Kit, PCR purification kit, gel extraction kit, and buffers P1 and P2

were purchased from Qiagen (Valencia, California). The dNTP mix was purchased from Bioline

(Taunton, Massachusetts). The oligonucleotide primers were purchased from IDT (Coralville,









Iowa). The 1 kb DNA ladder, pre-stained protein marker, restriction enzymes and buffers, T4

DNA ligase and buffer, and BSA were purchased from New England Biolabs (Ipswich,

Massachusetts). Criterion pre-cast protein gels, broad range molecular weight marker, and

Laemmeli sample buffer were purchased from BioRad (Hercules, California). The GelCode

Blue stain and Coomassie Plus Protein Assay Reagent were purchased from Pierce (Rockford,

Illinois). Renin substrate 1 (R-2931) and human recombinant renin (R-2779) were purchased

from Sigma (St. Louis, Missouri). Bovine serum albumin standards were purchased from

Thermo Scientific (Rockford, Illinois).

Methods

Cloning of prorenin

Table 5-1. Prorenin DNA sequence flanked (N-terminal) with NcoI restriction site and Factor
Xa cutsite and (C-terminal ) stop codons and BamHI restriction site.
ccatggatcgatggtcgcctgccaaccgatactactacttttaaacgcatcttcctgaaacgta
tgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggtcctgaatg
gagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatcctgactaactac
atggacacgcaatattacggcgaaattggcattggtaccccgccgcagaccttcaaggttgttt
ttgacaccggctctagcaacgtatgggtgccttcttccaagtgttctcgtctgtacactgcatg
cgtttaccacaaactgtttgatgcgtctgactcctctagctacaaacacaatggtaccgaactg
accctgcgttattctaccggtaccgtttctggtttcctgagccaagatatcattactgttggcg
gtatcaccgtaacgcagatgttcggcgaagttaccgaaatgccagcgctgccgttcctggctga
attcgacggtgttgtaggtatgggttttattgaacaagcgatcggtcgtgtaactccgatcttc
gacaacattattagccagggtgttctgaaagaagatgtgttctctttttactataaccgtgatt
ctgaaaactcccaatctctgggcggccagatcgtgctgggtggctctgatccgcagcactacga
gggcaactttcactacatcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtt
tctgttggctcttctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgcta
gctacatctccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacg
tctgttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg
ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttccaaaa
agctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacctgggcgct
gggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataaccgcatcggtttc
gctctggcgcgtggttcctaataa

The E. coli codon and expression optimized gene for prorenin (sequence given in Table 5-

1) was purchased from DNA 2.0 and received in pJ2:G02057 vector (Figure 5-3). The gene was

flanked with an NcoI restriction site (yellow) followed by a Factor Xa cutsite (green) on the 5'











end and two stop codons cyann) followed by a BamHI restriction site (red) on the 3' end. The

pJ2:G02057 vector features Kanamycin resistance gene and the pUC-ori origin of replication,

given in blue in Figure 5-3, and the prorenin gene flanked by Ncol and BamHI DNA restriction

sites, given in red in Figure 5-3.


Ncol(3371)*
P pJR(258)
SphI (30D0)




G02057 K*
S*Kman


Sapl (2500) *
Bbsrl 59)..-. pJ2:G02057
33B5 bp --Asel (8eB)
Narl(2400)--
BstU (2419) -



*- sHil (1161)
BamHI(2195)

Aflll (2080)
pJF (1974) W
ipUC-ori
*ApaLI (1535)


Figure 5-3. pJ2:G02057 storage vector in which prorenin DNA was obtained. Kanamycin
resistance gene and pUC-ori are shown in blue and prorenin gene is shown in red.
Prorenin gene was flanked by Ncol and BamHI cutsites. Figure courtesy of DNA2.0

The prorenin gene was removed from pJ2:G02057 via restriction digestion with Ncol and

BamHI cutsites by standard protocol. In addition, the pET32a vector (map shown in Figure 5-4)

was prepared for sub-cloning of prorenin by double-cleavage with the same enzymes. The

pET32 vector series is designed for cloning and high-level expression of proteins fused with the

109 amino acid Trx-tag thioredoxin protein. The products of both digestions were run on a 1%

agarose gel and the linearized vector and prorenin gene were subsequently excised from the gel

and purified via the Qiagen Gel Extraction and Purification Kit. The purified products were











ligated together using T4 DNA Ligase according to standard ligation procedure. This vector


sub-cloned with the prorenin gene construct will now be referred to as pET32a XaPR. This


plasmid was transformed into XL1Blue strain E. coli cells and subsequently purified using the


Qiagen mini-prep plasmid prep kit and checked by sequencing. Samples were run on a 1%


agarose gel to check size and purity of cloning product (shown in Figure 5-5). Figure 5-6


provides a cartoon representation of the fusion construct, showing the positions of the two fusion


partners, prorenin and thioredoxin, separated by Factor Xa and enterokinase cleavage sites, a


6His tag, and an S-tag.


Ava I1158)
Xho 1(156)
Eag I1 66)
Not 1(166)
SHind 111(173)
Sal 1(179)
S// ac 1(19o0
I 1 .. I EcoR 1(192)
BamH 1(i198
EcoR Vii-o
N. 11: 2 ,1

Dra 111(5658) ,n I '

,34-5889) Rsr11(589)
/ Xba I(729
Sea 1(4995) /SgrA 1(84o)
Pvu 1(4885) Sph 1(s 6)
7 EcoN I{oo56)
Pst 1(4760)N 1( )
-" ApaB 1(1205)
Bsa 1(4576)
Eam 1105 1(457
pET-32a(+) 1 lu I(152)
(5900bp) Bl 1(1535)
/ BstE 1I(1702)
\ ^ .Bmg 1(1730)
AlwN 1(4038) Apa 1(1732)
S/ /BssH 11(1932)
'-',, / i ~,F' 1(2027)

BspLU11 1(3622)
Sap 1(3506) PStiA 1(2366)
BstO 1(0 3393)
Tthl 11 1(3367) Psp5 11(2628)
BspG 1(3148)


Figure 5-4. pET-32a(+) vector map. TrxA gene, illustrated by a black arrow, is downstream of
the multiple cloning site that contains both the Ncol and BamHI restriction sites.
Figure courtesy of Novagen.









T~;.--~*---------

I '1 : 3 4 5 6 7
Size (I b -- -


I-
-





Figure 5-5. 1% agarose gel used to confirm purity ofpET32a XaPR plasmid prior to
sequencing. Lane 1: 2 ptL of 1kb DNA ladder. Lanes 2-7: 2 ptL of purified
pET32a XaPR.

S-TAG
HI-TAG ENTEROKNASE CUTSITE
| | FACTOR XACUTSITE

TIOREDOXIN
12 WPRORENIN 45 kD


Figure 5-6. Domain diagram of the prorenin-thioredoxin fusion construct.

Expression of prorenin-trx fusion construct

OrigamiB(DE3) and BL21(DE3) strain E. coli cells were transformed independently with

pET32aXaPR DNA by standard heat-shock methodology and subsequently plated on Luria-

Bertani (LB) agar plates containing 100 mg/mL ampicillin, 20 mg/mL tetracycline, and 10

mg/mL kanamycin (for Origami), and 100 mg/mL ampicillin (for BL21), for selection. 1 mL

liquid culture was grown in sterile LB media at 37 C and 200 RPM, to an approximate OD600 =

0.6, then added to a 3 L Fernbaugh flask containing 1 L sterile LB media. Cultures were grown

at 37 C until the OD600 was approximately 0.8. Cultures were induced for over-expression by

adding 1 mL of 0.8 M isopropyl-P-D-thiogalactoside (IPTG) at 20 OC and incubated for 24 hours

at 200 RPM.









Harvesting of cells and collection of soluble protein

After over-expression, the 1 L culture was centrifuged for 15 minutes at 5000 RPM at 4 C

in order to isolate the cell pellet. Harvested cells were resuspended in 30 mL buffer consisting of

0.02 M sodium monophosphate, 0.4 M sodium chloride, and 40 mM imidazole, pH 7.4, and 30

[IL low concentration protease inhibitor cocktail (set VII from Calbiochem) was added. Cells

were subsequently lysed using sonication in 5 second on/off pulses for approximately 7 minutes,

and passage through a French Pressure Cell (3x at 1000 psi). Pulsed sonication was performed

in an attempt to lyse the cells without causing significant shearing of the proteins. Lysed cells

were then centrifuged at 3000 RPM for 10 minutes and then again at 18,500 x g for 15 minutes at

4 C to remove unwanted cellular components and insoluble proteins. The supernatant, which

now contained the fusion construct and other soluble cell content, was collected for further

purification of prorenin-thioredoxin fusion construct.

Purification of fusion construct, gel electrophoresis, and protein concentration estimates

All chromatographic steps described in subsequent sections were performed using an

AKTA Prime (GE Healthcare) monitoring eluent absorbance at 280 nm and conductivity in

milli-Siemens (mS). Numerous purification schemes were examined. Reported herein are the

methods involved in the most successful of those schemes. All protein gel electrophoresis on

prorenin and prorenin-thioredoxin fusion construct was performed using 18% Tris-glycine pre-

cast Criterion gels, run at 180 volts. Protein concentrations were determined using Coomassie

Plus Protein Assay Reagent (Pierce) with bovine serum albumin (BSA) standards (Thermo

Scientific).

HiTrapTM Chelating HP affinity chromatography

The soluble protein mixture was loaded onto a 5 mL Ni-charged HiTrapTM Chelating HP-

column (Amersham Biosciences) and eluted over a 75 mL, 0.03 1 M imidazole gradient at 5









mL/min flow rate. 1 mL fractions were collected during elution. Fractionated eluted protein was

analyzed via SDS-PAGE (4-20% Tris-glycine pre-cast Criterion gel), run at 180 volts for

approximately one hour, and visualized with GelCode Blue stain (Pierce).

HiTrapTM Q HP anion exchange chromatography

Fractions containing the target protein were collected and pooled and added to 200 mL of

0.02 M Bis-tris, pH 8, for anion exchange chromatography. A 5 mL HiTrapTM Q HP anion

exchange column was equilibrated with 3 5 column volumes of 0.02 M Bis-tris, pH 8,

following by 3 5 volumes 1 M NaC1, 0.02 M Bis-tris, pH 8, and then 3 5 volumes 0.02 M

Bis-tris, pH 8. The protein mixture was loaded onto the column and eluted over a 75 mL, 0 to 1

M NaCl gradient at a flow rate of 5 mL/min and 1 mL fractions collected. Fractions were run on

SDS-PAGE using a 4-20% Tris-glycine pre-cast Criterion gel. This chromatographic step

typically resulted in prorenin-thioredoxin fusion construct of greater than 90% purity, as

estimated by SDS-PAGE.

Buffer exchange by HiPrepTM desalting column

In preparation for removal of the thioredoxin fusion partner by Factor Xa cleavage, the

protein solution was buffer exchanged into 100 mM NaC1, 50 mM Tris-HC1, 5 mM calcium

chloride, pH 8. All buffer exchange steps were carried out via a HiPrepTM desalting column and

standard buffer exchange procedure. The column was equilibrated with this buffer and the

protein solution was loaded onto the column at a rate of 10 mL/min and fractions collected.

Other methods of buffer exchanged were compared for efficacy, including dialysis, but none

were found to be as effective as the methodology first described above.

Concentrating of protein samples

In preparation for removal of the thioredoxin fusion partner by Factor Xa cleavage, the

protein solution was concentrated to the optimal concentration of approximately 0.5 mg/mL.









All concentration steps were carried out using the Millipore Amicon Ultra-4 Centrifugal Filter

Devices. Concentrations were determined using a standard Bradford assay. Centrifugation

steps were performed at 4000 RPM. Several different protein concentrations were analyzed

before settling on an optimum, including 0.25 3 mg/mL.

Cleavage of Fusion Construct

Approximately 1 mL of prorenin at an approximate concentration of 0.5 mg/mL was

incubated at 4 OC for 3 days with 6 30 p.L (dependent upon amount of fusion construct present,

1 Unit enzyme/10tg fusion construct) of NEB Factor Xa protease (1 mg/mL), 60 ptL of glycerol,

and 35 p.L of Bovine Serum Albumin (BSA) (2 mg/mL). Other conditions were tested, and these

were found most optimal. After completion of the reaction, the reaction products prorenin (45

kD) and thioredoxin (12 kD) were visualized using SDS-PAGE (18% Tris-glycine pre-cast

Criterion gel), and the amino acid sequence of prorenin is provided in Table 5-2.

Table 5-2. Prorenin amino acid sequence.
LPTDTTTFKRIFLKCRMPSIRESLKERGVDMARLGPEWSQPMKRLTLGNTTSSVILTNYMDTQY
YGEIGIGTPPQTFKWFDTGSSNVWVPSSKCSRLYTACVYHKLFDASDSSSYKHNGTELTLRYS
TGTVSGFLSQDIITVGGITVTQMFGEVTEMPALPFLAEFDGVVGMGFIEQAIGRVTPIFDNIIS
QGVLKEDVFSFYYNRDSENSQSLGGQIVLGGSDPQHYEGNFHYINLIKTVWQIQMKGVSVGSST
LLCEDGCLALVDTGASYSGSTSSIEKLMEALGAKKRLFDYVKCNEGPTLPDISFHLGGKEYTL
TSADYVFQESYSSKKLCTLAIHAMDIPPPTGPTWALGATFIRKFYTEFDRRNNRIGFALAR

Quenching Factor Xa reaction with Novagen Xarrest agarose

Excess Factor Xa enzyme was sequestered using Novagen Xarrest agarose at a ratio of

100 ptL of slurry per 4 units Factor Xa protease. The agarose was previously equilibrated in 100

mM NaC1, 50 mM Tris-HC1, 5 mM calcium chloride, pH 8. The products of the cleavage

reaction were added directly to the agarose and incubated at room temperature for approximately

10 minutes, at which time the reaction mixture was centrifuged at 1000 x g for 5 minutes and the









supernatant, containing thioredoxin and prorenin, was collected, while the Factor Xa remained

bound to the agarose.

Purification of prorenin from Factor Xa protease and thioredoxin

In a final purification step, prorenin was purified away from thioredoxin using His-bind

(Ni-NTA) resin. The supernatant, which contained the cleaved protein, was adjusted to pH 7.5

with 1M Tris-HC1, pH 8.0, and added directly to equilibrated Ni-NTA His-bind resin (2mL resin

slurry is required per 10mg His-tagged protein). The reaction mixture was allowed to equilibrate

for 10 minutes at room temperature, then centrifuged at 1000 x g for minute to pellet the resin.

The supernatant then contains the pure recombinant prorenin, while the thioredoxin fusion tag

remains bound to the resin.

Circular dichroism spectroscopy

Circular dichroism experiments with prorenin were run on an AVIV 202 Circular

Dichroism Spectrometer (Steve Hagen laboratory, University of Florida) at 25 C. The

monochromator was set to a wavelength of 260 nm 190 nm with a bandwidth of 1 nm and a

slitwidth of 0.569 nm. The protein was buffered in 50 mM phosphate and 2.5% glycerol, pH 7.0.

Activation of prorenin

Prorenin was reversibly activated by a pH switch to acidic conditions. At pH 4.5, the pro-

segment was liberated from the active site, providing renin activity. Upon a pH switch back to

8.0, renin activity is lost as the pro-segment forms salt bridges with enzyme portion of the

protein.

Prorenin activity assay

FRET-based renin activity assays were run at pH 4.5, where the zymogen is expected to be

in the acid-activated conformation (pro-segment liberated from active site), and also at pH 8.0,

where prorenin is expected to be in the inactive conformation (pro-segment blocking the active









site due to presence of salt bridges). Buffer for all activity assays was 0.1 M NaC1, 0.05 M Tris

base, pH 4.5 or pH 8.0. Lyophilized renin substrate 1 was dissolved in high-quality anhydrous

dimethylsulfoxide (DMSO) to make a stock solution of 500 [M substrate. Prior to activity

assay, a fresh solution of 2 tM Renin Substrate 1 was prepared in 100 mM NaCl and 50 mM

tris(hydroxymethyl) aminomethane (Tris base), pH 8.0. The substrate solution was dispensed

into a clean UV-pass fluorescence cuvette, and each assay was started by adding approximately

3% of the final volume of renin-containing solution diluted in the assay buffer. Excitation and

emission monochromators were set to 340 nm and 490 nm, respectively.

Results and Discussion

Sub-cloning of Prorenin Gene into pET32a Expression Vector

The E. coli codon and expression optimized gene for prorenin was purchased from DNA

2.0 and received in the pJ2:G02057 vector. For the purposes of sub-cloning the gene into the

pET32a vector, the DNA sequence was flanked with an Ncol restriction site (ccatgg) on the 5'

end and a BamHI restriction site (ggatcc) on the 3' end. After digestion with Ncol and BamHI,

ligation was carried out using standard procedures. Ligation was confirmed using DNA gel

electrophoresis and subsequent sequencing, thus sub-cloning was successful.

Over-expression and Purification of Prorenin-thioredoxin Fusion Construct

pET32aXaPR was transformed separately into both BL21(DE3) and OrigamiB(DE3)

strain E. coli cells for a pilot-scale expression experiment. Given in Figures 5-7 and 5-8 are

pictures of the SDS-PAGE gels showing the results of these experiments. The levels of

expression of the prorenin-thioredoxin fusion construct from BL21(DE3) and OrigamiB(DE3)

are comparable. Scaling up of the expression procedure was carried out, from both strains, in 1

L LB and 24 hours of induction time at 20 OC.









Size (kD)


Figure 5-7. Pilot expression of prorenin-thioredoxin fusion construct in BL21(DE3) strain E.
coli cells. Lane 1: broad range protein ladder, Lanes 2 9: NOT INDUCED WITH
IPTG, time points taken at t = 0, 1, 2, 4, 6, 8, 12, and 24 hours, respectively, Lanes 10
17: INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 6, 8, 12, and 24
hours, respectively. The apparent shift in size of the over-expression product is an
artifact created by the curvature of the gel during the photography step, as opposed to
an increase in size of the protein itself.


Figure 5-8. Pilot expression of prorenin-thioredoxin fusion construct in OrigamiB(DE3) strain
E. coli cells. Lanes 1 -7: NOT INDUCED WITH IPTG, time points taken at t = 0, 1,
2, 4, 8, 12, and 24 hours, respectively, Lane 8: broad range protein ladder, Lanes 9 -
15: INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 8, 12, and 24 hours,
respectively. The apparent shift in size of the over-expression product is an artifact
created by the curvature of the gel during the photography step, as opposed to an
increase in size of the protein itself.












The purification of the prorenin-thioredoxin fusion construct was carried out via sequential


chromatography using a nickel-charged HiTrapTM Chelating HP affinity column followed by a


HiTrapTM Q HP anion exchange column. Figures 5-9 and 5-10 show typical chromatograms


corresponding to each of these separation steps. Because the fusion construct harbors a 6-His


tag, it has high affinity for nickel-charged column; as such, the protein is retained on the column


and eluted over a slow gradient of the competitive inhibitor imidazole. Fractions containing the


fusion construct were pooled and loaded onto an anion exchange column equilibrated according


to the isoelectric point of the protein. After these steps, prorenin-thioredoxin was estimated to be


>95% pure, as shown in Figure 5-11.

AT2006Jul11no00210 UV AT2006Juillno00210 Cond AT2006Jul11no00210 Fractions
AT2006Jul 11no002 10 Loabook
mAu
1600


1400


1200


1000


800


600


400


200


S5 Break oint Breakpoint 7 Break o, 8 Break pomt 9

00 50 100 150 200 250 300 mm


Figure 5-9. Typical chromatogram from HiTrapTM Chelating HP nickel affinity column. Green
trace shows progression of imidazole gradient. Red trace shows conductivity line in
units of mS. Blue line shows UV at 280 nm. Fractions 10 17 were collected for
further purification.






















100








60




40




20


F1 7 0 .mln Break Bntr- B-eakpo lT-it / Break point 8 -------- --B reak 'mgat__
,0 F 1 1 2 3 4 5 6 7 8 9 10 111213 14 15 16 171819 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 3 33 7 Waste
00 100 200 300 400 500 mm




Figure 5-10. Typical chromatogram from HiTrapTM Q HP anion exchange column. Green trace

shows progression of NaCl gradient. Red trace shows conductivity line in units of

mS. Blue line shows UV at 280 nm.



Size (kD)


**
l'

-i


1 2


Figure 5-11. 18% Tris-glycine SDS-PAGE gel demonstrating purity of prorenin-thioredoxin

fusion construct following sequential chromatographic steps.


AT20016Dec13nno0 210 IIV
AT2006Dec13nno0 210 Iniect


AT2006Dec13nn002?10 Cond
-AT2n006Dec13nnon210 Inhnnk


AT2700n6Dec13nnon00210 Fractons










Enzymatic Removal of Thioredoxin

Enzymatic removal of thioredoxin presented many difficulties. The prorenin-thioredoxin

fusion construct contains cleavage sites for both enterokinase and Factor Xa. The enterokinase

site is found in the native pET32a vector sequence, and the Factor Xa site was incorporated by

including the corresponding sequence in the gene that was sub-cloned into the pET32a vector.

All initial work was performed using enterokinase. It was demonstrated, however, that

enterokinase resulted in non-specific cleavage of the prorenin-thioredoxin fusion protein. As

such, all subsequent work was performed using Factor Xa. Incubation with Factor Xa was

successful in liberating prorenin from the thioredoxin fusion partner, as shown in Figure 5-12;

however, an estimated 80 90% of prorenin remaining in the sample became insoluble upon

cleavage from thioredoxin, drastically lowering the overall protein yield. Evidence was

collected, however, suggesting that the remaining prorenin was properly folded and activatable

upon a pH switch to acidic conditions. Following the Factor Xa reaction, thioredoxin (with a 6-

His tag) was successfully removed from the reaction tube using loose nickel resin.


Size kD
20

11
97

66


45


36


Figure 5-12. 18% Tris-glycine SDS-PAGE gel demonstrating purity of prorenin-thioredoxin
fusion construct (lane 2) and prorenin (lane 3) after separation from thioredoxin.
Broad range protein marker is shown in lane 1 for size reference.









Evidence of Proper Folding

A circular dichroism spectrum was obtained in order to determine the relative secondary

structure of recombinant wild-type prorenin; spectrum is shown in Figure 5-13 and results of

secondary structure analysis are reported in Table 5-3. This data reports a secondary structure

consisting of 17.4% a-helix 30.4% P-sheet, 22% turn, and 29.5% unstructured (51.5% random

coil). To date, no crystal structure of prorenin exists for comparison; however, a prediction from

EXPASY Secondary Structure Prediction program (http://www.expasy.ch/tools) reports a similar

theoretical secondary structure.





I.2








Figure 5-13. Circular dichroism spectrum of prorenin after liberation from thioredoxin fusion
construct and separation from thioredoxin.

Table 5-3. Secondary structural data for prorenin (obtained from circular dichroism analysis),
renin, pepsinogen, and pepsin (each from analysis of crystal structure data), and
theoretical prorenin structure analysis (as determined by EXPASY secondary
structure prediction tool).
Prorenin Prorenin Renin Pepsinogen Pepsin
(Experimental) (Theoretical)
a-helix 17% 14% 10% 26% 15%
P-sheet 30% 32% 40% 20% 46%
Coil/turn 52% 54% 50% 54% 39%

Comparisons were also been made with the crystal structures of renin, pepsin (a similar

aspartic protease), and pepsinogen (the inactive zymogen of pepsin). The similarity of these

values provides strong evidence that the thioredoxin fusion system is successful in producing

soluble, properly folded prorenin from a bacterial system.










Renin Activity Measurements of pH-activated Prorenin

Renin activity in pH-activated prorenin was measured by monitoring the cleavage kinetics

of the fluorescently labeled peptide substrate analog Renin Substrate I (R2931), purchased from

Sigma. The assay utilizes fluorescence resonance energy transfer (FRET) to produce a

spectroscopic response to the enzymatic cleavage of the substrate by pH-activated prorenin.

Renin Substrate I is the small peptide Arg-Glu-(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-

Thr-Lys-(DABCYL)-Arg. The peptide is the normal substrate for renin, with the fluorophore 5-

(aminoethyl)aminonaphthalene sulfonate (EDANS) linked at one end and the non-fluorescent

chromophore dimethylaminoazo-benzene-4-carboxylate (DABCYL) linked at the other end. The

absorbance of the DABCYL chromophore overlaps with the excited-state fluorescence of the

EDANS fluorophore thereby quenching the EDANS via a FRET response, as shown in Figure 5-

14. Proteolytic cleavage of the substrate results in spatial separation of the fluorophore and

acceptor, restoring the fluorescence of the EDANS. As such, a gain in fluorescence over time

can be exploited as a means to assess activated prorenin/renin activity.


dabeyl an





SEDANS
r absorbance \\
I o
0 EDANS \
fluoreeene \ B


300 400 500 600
Wavelength (nm)


Figure 5-14. DABCYL absorption overlaps with the EDANS fluorescence thereby quenching
the fluorescence through fluorescence resonance energy transfer. Figure courtesy of
Sigma.









Assays were run at pH 4.5 where prorenin is expected to be in active conformation (pro-

segment liberated from active site), as well as pH 8.0, where the pro-segment is thought to be

physically blocking the active site, thus rendering the protease inactive, and the results are shown

in Figure 5-15. At pH 8.0, no increase in fluorescence signal is seen.

A B









0 100 200 300 400 500 600 0 100 200 300 400 500 600
Time (s) Time (s)
6 1 20 z60 360 400 500 600 6 160 200 360 400 500 600
Time (s) Time (s)

Figure 5-15. Renin Substrate I activity screening; A) activity screen at pH 8.0, where prorenin is
thought to be in its inactive conformation. No increase in fluorescence is seen. B)
Activity screen at pH 4.5, where the pro-segment is liberated from the active site of
the enzyme. An increase in fluorescence demonstrates that prorenin can be activated
at acidic pH.

Upon lowering the pH to 4.5, the increase seen in fluorescence signal over time

demonstrates that this system produces prorenin that can be activated at acidic pH. In conjuction

with CD data, the results from the activity assay suggest that the thioredoxin-fusion construct

expression methodology is successful in producing active, properly folded prorenin, albeit in

small yield at the present time.

Cysteine Mutagenesis of Prorenin DNA for Possible EPR Studies

The site-directed spin-labeling EPR methodology may be useful to characterize the

conformational changes in the pro-segment of prorenin that occur as a function of either pH or

site-specific mutations. Analysis of the EPR line shapes from spin labels incorporated into the

pro-segment could be used to validate the hypothesis that prorenin can undergo a reversible, non-









proteolytic acid-induced activation, which, based upon enzymatic assays, suggests that the pro-

segment undergoes a conformational change thus exposing the active site of the enzyme. No

structural evidence (X-ray or spectroscopic) of this conformational change has ever been

presented. To this end, numerous CYS mutants were generated by site-directed mutagenesis

using the Eppendorf Mastercycler Personal thermocycler. All primers were designed using

PrimerX (http://www.bioinformatics.org/primerx/cgi-bin/DNA 1.cgi) and synthesized and

purchased from Integrated DNA Technology (IDT DNA, http://www.idtdna.com). Careful

attention was paid to primer length (30 45 bases), GC content (40 50%), and melting

temperature (Tm) (60 700 C). Table 5-4 shows the specific sequences, lengths, GC content, and

Tm of each of the primers used in these experiments, and table 5-5 shows the thermal cycling

parameters used for mutagenesis reactions. The following mutants were successfully generated

by the procedure described above: T7C, F8C, L13C, V28C, and G35C. All successful reactions

were analyzed via DNA gel electrophoresis using 1% w/v agarose gel and DNA sequencing at

University of Florida DNA Sequencing Core.

Table 5-4. PCR primers utilized to introduce mutations to prorenin.
Mutation Primer (5' 3') Tm (C) m.w. %GC
G35C
Forward GTAGATATGGCACGTRCTGTGTCCTGAATGGAGCCAAC 65.7 11,429.4 51.3
Reverse GTTGGCTCCATTCAGGACACAGACGTGCCATATCTAC 65.7 11,309.4 51.3
L13C
Forward CTTTTAAACGCATCTTCTGTAAACGTATGCCTTCCATCCG 63.9 13,038.5 41.8
Reverse CGGATGGAAGGCATACGTTTACAGAAGATGCGTTTAAAAG 63.9 13,038.5 41.8
V28C
Forward CTCTGAAAGAGCGTGGTTGTGATATGGCACCTCTGGG 67.1 11,516.5 54.0
Reverse CCCAGACGTGCCATATCACAACCACGCTCTTTCAGAG 67.1 11,223.3 54.0
T7C
Forward CTGCCAACCCATACTACTTGTTTTAAACGCATCTTCCTG 63.6 11,827.7 40.3
Reverse CAGGAAGATGCGTTTAAAACAAGTAGTATCGGTTGGCAG 63.6 12,143.9 40.3
F8C
Forward CCGATACTACTACTTGTAAACGCATCTTCCTG 59.5 9,694.3 43.8
Reverse CAGGAACATGCGTTTACAAGTAGTAGTATCGC 59.5 9,952.5 43.8









Table 5-5. Thermal cycling parameters for site-directed mutagenesis reactions on prorenin.
Segment Cycles Temperature Time
1 1 950C 5 minutes
2 18 950C 1 minute
500C 1 minute
680C 8 minutes

Expression and Purification of V28C Mutant Prorenin

V28C prorenin mutant was expressed and purified using the procedure described

previously. Figure 5-16 shows an SDS-PAGE gel indicating the high purity of the prorenin-

thioredoxin fusion construct following anion exchange chromatography. However, in similar

fashion to wild-type, upon enzymatic removal of thioredoxin, a majority of the V28C prorenin

mutant protein became insoluble, thus substantially lowering the final protein yield.

Size (kD)















Figure 5-16. SDS-PAGE gel showing purity of V28C mutant following anion exchange
chromatography. Lane 1: broad range protein molecular weight marker, Lanes 2-12:
fractions 7-18, respectively, Lanes 13-18: fractions 28 through 33, respectively, and
corresponding to prorenin-thioredoxin fusion construct.

Conclusions

An innovative system to express soluble, properly folded prorenin from E. coli was

evaluated. Prorenin-thioredoxin fusion construct was successfully expressed and purified to 90 -

95% using sequential affinity and anion exchange chromatographic steps. Thioredoxin was









successfully liberated from the fusion construct using Factor Xa protease; however, at this time a

substantial portion of the protein sample became insoluble, thus lowering the final protein yield

to very small levels. The remaining prorenin was examined for proper folding by circular

dichroism spectroscopy and for renin activity via a FRET-based assay. In conclusion, though

total yield per liter E. coli growth is relatively low, this methodology allows for the production of

soluble, properly folded, activatable prorenin without the need for complicated denaturation and

refolding steps which often yield misfolded or inactive protein. Circular dichroism studies

confirmed proper folding of the protein, and activity assays showed that prorenin was activatable

at low pH.









CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTIONS

Conclusions

Prorenin and HIV-1 Protease (HIV-1PR) are both aspartic proteases with broad

implications in the medical field. HIV-1PR is a viral protein necessary for HIV-1 maturation and

prorenin is a zymogen whose activation is vital to the regulation of blood pressure via its role in

the Renin-Angiotensin System (RAS). As of 2008, the World Health Organization (WHO) has

reported that the number of people living with HIV or AIDS was approximately 33.4 million,

and the American Heart Association reported that the percentage of adults over the age of 20

living with hypertension was approximately 32% with approximately 24,000 deaths in the

United States alone (Clemente, Moose et al. 2004). Chapter 1 provides detailed background

information relevant to both HIV-1PR and renin/prorenin. Techniques used to examine these

proteases include circular dichroism spectroscopy (CD), mass spectrometry, continuous wave

(CW) EPR and pulsed double electron-electron resonance (DEER) spectroscopy. Detailed

descriptions on each of these methodologies are given in Chapter 2.

Chapter 3 describes results from CW EPR experiments of HIV-1PR. CW EPR

investigations provided a means to monitor the autoproteolytic degradation of the active protease

constructs, Subtype F and CRF01_A/E. Currently, unpublished work performed by graduate

student Angelo Mike Veloro demonstrated that substantial autoproteolysis of the active proteases

V6 and MDR769, without the three stabilizing mutations Q7K, L33I, and L63I, takes place

during the course of the purification; thus, to date, most of our structural work has been focused

on protease constructs that have incorporated the Q7K, L33I, and L63I substitutions. In order to

continue investigations of active protease, we needed to understand how conditions during

sample preparation and storage affect the autoproteolytic process. As such, the rate of









autoproteolysis of active spin labeled HIV-1PR constructs was monitored via EPR spectroscopy.

Over time, a sharp spectral component appeared in the high-field resonance line of the EPR

spectrum. This signal is attributed to a change in correlation time and provides a direct means of

monitoring protein degradation. Quantitative analysis of the normalized intensity of the high

field line provided a value proportional to the concentration of the proteolysed peptide fragment

containing the spin label. This methodology has the advantage over HPLC or SDS-PAGE that is

non-destructive and requires very little protein sample. Additionally, we were able to

demonstrate that with a quick purification and timely addition of inhibitor, autoproteolysis can be

greatly reduced, implying that DEER experiments can likely be repeating using active protease.

Pulsed EPR methodologies and site-directed spin-labeling (SDSL) were employed in order

to characterize the conformational ensembles of HIV-1PR from various subtypes and patient

isolates, including Subtypes B, C, F, CRF01_A/E, V6 and MDR769, as described in Chapter 4.

We find that the CW line shapes are dominated by the intrinsic motion of the spin label, and as

such do not report on changes in flap motions and dynamics. However, the pulsed EPR

technique DEER, also known as pulsed electron double resonance (PELDOR), yielded dipolar

modulated echo curves that provided detailed information regarding changes in flap

conformations and flexibility. Results were analyzed to provide distance distribution profiles

that provided information regarding the distances between spin labels at positions K55C and

K55C' which could then be quantitatively analyzed in order to describe the conformational

ensemble of the protease flaps, as well as the flexibility and dynamics of the flaps in the apo

form and in the presence of FDA-approved protease inhibitors and a non-hydrolyzable substrate

mimic CA-p2.









In addition to the work done on HIV-1PR, a significant portion of my graduate research

experience was dedicated to developing a soluble expression system for prorenin using the

thioredoxin fusion methodology, with partial success, as described in Chapter 5. The zymogen

was successfully over-expressed from both BL21(DE3) and OrigamiB(DE3) strain E. coli cells

and the fusion construct was successfully purified using a series of chromatographic steps. The

cleavage of thioredoxin from prorenin seemingly rendered prorenin unstable, causing the protein

to consistently crash from solution, substantially lowering the total protein yield. Soluble

prorenin remaining after separation from thioredoxin fusion partner was examined using circular

dichroism spectroscopy, and secondary structures were calculated. No crystal structure of

prorenin exists to date, but the secondary structural composition was comparable to that of

pepsinogen, a similar aspartic protease zymogen, leading to the conclusion that the small amount

of prorenin remaining after cleavage from the fusion partner is likely properly folded.

Additionally, a FRET-based assay was used to show that the protein was indeed activated by pH.

Overall, the method was successful in providing a small amount of properly folded, pH

activatable prorenin; however, the purification and cleavage processes need to be further

optimized for future studies aimed at SDSL EPR, NMR, and crystallography.

Future Directions

A Site-Directed Spin-labeling Approach to Studying HIV-1 Protease

To date, research in the group has focused on proteases from three subtypes, a circulating

recombinant form, and two patient isolates, namely Subtype B, C, F, CRF01_A/E, and V6 and

MDR769. However, this research should be expanded to include additional subtypes and patient

isolates, including but not limited to Subtype D, G, H, J, and K. Additionally, DEER

experiments should be repeated using active constructs for comparison to inactive construct data.

CW EPR experiments showed that if the active protease is purified quickly with prompt addition









of inhibitor, autoproteolysis is slowed sufficiently to perform analyses. Relevant single point

mutations should be probed to determine the effect of secondary polymorphisms on the

conformational ensemble of the protease and the development of drug resistance. Each of these

results should be compared with results from other techniques, including isothermal titration

calorimetry, differential scanning calorimetry, and nuclear magnetic resonance.

A crucial step in marking the significance of this work would be to correlate results on the

conformation ensemble, or more specifically the relative percentage closed conformation, to in

vivo work, particularly IC50 values, viral fitness, and drug resistance. IC50 is defined as the half

maximal inhibitory concentration and is a measure of the effectiveness of a compound in

inhibiting biological or biochemical function. Viral fitness refers to the relative replication

competence of a virus. Viral fitness can be assessed directly, by several methods, in tissue

culture systems.

Recombinant Bacterial Expression and Biophysical Characterization of the Aspartic Acid
Zymogen Prorenin

After further optimization of the purification and subsequent liberation of prorenin from

the fusion partner thioredoxin, EPR and NMR techniques can be used to better understand the

activation mechanism of prorenin by obtaining direct spectroscopic evidence of conformational

changes of the pro-segment. The conformations and/or conformational changes of the pro-

segment of prorenin can be characterized in both the active and inactive states, and the

reversibility of the acid-induced activation can be studied.

As mentioned previously, no crystal structure of prorenin currently exists. Thus, a future

aim focuses on obtaining a high resolution X-ray crystal structure of prorenin in its inactive state.

X-Ray crystallography is capable of obtaining atomic resolution structure of proteins, though it

requires relatively high amounts of protein. Current yields will likely provide sufficient amounts









of protein for SDSL EPR investigations of prorenin; however, a continuing goal is to optimize

our method of protein expression to improve yield for X-Ray crystallography and NMR

investigations.










APPENDIX A
PRORENIN DNA AND AMINO ACID SEQUENCE

Appendix A provides the deoxyribonucleic acid (DNA) and amino acid sequences for the

prorenin construct produced for the research in this dissertation, as well as each of the mutant

prorenin constructs successfully created via site-directed mutagenesis (T7C, F8C, L13C, V28C,

and G35C). pET32a(+) expression vector. The DNA for the Escherichia coli (E. coli) codon

optimized prorenin was synthesized by DNA2.0. The DNA sequence is flanked with an Ncol

restriction site (yellow) and a FactorXa cutsite (teal) on the N-terminal end and two stop codons

(magenta) followed by a BamHI restriction site (green) on the C-terminal end for sub-cloning

into the pET32a vector. The start codon (ATG) is located upstream of the sequence encoding for

thioredoxin, which is found within the sequence of the pET32a vector (Figure 5-x). Residue one

of prorenin is underlined for clarity. Sites of engineered CYS residues are highlighted in grey.

Table A-1. E. coli codon-optimized prorenin sequence with Factor Xa cutsite.
ccatggatcgatggtcgcctgccaaccgatactactacttttaaacgcatcttcctgaaa
P W I E G R L P T D T T T F K R I F L K
cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggt
R M P S I R E S L K E R G V D M A R L G
cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc
P E W S Q PM KR L T L G NT T S S V I
ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag
L T N Y M D T Q Y Y G E I G I G T P P Q
accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt
T F K V V F D T G S S N V W V P S S K C
tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc
S R L Y T A C V Y H K L F D A S D S S S
tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc
Y K H N G T E L T L R Y S T G T V S G F
ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt
L S Q D I I T V G G I T V T Q M F G E V
accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt
T E M P A L P F L A E F D G V V G M G F
attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt
I E Q A I G R V T P I F D N I I S Q G V
ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg
L K E D V F S F Y Y N R D S E N S Q S L










Table A-1. Continued.
ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac
G G Q I V L G G S D P Q H Y E G N F H Y
atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct
I N L I K T G V W Q I Q M K G V S V G S
tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc
S T L L C E D G C L A L V D T G A S Y I
tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg
S G S T S S I E K L M E A L G A K K R L
ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg
F D Y V V K C N E G P T L P D I S F H L
ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc
G G K E Y T L T S A D Y V F Q ES Y S S
aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc
K K L C T L A I H A M D I P P P T G P T
tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac
W A L G A T F I R K F Y T E F D R R N N
cgcatcggtttcgctctggcgcgtggttcc ggatcc
RIG F A L A R G S G S

Table A-2. E. coli codon-optimized prorenin T7C sequence with Factor Xa cutsite.
ccatggatcgatggtcgcctgccaaccgatactactt,:ittttaaacgcatcttcctgaaa
P W I E G R L P T D T T C F K R I F L K
cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggt
R M P S I R E S L K E R G V D M A R L G
cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc
P E W S Q PM KR L T L G NT T SS V I
ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag
L T N Y M D T Q Y Y G E I G I G T P P Q
accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt
T F K V V F D T G S S N V W V P S S K C
tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc
S R L Y T A C V Y H K L F D A S D S S S
tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc
Y K H N G T E L T L R Y S T G T V S G F
ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt
L S Q D I I T V G G I T V T Q M F G E V
accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt
T E M P A L P F L A E F D G V V G M G F
attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt
I E Q A I G R V T P I F D N I I S Q G V
ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg
L K E D V F S F Y Y N R D S E N S Q S L
ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac
G G Q I V L G G S D P Q H Y E G N F H Y
atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct
I N L I K T G V W Q I Q M K G V S V G S










Table A-2. Continued.
tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc
S T L L C E D G C L A L V D T G A S Y I
tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg
S G S T S S I E K L M E A L G A K K R L
ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg
F D Y V V K C N E G P T L P D I S F H L
ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc
G G K E Y T L T S A D Y V F Q ES Y SS
aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc
K K L C T L A I H A M D I P P P T G P T
tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac
W A L G A T F I R K F Y T E F D R R N N
cgcatcggtttcgctctggcgcgtggttcc ggatcc
RIG F A L A R G S G S

Table A-3. E. coli codon-optimized prorenin F8C sequence with Factor Xa cutsite.
ccatggatcgatggtcgcctgccaaccgatactactactt I-i-icgcatcttcctgaaa
P W I E G R L P T D T T T C K R I F L K
cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggt
R M P S I R E S L K E R G V D M A R L G
cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc
P E W S Q PM KR L T L G NT T SS V I
ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag
L T N Y M D T Q Y Y G E I G I G T P P Q
accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt
T F K V V F D T G S S N V W V P S S K C
tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc
S R L Y T A C V Y H K L F D A S D S S S
tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc
Y K H N G T E L T L R Y S T G T V S G F
ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt
L S Q D I I T V G G I T V T Q M F G E V
accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt
T E M P A L P F L A E F D G V V G M G F
attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt
I E Q A I G R V T P I F D N I I S Q G V
ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg
L K E D V F S F Y Y N R D S E N S Q S L
ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac
G G Q I V L G G S D P Q H Y E G N F H Y
atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct
I N L I K T G V W Q I Q M K G V S V G S
tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc
S T L L C E D G C L A L V D T G A S Y I
tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg
S G S T S S I E K L M E A L G A K K R L










Table A-3. Continued.
ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg
F D Y V V K C N E G P T L P D I S F H L
ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc
G G K E Y T L T S A D Y V F Q ES Y SS
aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc
K K L C T L A I H A M D I P P P T G P T
tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac
W A L G A T F I R K F Y T E F D R R N N
cgcatcggtttcgctctggcgcgtggttcc ggatcc
RIG F A L A R G S G S

Table A-4. E. coli codon-optimized prorenin L13C sequence with Factor Xa cutsite.
ccatggatcgatggtcgcctgccaaccgatactactatttaaacgcatcttc t : i -i -
P W I E G R L P T D T T T F K R I F C K
cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggt
R M P S I R E S L K E R G V D M A R L G
cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc
P E W S Q PM KR L T L G NT T SS V I
ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag
L T N Y M D T Q Y Y G E I G I G T P P Q
accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt
T F K V V F D T G S S N V W V P S S K C
tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc
S R L Y T A C V Y H K L F D A S D S S S
tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc
Y K H N G T E L T L R Y S T G T V S G F
ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt
L S Q D I I T V G G I T V T Q M F G E V
accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt
T E M P A L P F L A E F D G V V G M G F
attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt
I E Q A I G R V T P I F D N I I S Q G V
ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg
L K E D V F S F Y Y N R D S E N S Q S L
ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac
G G Q I V L G G S D P Q H Y E G N F H Y
atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct
I N L I K T G V W Q I Q M K G V S V G S
tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc
S T L L C E D G C L A L V D T G A S Y I
tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg
S G S T S S I E K L M E A L G A K K R L
ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg
F D Y V V K C N E G P T L P D I S F H L
ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc
G G K E Y T L T S A D Y V F Q ES Y SS










Table A-4. Continued.
aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc
K K L C T L A I H A M D I P P P T G P T
tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac
W A L G A T F I R K F Y T E F D R R N N
cgcatcggtttcgctctggcgcgtggttcc ggatcc
RIG F A L A R G S G S

Table A-5. E. coli codon-optimized prorenin V28C sequence with Factor Xa cutsite.
ccatggatcgatggtcgcctgccaaccgatactactacttttaaacgcatcttcctgaaa
P W I E G R L P T D T T T F K R I F L K
cgtatgccttccatccgtgaatctctgaaagagcgtgci t :it gitatggcacgtctgggt
R M P S I R E S L K E R G C D M A R L G
cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc
P E W S Q PM KR L T L G NT T S S V I
ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag
L T N Y M D T Q Y Y G E I G I G T P P Q
accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt
T F K V V F D T G S S N V W V P S S K C
tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc
S R L Y T A C V Y H K L F D A S D S S S
tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc
Y K H N G T E L T L R Y S T G T V S G F
ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt
L S Q D I I T V G G I T V T Q M F G E V
accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt
T E M P A L P F L A E F D G V V G M G F
attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt
I E Q A I G R V T P I F D N I I S Q G V
ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg
L K E D V F S F Y Y N R D S E N S Q S L
ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac
G G Q I V L G G S D P Q H Y E G N F H Y
atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct
I N L I K T G V W Q I Q M K G V S V G S
tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc
S T L L C E D G C L A L V D T G A S Y I
tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg
S G S T S S I E K L M E A L G A K K R L
ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg
F D Y V V K C N E G P T L P D I S F H L
ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc
G G K E Y T L T S A D Y V F Q ES Y SS
aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc
K K L C T L A I H A M D I P P P T G P T
tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac
W A L G A T F I R K F Y T E F D R R N N










Table A-5. Continued.
cgcatcggtttcgctctggcgcgtggttcc ggatcc
RI G F A L A R G S -- G S

Table A-6. E. coli codon-optimized prorenin G35C sequence with Factor Xa cutsite.
ccatggatcgatggtcgcctgccaaccgatactactacttttaaacgcatcttcctgaaa
P W I E G R L P T D T T T F K R I F L K
cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctct:it
R M P S I R E S L K E R G V D M A R L C
cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc
P E W S Q PM KR L T L G NT T S S V I
ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag
L T N Y M D T Q Y Y G E I G I G T P P Q
accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt
T F K V V F D T G S S N V W V P S S K C
tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc
S R L Y T A C V Y H K L F D A S D S S S
tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc
Y K H N G T E L T L R Y S T G T V S G F
ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt
L S Q D I I T V G G I T V T Q M F G E V
accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt
T E M P A L P F L A E F D G V V G M G F
attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt
I E Q A I G R V T P I F D N I I S Q G V
ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg
L K E D V F S F Y Y N R D S E N S Q S L
ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac
G G Q I V L G G S D P Q H Y E G N F H Y
atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct
I N L I K T G V W Q I Q M K G V S V G S
tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc
S T L L C E D G C L A L V D T G A S Y I
tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg
S G S T S S I E K L M E A L G A K K R L
ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg
F D Y V V K C N E G P T L P D I S F H L
ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc
G G K E Y T L T S A D Y V F Q ES Y SS
aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc
K K L C T L A I H A M D I P P P T G P T
tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac
W A L G A T F I R K F Y T E F D R R N N
cgcatcggtttcgctctggcgcgtggttcc ggatcc
R I G F A L A R G S G S










APPENDIX B
YEAST PROTEINASE A DNA AND AMINO ACID SEQUENCES

Soluble expression and purification methods were employed with Yeast Proteinase A (data

not included in dissertation). Appendix B provides the DNA and amino acid sequences for the

inactive (D215N) pro-yeast proteinase A (pro-YPRA) construct, as well as the vector map. DNA

for the E. coli codon optimized pro-YPRA was synthesized by DNA2.0. The DNA sequence is

flanked with an Ncol restriction site (yellow) and a FactorXa cutsite (teal) on the N-terminal end

and two stop codons (magenta) followed by a BamHI restriction site (green) on the C-terminal

end for subcloning into the pET32a vector. The start codon (ATG) is located upstream of the

sequence encoding for thioredoxin, which is found within the sequence of the pET32a vector

(Figure 5-x). Residue one of prorenin is underlined for clarity. Sites of engineered CYS

residues are highlighted in grey. Mutated active site reside D215N is highlighted in dark yellow.

Table B-1. E. coli codon-optimized Pro-YPRA D215N sequence with Factor Xa cutsite.
ccatggctatcgatggtcgcaaggttcacaaggcaaagatttacaaacatgaactgagcgat
MAID G R K V H K A K I Y K H EL S D
gaaatgaaagaggtcaccttcgagcagcacctggcgcatttgggtcaaaaatacctgacc
EM K E VT F E Q H LA H L G Q KY L T
cagttcgagaaagctaatccggaggtcgttttcagccgcgagcacccgtttttcacggaa
Q FE K A N P E V V F SR E H P F FT E
ggcggtcacgatgttccgctgaccaattatctgaatgcccaatactataccgacatcacg
G G H DV P L TN Y L N A Q Y Y T D I T
ttgggcaccccaccgcaaaactttaaggttatcctggacacgggtagcagcaatttgtgg
L G T P P Q N F K V I L D T G S S N L W
gttcctagcaacgaatgtggtagcttggcctgctttctgcactccaaatatgaccatgag
V P SNE C G S LA C FL H SKY D HE
gcgtcgagcagctacaaggccaacggtacggaatttgccatccagtacggcaccggtagc
A S S S Y K A N G T E F A I Q Y G T G S
ctggaaggctatatcagccaagatacgctgagcatcggcgatctgactatcccgaagcag
L E G Y I S Q D T L S I G D L T I P K Q
gatttcgcagaagccaccagcgagccgggtctgaccttcgctttcggtaaatttgatggt
D F A E A T S E PG L T F A F G K F D G
attctgggtctgggttacgacacgatcagcgtcgacaaagtggtcccaccgttttataac
IL G L G Y D T I S V D K V V P P F Y N
gcaattcagcaggacctgctggatgaaaaacgcttcgcgttctatctgggtgacacgtcg
A I Q Q DL L D E KR F A F Y L GD T S











Table B-1. Continued.
aaggacaccgag ggtggtgaggccacctttggcggtatcgatgagagcaagtttaag
K D T E N G G E A T F G G I D E S K F K
ggcgacattacttggctgccggtccgccgtaaggcgtactgggaggtcaagttcgagggt
G D I T W L P V R R K A Y W E V K F E G
atcggcttgggtgatgagtacgccgagctggagtctcatggtgcagcgatcaacaccggc
I G L G D E Y A E L E S H G A A I N T G
acgagcctgatcacgctgccgtctggtttggccgagatgatcaacgcggagattggtgca
T S L I T L P S G L A E M I N A E I G A
aagaagggttggactggtcagtacacgctggattgtaatacccgtgataacttgccggat
K K G W T G Q Y T L D C N T R D N L P D
ctgatcttcaatttcaacggctacaatttcaccatcggcccgtacgactacacgttggag
L I F N F N G Y N F T I G P Y D Y T L E
gtgagcggcagctgtatcagcgcgatcaccccgatggacttcccggagccggttggcccg
V S G S C I S A I T P M D F P E P V G P
ctggcgattgttggtgatgcgtttctgcgcaaatactacagcatttatgatctgggtaac
L A I V G D A F L R K Y Y S I Y D L G N
aatgccgtgggcctggccaaagcgatc ggatcc
N A V G LA K AI G S





?Pvul (381)
S.KanR
ApaLI (3363)
pUCori







pJ201:19864
D215NPro-YPRA
Apl,' 33823 bp
Apal ,2'.i .
'mTxn termin;
KasI (2688) Xmal (1150)
N col1 (1183)
rxn terminator

/ ,BspEI (1322)
EcoRV (2467) ', p (322)

BamHl(2358)

PvuIll (2213) Agel (1596)
\nsert:19864
Sall(1755)



Figure B-1. Map of pJ201:19864 with D215N pro-yeast proteinase A insert (red).









APPENDIX C
HIV-1 PROTEASE DNA AND AMINO ACID SEQUENCES

Appendix C provides the DNA and amino acid sequences for the HIV-1 Protease

constructs produced for the dissertation research. The DNA for the E. coli codon optimized

HIV-1 Protease (HIV-1PR) subtype Fsi K45C and E. coli codon optimized CRF01_A/Esi K55C

was synthesized and ordered by DNA2.0. Chapter 1 provided a more detailed discussion on

construct nomenclature and amino acid substitution code. In our naming scheme, a subscript "s"

refers to sequences that have incorporated the Q7K, L33I, and L63I substitutions that stabilize

against autoproteolysis. The subscript "i" refers to inactive protease (D25N). Each of the other

constructs listed in this appendix were produced via site-directed spin-labeling using the primers

listed in Appendix E. All DNA sequences are flanked with an Ndel restriction site (yellow) on

the N-terminal end and two stop codons (magenta) followed by a BamHI restriction site (green)

on the C-terminal end for subcloning into the pET23 expression vector. The start codon (ATG)

is located, in frame, within the Ndel restriction site. Residue one of HIV-1PR is underlined for

clarity. Sites of engineered CYS residues are highlighted in grey. Active site residue D25 (for

active constructs) or D25N (for inactive constructs) is highlighted in dark yellow.

Subtype F Construct Sequences

Table C-1. E. coli codon-optimized HIV-1PR subtype Fsi K45C sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L N T G A D D T V I E D M N L
ccgggtaagtggaaacccit.;: -tgattggcggtattggtggtttcatcaaagtcaagcaa
P G K W K P C M I G G I G G F I K V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc
Y D Q I I I E I A G H K A I G T V L V G
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S










Table C-2. E. coli codon-optimized HIV-1PR subtype F, K45C sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L D T G A D D T V I E D M N L
ccgggtaagtggaaacccigt.i: -tgattggcggtattggtggtttcatcaaagtcaagcaa
P G K W K P C M I G G I G G F I K V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc
Y D Q I I I E I A G H K A I G T V L V G
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S

Table C-3. E. coli codon-optimized HIV-1PR subtype Fsi K55C sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L N T G A D D T V I E D M N L
ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatc ti.gtcaagcaa
P G K W K P K M I G G I G G F I C V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc
Y D Q I I I E I A G H K A I G T V L V G
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S

Table C-4. E. coli codon-optimized HIV-1PR subtype Fs K55C sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L D T G A D D T V I E D M N L
ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatcti:,ticaagcaa
P G K W K P K M I G G I G G F I C V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc
Y D Q I I I E I A G H K A I G T V L V G
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S

Table C-5. E. coli codon-optimized HIV-1PR subtype Fsi T74C sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L N T G A D D T V I E D M N L










TableC-5. Continued.
ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatcaaagtcaagcaa
P G K W K P K M I G G I G G F I K V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcgci t ti.:gttctggttggc
Y D Q I I I E I A G H K A I G C V L V G
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S

Table C-6. E. coli codon-optimized HIV-1PR subtype Fs T74C sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L D T G A D D T V I E D M N L
ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatcaaagtcaagcaa
P G K W K P K M I G G I G G F I K V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcgci t t l::lt: ctggttggc
Y D Q I I I E I A G H K A I G C V L V G
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S

Table C-7. E. coli codon-optimized HIV-1PR subtype Fg sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L N T G A D D T V I E D M N L
ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatcaaagtcaagcaa
P G K W K P K M I G G I G G F I K V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc
Y D Q I I I E I A G H K A I G T V L V G
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S

Table C-8. E. coli codon-optimized HIV-1PR subtype Fs sequence.
catatgccgcagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa
H M P Q I T L W K R P L V T I K V G G Q
ttgaaggaggccctgctg accggtgcggacgataccgtgattgaggacatgaatctg
L K E A L L D T G A D D T V I E D M N L
ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatcaaagtcaagcaa
P G K W K P K M I G G I G G F I K V K Q
tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc
Y D Q I I I E I A G H K A I G T V L V G










Table C-8. Continued.
ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac
P T P V N I I G R N L L T Q I G A T L N
ttc ggatcc
F G S

CRF01_A/E Construct Sequences

Table C-9. E. coli codon-optimized HIV-1PR subtype A/Es, K55C sequence.
catatgccgcagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa
H M P Q I T L W K R P L V T V K I G G Q
ctgaaagaagcgctgctg accggtgcggatgatacggtcattgaggacatcaatctg
L K E A L L N T G A D D T V I E D I N L
ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatc ti.:gtgcgccaa
P G K W K P K M I G G I G G F I C V R Q
tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttggt
Y D Q I I I E I A G K K A I G T V L V G
ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac
P T P V N I I G R N M L T Q I G A T L N
ttc ggatcc
F G S

Table C-10. E. coli codon-optimized HIV-1PR subtype A/E, PMPR K55C sequence.
catatgccgcagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa
H M P Q I T L W K R P L V T V K I G G Q
ctgaaagaagcgctgctg accggtgcggatgatacggtcattgaggacatcaatctg
L K E A L L D T G A D D T V I E D I N L
ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatcti:i tgcgccaa
P G K W K P K M I G G I G G F I C V R Q
tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttggt
Y D Q I I I E I A G K K A I G T V L V G
ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac
P T P V N I I G R N M L T Q I G A T L N
ttc ggatcc
F G S

Table C- 1. E. coli codon-optimized HIV-1PR subtype A/Esi T74C sequence.
catatgccgcagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa
H M P Q I T L W K R P L V T V K I G G Q
ctgaaagaagcgctgctg accggtgcggatgatacggtcattgaggacatcaatctg
L K E A L L N T G A D D T V I E D I N L
ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatcaaagtgcgccaa
P G K W K P K M I G G I G G F I K V R Q
tacgaccagatcattatcgagattgctggtaagaaggcaattggc;it::itcttggttggt
Y D Q I I I E I A G K K A I G C V L V G
ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac
P T P V N I I G R N M L T Q I G A T L N










Table C- 1. Continued.
ttc ggatcc
F G S

Table C-12. E. coli codon-optimized HIV-1PR subtype A/Es T74C sequence.
catatgccgcagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa
H M P Q I T L W K R P L V T V K I G G Q
ctgaaagaagcgctgctg accggtgcggatgatacggtcattgaggacatcaatctg
L K E A L L D T G A D D T V I E D I N L
ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatcaaagtgcgccaa
P G K W K P K M I G G I G G F I K V R Q
tacgaccagatcattatcgagattgctggtaagaaggcaattggc ti.:gtcttggttggt
Y D Q I I I E I A G K K A I G C V L V G
ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac
P T P V N I I G R N M L T Q I G A T L N
ttc ggatcc
F G S

Table C-13. E. coli codon-optimized HIV-1PR subtype A/Esi sequence.
catatgccgcagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa
H M P Q I T L W K R P L V T V K I G G Q
ctgaaagaagcgctgctg accggtgcggatgatacggtcattgaggacatcaatctg
L K E A L L N T G A D D T V I E D I N L
ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatcaaagtgcgccaa
P G K W K P K M I G G I G G F I K V R Q
tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttggt
Y D Q I I I E I A G K K A I G T V L V G
ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac
P T P V N I I G R N M L T Q I G A T L N
ttc ggatcc
F G S

Table C-14. E. coli codon-optimized HIV-1PR subtype A/Es sequence.
catatgccgcagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa
H M P Q I T L W K R P L V T V K I G G Q
ctgaaagaagcgctgctg accggtgcggatgatacggtcattgaggacatcaatctg
L K E A L L D T G A D D T V I E D I N L
ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatcaaagtgcgccaa
P G K W K P K M I G G I G G F I K V R Q
tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttggt
Y D Q I I I E I A G K K A I G T V L V G
ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac
P T P V N I I G R N M L T Q I G A T L N
ttc ggatcc
F G S









APPENEDIX D
A SOLUBLE EXPRESSION SYSTEM FOR GM2 ACTIVATOR PROTEIN

The thioredoxin-fusion methodology was employed for GM2 activator protein (GM2AP)

in an attempt to find a better, cheaper, less time-consuming purification scheme. The overall

results of that project will be discussed here in Appendix D. The gene for GM2AP was removed

from pET16 expression vector and ligated into pET32a to facilitate thioredoxin-fusion

expression via standard restriction digestion and ligation procedures. Figure D-1 shows the

results of a pilot expression of the GM2AP thioredoxin fusion construct in OrigamiB(DE3) E.

coli cells and subsequently purified according to the procedure described in Chapter 5. A typical

affinity column chromatogram is shown in Figure D-2. Figure D-3 shows a sodium dodecyl

sulfate polyacrylamide gel electrophorsis (SDS PAGE) gel with >90% pure GM2AP-trx, and

the results of enzymatic cleavage of GM2AP from thioredoxin.

Size (kDa)























taken at t 0, 1, 2, 4, 6, 8, 12, and 24 hours, respectively.
200














10 1-1-2 13 14 151617


Figure D-1. SDS-PAGE gel showing pilot expression of prorenin GM2AP-thioredoxin fusion
construct in OrigamiB(DE3) strain E. coli cells. Lanes 1-8: NOT INDUCED WITH
IPTG, time points taken at t = 0, 1, 2, 4, 6, 8, 12, and 24 hours, respectively, Lane 9:
broad range protein marker, Lanes 10 17: INDUCED WITH IPTG, time points
taken at t = 0, 1, 2, 4, 6, 8, 12, and 24 hours, respectively.














AT2006Nov13no00210 UV
AT2006Nov13no00210 Inlect


AT2006Nov13no002 10 Cond
-AT2006Nov13no00210 Lobook


AT2006Nov13no002 10 Fractions


Brek pint I 5 mImn Flow 1 0 mI/mi

00 200


iak 1 1n 7 Break omt 8 -eak omt 9
|123456789 4 | | Wae..
400 600 800 mm


Figure D-2. Typical chromatogram for GM2AP-trx fusion construct using 5 mL nickel-charged
HiTrap HP affinity column.


Wci---'

rkL


Figure D-3. SDS-PAGE gel showing in purity of GM2AP-trx fusion construct following
senquential affinity and anion exchange chromatography steps and successful
cleavage of GM2AP-trx fusion construct into GM2AP and thioredoxin (the third band
is likely the FactorXa enzyme).











Additionally, a fluorescence resonance energy transfer (FRET)-based assay was performed


in order to assess the functionality of the protein. Shown in Figure D-4 are the results of a lipid


vesicle-binding assay with 3 mg/mL GM2AP-trx fusion construct at pH 8.0 and pH 4.5,


respectively. Vesicle composition was 10 [M POPC:dansyl-PHPE 9:1 in 50tM ammonium


acetate buffer. Sample was excited at 280 nm with a slit width of 2 nm. GM2AP is known to


bind to membranes at pH 4.5. At pH 4.5 an increase in fluorescence is seen around 510nm. That


increase in the fluorescence signal is indicative of GM2AP function. At pH 8.0, where GM2AP


does not bind to membranes, no increase in fluorescence is seen. Thus, the GM2AP-trx fusion


construct is binding to lipid vesicles.


A B

70oooo- pH 8.0 40000 pH 4.5

600000 400000-
vehicles only
500000, ---vesicles + GM2AP-trx 350000- vehicles only
40000..--. vehicles + GM2AP-trx
S300000
-, 4 00 O 250000 "
4 300000. 200000

12050000


^ 100000 L ,
250 300 350 400 450 500 550 250 300 350 400 450 500 550
wavelength wavelength


Figure D-4. Results of a FRET-based functional assay, where the increase in fluorescence signal
is indicative of GM2AP function.









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BIOGRAPHICAL SKETCH

Jamie Laura Kear was born in 1982 in Carbondale, Illinois, and moved to Bloomington,

Illinois in 1989 where she lived until 2002. She graduated from Bloomington High School in

2000, and subsequently attended Illinois State University until December 2001. She transferred

to Southern Illinois University in January 2002, where she graduated with honors with both a

bachelors of science (B.S) degree in chemistry and a bachelors of science (B.S.) degree in

biological sciences. She was admitted to the Department of Chemistry graduate program at the

University of Florida in 2005, where she joined the research group of Dr. Gail E. Fanucci. She

defended her dissertation in April 2010 and obtained her Ph.D. in Biochemistry in August 2010.





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1 A N ELECTRON PARAMAGNETIC RESONANCE STUDY OF HIV 1 PROTEASE AND THE DEVELOPMENT OF A SOLUBLE EXPRESSION SYSTEM FOR PRORENIN By JAMIE LAURA KEAR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Jamie Laura Kear

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3 To my parents David and Gail Kear and to my broth er Sergeant Daniel (Danny) Kea r With a special dedication to my grandfather Norman Kear

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4 ACKNOWLEDGMENTS I would first and foremost like to thank my parents David and Gail Kear for their endless encouragement, love and support, my brother S ergeant Daniel Kear for his services to our country and jus t for being the wonderful friend that he is. I would also like to offer a special thanks to my grandfather and friend Norman Kear for his unconditional love and so many words of wisdom which I will carry with me forever. Secondly, I n eed to express my most sincere gratitude to my advisor and mentor Doctor Gail E. Fanucci for her patience and encouragement, for countless opportunities to present my researc h at national conferences, and for her guidance and support. I would also like to thank all the other members of my doctoral committee, Doctors Jon Stewart, Nicole Hore nstein, Alex Angerhofer, Distinguished Professor Ben Dunn of the Department of Biochemistry and Molecular Biology within the University of Florida College of Medicine and Maureen Goodenow of the Department of Pathology, Immunology, and Laboratory Medicine at the University of Florida College of Medicine I would like to thank Ben Dunn and Maureen Goodenow for many helpful discussions and for their collaborations with the HIV 1 Protease project, Alex Angerhofer for the time on the E 580 instrument by which a substantial portion of the data in this dissertation was obtained, Nicole Horenstein for always being available when I wanted to chat about science, and Jon Stewart for the opportunity to teach alongside him for many semesters of Biochemistry Lab, during which time I was given the University of Florida Graduate Teaching Award. I owe this award in part, to him. I would like to thank all past and present members of the Fanucci group for their friendship, motivation, help and support, particularly Austin Turner, Natasha Pirman, Jeffrey Carter, Stacey Ann Benjamin, Ian Mitchelle de Vera, Star Gonzales, Mike Veloro, and former member s Doctors Luis Galiano, Jordan Mathi as and Mandy Blackburn Each of you played a part in making graduate school much more enjoyable for me and provided me with memories

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5 that will last forever. In addition, thanks go to several undergraduates who studied under me and helped perform experime nts while doing undergraduate research in the Fanucci group, including Justin Hewlett, Lisette Fred, and Alvancin Louis I would like to thank the Department of Chemistry and Biochemistry and the Department of Biological Sciences at Southern Illinois Uni versity Carbondale, where I worked to obtain my B achelor of Science (B.S.) degre es in both c hemistry and b iological Sciences In particular, I want to exp ress my deepest gratitude to Doctors Matt hew McCarroll and Boyd Goodson. D octor McCarroll provided m e with the opportunity to do research in his lab as an undergraduate, an invaluable experience th at was greatly appreciated. Doctor Goodson, an absolutely amazing teacher, cemented my love of chemistry. The two of them were ultimately the reason I ch ose I pursue a Ph.D. in Chemistry. I would like to thank the American Heart Associati on for support via a 2 year pre doctoral fellowship, the N ational I nstitutes of H ealth (NIH) Acquired Immunodeficiency S yndrome (A IDS ) Reagents Program for free acce ss to h um an immunodeficiency virus type 1 (H IV 1 ) protease inhibitors, and the National High Magnetic Field Lab (NHMFL) in house research proposal ( IHRP ) the NIH and National Science Foundation ( NSF ) and U niversity of Florida (UF) Startup for funding. This work was supported by NSF MBC 0746533 and ARI DMR 9601864 NIH R37 AI28571, AHA 0815102E the UF Center for AIDS Research and NHMFL IHRP.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ......................... 11 LIST OF FIGURES ................................ ................................ ................................ ....................... 15 LIST OF AMINO ACIDS AND AMINO ACID ABBREVIATIONS ................................ ......... 23 LIST OF H UMAN I MMUNODEFICIENCY V IRUS TYPE 1 (HIV 1) PROTEASE (HIV 1PR) INHIBITORS AND ABBREVIATIONS ................................ ................................ ...... 24 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 25 CHAPTER 1 INTRODUCTION TO ASPARTIC PROTEASES ................................ ................................ 34 Introduction to Proteases ................................ ................................ ................................ ........ 34 Intr oduction to Aspartic Proteases ................................ ................................ ................... 35 General Mechanism of Catalysis by Aspartic Proteases ................................ ................. 36 Introduction to HIV 1 Protease ................................ ................................ .............................. 37 HIV as a World Pandemic ................................ ................................ ............................... 37 Introduction to HIV 1 ................................ ................................ ................................ ...... 39 HIV 1 Viral Life Cycle ................................ ................................ ................................ ... 42 The HIV 1 Viral Genome ................................ ................................ ................................ 44 Structure and Function of HIV 1 Protease ................................ ................................ ...... 45 Conformational Sampling and the Conformational Ensemble of the HIV 1 Protease Flaps ................................ ................................ ................................ ............................. 50 Hydrophobic Sliding Mechanism ................................ ................................ .................... 50 HIV 1 Protease Construct Terminology ................................ ................................ .......... 51 HIV 1 Subtype B Protease ................................ ................................ .............................. 52 HIV 1 Subtype F Protease ................................ ................................ ............................... 53 HIV 1 Subtype C Protease ................................ ................................ .............................. 54 HIV 1 Protease Recombinant Form CRF01_A/E ................................ ........................... 55 Therapeutic App roaches to HIV 1 Infection and Inhibition of HIV 1 Protease ............. 56 Drug Resistance in Protease Inhibitor Exposed Patient Isolates of HIV 1 Protease ...... 62 Multi drug Resistant Patient Isolate MDR769 ................................ ................................ 64 Drug Resistant Patient Isolate V6 ................................ ................................ .................... 65 Introduction to Prorenin ................................ ................................ ................................ .......... 65 Hypertension and Its Impact on Society ................................ ................................ .......... 65 Renin, Prorenin, and the Renin Angiotensin System ................................ ...................... 66 Biosynthesis and Intercellular Processing ................................ ................................ ....... 67 Structure and Function of Prorenin ................................ ................................ ................. 69

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7 Difficultie s in Expression and Purification of Prorenin ................................ .................. 70 Scope of the Dissertation ................................ ................................ ................................ ........ 71 2 BACKGROUND FOR TECHNIQUES AND METHODOLOGIES ................................ .... 75 Introduction ................................ ................................ ................................ ............................. 75 Setting up a Protein Expression System for Escherichia coli ................................ ................ 75 Inclusion Body Isolation and Protein Refolding ................................ ................................ .... 81 Common Methods of Protein Separation ................................ ................................ ............... 83 Introduction ................................ ................................ ................................ ..................... 83 Separation Based Upon Size ................................ ................................ ........................... 83 Separation Based Upon Charge or Isoelectric Point ................................ ....................... 84 Separation Based Upon Binding Affinity ................................ ................................ ........ 85 Circular Dichroism Spectroscopy ................................ ................................ ........................... 86 Site Directed Spin Labeling (SDSL) ................................ ................................ ...................... 88 Introduction ................................ ................................ ................................ ..................... 88 Choice of Spin Label ................................ ................................ ................................ ....... 90 Spin Label Conformations an d the 4/ 5 Model for (1 oxyl 2,2,5,5 tetramethyl 3 pyrroline 3 methyl)methanethiosulfonate ( MTSL ) ................................ ..................... 91 Continuous Wave Electron Paramagnetic Resonance (CW EPR) Spectroscopy ................... 92 Introduction ................................ ................................ ................................ ..................... 92 Nitroxide Spectral Line Shapes ................................ ................................ ....................... 95 Protein Requirements for CW EPR ................................ ................................ ................. 96 CW EPR Data Analysis ................................ ................................ ................................ ... 96 Pulsed Electron Paramagnetic Resonance (EPR) Spectroscopy ................................ ............ 98 Introduction ................................ ................................ ................................ ..................... 98 Phase Memory Time, T m ................................ ................................ ............................... 102 Protein Requirements for Pulsed EPR Experiments ................................ ..................... 104 Analysis of Double Electron Electron Resonance ( DEER ) Data ................................ .. 104 Direct Fourier transform ................................ ................................ ......................... 105 Curve fitting and Monte Carlo analysis ................................ ................................ 105 Tikhonov regularization ................................ ................................ ......................... 106 Zero time selection ................................ ................................ ................................ 108 Self consistent analysis ................................ ................................ .......................... 109 Interpretation of distance distribution profiles ................................ ....................... 110 G aussian reconstruction process ................................ ................................ ............ 111 Error analysis by population suppression and validation ................................ ....... 112 3 CONTINUOUS WAVE ELECTRON PARAMA GNETIC RESONANCE STUDIES OF HIV 1 PROTEASE ................................ ................................ ................................ ......... 114 Introduction ................................ ................................ ................................ ........................... 114 Materials and Methods ................................ ................................ ................................ ......... 119 Materials ................................ ................................ ................................ ........................ 119 Methods ................................ ................................ ................................ ......................... 120 Cloning of HIV 1 protease ................................ ................................ ..................... 120 Site directed mutagenesis of HIV 1 protease constructs ................................ ....... 121

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8 Expression of HIV 1 protease constructs ................................ ............................... 125 Details of protease constructs ................................ ................................ ................. 125 HIV 1 protease purification buffers ................................ ................................ ....... 126 HIV 1 protease purification ................................ ................................ ................... 128 Spin labeling ................................ ................................ ................................ .......... 132 Circular dichroism spectroscopy ................................ ................................ ............ 133 Sample preparation for EPR data collec tion ................................ .......................... 134 CW EPR measurements ................................ ................................ ......................... 135 Mass spectrometry experiments ................................ ................................ ............. 136 Results and Discussion ................................ ................................ ................................ ......... 137 Affect of Inhibitors on CW EPR Line Shapes of HIV 1PR Subtype F and CRF01_A/E ................................ ................................ ................................ ................ 137 Monitoring the A utoproteolysis of HIV 1 Protease by SDSL EPR and Mass Spectrometry ................................ ................................ ................................ .............. 139 Conclusions ................................ ................................ ................................ ........................... 146 Affect of Inhibitors on CW EPR Line Shape s of HIV 1PR Subtype F and CRF01_A/E ................................ ................................ ................................ ................ 146 Monitoring the Autoproteolysis of HIV 1 Protease by SDSL EPR and Mass Spectrometry ................................ ................................ ................................ .............. 147 4 PULSED ELECTRON PARAMAGNETIC RESONANCE STUDIES OF HIV 1 PROTEASE ................................ ................................ ................................ .......................... 154 Introduction ................................ ................................ ................................ ........................... 154 Previous Work ................................ ................................ ................................ ...................... 156 Materials and Methods ................................ ................................ ................................ ......... 161 Materials ................................ ................................ ................................ ........................ 161 Methods ................................ ................................ ................................ ......................... 162 Details of protein constructs ................................ ................................ ................... 162 Expression of HIV 1 protease ................................ ................................ ................ 164 Purification of HIV 1 protease ................................ ................................ ............... 164 Spin labeling ................................ ................................ ................................ .......... 164 Buffer requirements ................................ ................................ ................................ 165 Circular dichroism spectroscopy ................................ ................................ ............ 165 DEER experiments ................................ ................................ ................................ 166 DEER data analysis ................................ ................................ ................................ 166 Po pulation validation ................................ ................................ .............................. 167 Results and Discussion ................................ ................................ ................................ ......... 167 Subtype Polymorphisms Found Among Subtypes B, C, F, CRF01_A/E and Patient Iso lates V6 and MDR769 Confer Altered Flap Conformations and Flexibility in the Apo Protease ................................ ................................ ................................ ........ 167 Introduction ................................ ................................ ................................ ............ 167 Zero time selecti on ................................ ................................ ................................ 168 Background subtracted dipolar modulated echo curves ................................ ......... 169 Data analysis and population validation process: Subtype B si ............................... 171 Data analysis and population validation process: Subtype C si ............................... 173 Data analysis and population validation process: Subtype F si ............................... 175

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9 Data analysis and population validation: CRF01_A/E ................................ .......... 177 Data analysis and population validation: V6 i ................................ ......................... 179 Data analysis and population validation: MDR769 i ................................ ............... 181 Polymorphism induced shifts in the conformational ensemble ............................. 183 Inhibitor Induced Flap Closure in CRF01_A/E Constructs ................................ .......... 188 CRF01_A/E si dipolar modulation zero time determination ................................ ... 188 CRF01_A/E si with CA p2 substrate ................................ ................................ ....... 190 CRF01_A/E si with Nelfinavir ( NFV ) ................................ ................................ ..... 192 CRF01_A/E si with Tipranavir ( TPV ) ................................ ................................ ..... 194 CRF01_A/E si with Lopinavir ( LPV ) ................................ ................................ ...... 196 CRF01_A/E si with Saquinavir ( SQV ) ................................ ................................ .... 198 CRF01_A/E si with Atazanavir ( ATV ) ................................ ................................ .... 200 CRF01_A/E si with Darunavir ( DRV ) ................................ ................................ ..... 202 CRF01_A/E si with Amprenavir ( APV ) ................................ ................................ .. 204 CRF01_A/E si with Ritonavir ( RTV ) ................................ ................................ ...... 206 CRF01_A/E si with Indinavir ( IDV ) ................................ ................................ ........ 208 A comparison of distance profiles from CRF01_A/E si with various inhibitors ..... 209 Inhibitor Induced Flap Closure in Subtype F si ................................ .............................. 211 Subtype F si dipolar modulation zero time determination ................................ ....... 211 Subtype F si with RTV ................................ ................................ ............................. 213 Subtype F si with IDV ................................ ................................ .............................. 215 Subtype F si with LPV ................................ ................................ ............................. 217 Subtype F si with TPV ................................ ................................ ............................. 219 Subtype F si with SQV ................................ ................................ ............................. 221 Subtype F si with DRV ................................ ................................ ............................ 223 Subtype F si with NFV ................................ ................................ ............................. 225 Subtype F s i with ATV ................................ ................................ ............................. 227 Subtype Fsi with APV ................................ ................................ ............................ 229 Subtype F si with CA p2 ................................ ................................ .......................... 231 A comparison of distance profiles from Subtype F with various inhibitors .......... 233 Conclusions ................................ ................................ ................................ ........................... 233 5 SOLUBLE EXPRESSION AND PURIFIC ATION OF MULTIPLY DISULFIDE BONDED PROTEINS FROM ESCHERICHIA COLI ................................ ....................... 235 Introduction ................................ ................................ ................................ ........................... 235 Materials and Methods ................................ ................................ ................................ ......... 239 Materials ................................ ................................ ................................ ........................ 239 Methods ................................ ................................ ................................ ......................... 240 Cloning of prorenin ................................ ................................ ................................ 240 Expression of prorenin t hioredoxin fusion construct ................................ ............. 243 Harvesting of cells and collection of soluble protein ................................ ............. 244 Purification of fusion construct, gel electrophoresis, and protein concentration estimates ................................ ................................ ................................ ............. 244 HiTrap TM Chelating HP affinity chromatography ................................ .................. 244 HiTrap TM Q HP anion exchange chromatography ................................ ................. 2 45 ................................ .................... 245

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10 Co ncentrating of protein samples ................................ ................................ ........... 245 Cleavage of Fusion Construct ................................ ................................ ................ 246 Quenching Factor Xa reaction with Novagen Xarrest agarose .............................. 246 Purification of prorenin from Factor Xa protease and thioredoxin ........................ 247 Circular dichroism spectroscopy ................................ ................................ ............ 247 Activation of prorenin ................................ ................................ ............................ 247 Prorenin activity assay ................................ ................................ ........................... 247 Results and Discussion ................................ ................................ ................................ ......... 248 Sub cloning of Prorenin Gene into pET32a Expression Vector ................................ ... 248 Over expression and Purification of Prorenin thioredoxin Fusion Cons truct ............... 248 Enzymatic Removal of Thioredoxin ................................ ................................ ............. 252 Evidence of Proper Folding ................................ ................................ ........................... 253 Renin Activity Measurements of pH activated Prorenin ................................ .............. 254 Cysteine Mutagenesis of Prorenin D eoxyribo n ucleic A cid (DNA) for Possible EPR Studies ................................ ................................ ................................ ........................ 255 Expression and Purification of V28C Mutant Prorenin ................................ ................ 257 Conclusions ................................ ................................ ................................ ........................... 257 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ ............................... 259 Conclusions ................................ ................................ ................................ ........................... 259 Future Directions ................................ ................................ ................................ .................. 261 A Site Directed Spin labeling App roach to Studying HIV 1 Protease ......................... 261 Recombinant Bacterial Expression and Biophysical Characterization of the Aspartic Acid Zymogen Prorenin ................................ ................................ .............. 262 APPENDIX A PRORENIN DNA AND AMINO ACID SEQUEN CE ................................ ........................ 264 B YEAST PROTEINASE A DNA AND AMINO ACID SEQUENCES ............................... 270 C HIV 1 PROTEASE DNA AND AMINO ACID SEQUENCES ................................ .......... 272 Subtype F Construct Sequences ................................ ................................ ............................ 272 CRF01_A/E Construct Sequences ................................ ................................ ........................ 275 D A SOLUBLE EXPRESSION SYSTEM FOR GM2 ACTIVATOR PROTEIN .................. 277 LIST OF REFERENCES ................................ ................................ ................................ ............. 280 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 291

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11 LIST OF TABLES Table page 1 1 Representative aspartic proteases by name, function, source, size, and structure. ................. 36 1 2 Primary geographic prevalence of subgroups of human immunodeficiency virus type 1 ( HIV 1 ) ................................ ................................ ................................ ............................. 41 1 3 HIV 1 p rotease (HIV 1PR) polyprotein process ing sites. ................................ ...................... 46 1 4 HIV 1PR construct abbreviations and descriptions. ................................ ............................... 52 1 5 Food and Drug Administration ( FDA ) approved drugs f or the treatment of HIV. ................ 57 1 6 FDA a pproved protease inhibitors for HIV 1 highly active antiretroviral therapy ( HAART ) treatment. ................................ ................................ ................................ .......... 58 1 7 Classifications of hypertension and respective systolic and diastolic pressure ranges .......... 66 1 8 Pro peptide sequences of several aspartic protease zymogens. ................................ .............. 68 2 1 Differential codon usage in Homo sapiens and Escherichia col i (E. col) i cells. ................... 76 2 2 Common microwave bands and frequencies used in continuous wave (CW) electron paramagnetic resonance ( EPR ) spectroscopy ................................ ................................ ... 95 2 3 Standard pulse table used for double electron electron resonance ( DEER ) experiments. ... 100 2 4 Standard pulsed EPR parameters used in this study. ................................ ............................ 101 2 5 Typical parameters used for the echo decay experiment for determination of T m .............. 103 3 1 P olymerase chain reaction (P CR ) p rimers utilized to introduce mutations to HIV 1 PR CRF01_A/E. ................................ ................................ ................................ ..................... 122 3 2 PCR p rimers utilized to introduce mutations to HIV 1 PR s ubtype F. ................................ 122 3 3 Thermal c ycling parameters for HIV 1 PR site directed mutagenesis reactions. ................. 122 3 4 E coli codon optimized HIV 1PR Subtype F si K45C deoxyribonucleic acid ( DNA ) and amino acid sequences. ................................ ................................ ................................ ...... 122 3 5 E. coli codon optimized HIV 1PR Subtype F si K55C DNA and amino acid sequences. ..... 123 3 6 E. coli codon optimized HIV 1PR Subtype F s K55C DNA and amino acid sequences. ..... 123 3 7 E. coli codon optimized HIV 1PR CR F01_ A/E si K55C DNA and amino acid sequences. ................................ ................................ ................................ ........................ 124

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12 3 8 E. coli codon optimized HIV 1PR A/E s K55C DNA and amino acid sequences. ............... 124 3 9 Luria Bertani (LB) Media. ................................ ................................ ................................ .... 125 3 10 HIV 1PR purification buffers. ................................ ................................ ............................ 127 3 11 Standard parameters used for circular dichroi sm (CD) experiments ................................ 133 3 12 Standard CW EPR parameters used in this study. ................................ .............................. 136 3 13 CW EPR data analysis for Subtype F si K55M TSL. ................................ ............................ 138 3 14 CW EPR data analysis for A/E si K55MTSL. ................................ ................................ ..... 139 4 1 Comparison of relative percentage of closed flap conformation of HIV 1PR subtype B to p ublished v alues of K i K D and the number of non water mediated hydrogen .......... 160 4 2 HIV 1PR v ariant s equence alignment r esidues 1 50. ................................ ........................... 163 4 3 HIV 1PR v ariant s equence a lignment r esidues 51 99. ................................ ......................... 163 4 4 Zero times chosen for apo data analysis. ................................ ................................ .............. 169 4 5 Values of signal:noise ratios (SNRs) for background subtracted echo data. ........................ 170 4 6 Results of Gaussian reconstruction and population validation procedures for Bsi. ............. 172 4 7 Results of Gaussian reconstruction and population validation procedures for Csi. ............. 174 4 8 Distance distribution profile for apo H IV 1PR Fsi. ................................ ............................. 176 4 9 Distance distribution profile for HIV 1PR A/Esi. ................................ ................................ 178 4 10 Distance distribution profile for HIV 1PR V6i. ................................ ................................ 180 4 11 Distance distribution profile for HIV 1PR MDR769i. ................................ ....................... 183 4 12 Summary of distance parameters obtained from DEER distance profiles of HIV 1PR ..... 185 4 13 Zero times chosen for CRF01_A/Esi data analysis. ................................ ........................... 189 4 14 Distance distribution profile for CRF0 1_A/E with CA p2. ................................ ............... 191 4 15 Distance distribution profile for CRF01_A/E Nelfinavir ( NFV ) ................................ ...... 193 4 16 Distance distribution profil e for CRF01_A/E with Tipranavir ( TPV ) ............................... 195 4 17 Distance distribution profile for CRF01_A/E Lopinavir ( LPV ) ................................ ....... 197 4 1 Distance d istribution profile for CRF01_A/E Saquinavir ( SQV ) ................................ ....... 199

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13 4 19 Distance distribution profile for CRF01_A/E Atazanavir ( ATV ) ................................ ..... 201 4 20 Distance distribution profile for CRF01_A/E Darunavir ( DRV ) ................................ ...... 203 4 21 Distance distribution profile for CRF01_A/E Amprenavir ( APV ) ................................ ... 205 4 22 Distance distribution profile for CRF01_A/Esi with Ritonavir ( RTV ) ............................. 207 4 23 Distance distribution profile for CRF01_A/Esi with Indinavir ( IDV ) .............................. 209 4 24 Zero times chosen for F si data analysis. ................................ ................................ ............. 211 4 25 Distance distribution profile for Subtype Fsi with RTV. ................................ ................... 214 4 26 Distance distribution profile for Subtype Fsi with IDV. ................................ .................... 216 4 27 Distance distribution profile for Subtype Fsi with LPV. ................................ .................... 218 4 28 Distance distribution profile for Subtype Fsi with TPV ................................ ..................... 220 4 29 Distance distribution profile for Subtype Fsi with SQV. ................................ ................... 222 4 30 Distance distribution profile for Subtype Fsi with DRV. ................................ ................... 224 4 31 Distance distribution profile for Subtype Fsi with NFV. ................................ ................... 226 4 32 Distance distribution profile for Subtype Fsi with ATV. ................................ ................... 228 4 33 Distance distribution profile for Subtype Fsi APV. ................................ ........................... 230 4 34 Distance distribution profile for Subtype Fsi with CA p2. ................................ ................ 232 5 1 Prorenin DNA sequence with restriction site s, stop codons, and Factor Xa cuts ite ............. 240 5 2 Prorenin amino acid sequence. ................................ ................................ ............................. 246 5 3 Seconda ry structural data for prorenin ................................ ................................ ................. 253 5 4 PCR Primers utilized to introduce mutations to prorenin. ................................ .................... 256 A 1 E. coli codon optimized prorenin sequence with Factor Xa cutsite. ................................ ... 264 A 2 E. coli codon optimized prorenin T7C sequence with Factor Xa cutsite. ........................... 265 A 3 E. coli codon optimized prorenin F8C sequence with Factor Xa cut site. ........................... 266 A 4 E. coli codon optimized prorenin L13C sequence with Factor Xa cutsite. ......................... 267 A 5 E. coli codon optimized prorenin V28C sequence with Factor Xa cutsite. ......................... 268

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14 A 6 E. coli codon optimized prorenin G35C sequence with Factor Xa cutsite. ......................... 269 B 1 E coli codon optimized Pro YPRA D215N sequence with Factor Xa cutsite. ................... 270 C 1 E. coli codon optimized HIV 1 Protease subtype F si K45C sequence. ............................... 272 C 2 E. coli codon optimized HIV 1 Protease subtype F s K45C sequence. ................................ 273 C 3 E. coli codon optimized HIV 1 Protease subtype F si K55C sequence. ............................... 273 C 4 E. coli codon optimized HIV 1 Protease subtype F s K55C sequence. ................................ 273 C 5 E. coli codon optimized HIV 1 Protease subtype F si T74C sequence. ................................ 273 C 6 E. coli codon optimized HIV 1 Protease subtype F s T74C sequence. ................................ 274 C 7 E. coli codon optimized HIV 1 Protease subtype F s i sequence. ................................ .......... 274 C 8 E. coli codon optimized HIV 1 Protease subtype F s sequence. ................................ ........... 274 C 9 E. coli codon optimized HIV 1 Protease subtype A/E si K55C sequence. ........................... 275 C 10 E. coli codon optimized HIV 1 Protease subtype A/E s PMPR K55C sequence. .............. 275 C 11 E. col i codon optimized HIV 1 Protease subtype A/E si T74C sequence. .......................... 275 C 12 E. coli codon optimized HIV 1 Protease subtype A/E s T74C sequence. .......................... 276 C 13 E. coli codon optimized HIV 1 Protease subtype A/E si sequence. ................................ .... 276 C 14 E. coli codon optimized HIV 1 Protease subtype A/E s sequence. ................................ .... 276

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15 LIST OF FIGURES Figure page 1 1 Scheme of hydrolysis of a polypeptide chain.. ................................ ................................ ....... 34 1 2 Generally accepted mechanism of catalysis by aspartic proteases. ................................ ........ 36 1 3 Map showing percent of adult population living with human immunodeficiency virus ( HIV ) infection in 2007. ................................ ................................ ................................ .... 38 1 4 Cartoon representation of the structure of the HIV 1 virus. ................................ ................... 39 1 5 Phylogenic tree for HIV 1 and HIV 2, groups, subtypes, and CRFs. ................................ .... 40 1 6 Cartoon representation of the HIV 1 life cycle. ................................ ................................ ..... 43 1 7 Cartoon representation of the HIV 1 viral genome.. ................................ .............................. 44 1 8 Structure of Subtype B HIV 1 Protease. ................................ ................................ ................ 45 1 9 Ribbon diagram of HIV 1 protease ( HIV 1PR ) bound to the non hydrolyzable substrate mimic CA p2. ................................ ................................ ................................ .................... 47 1 10 Structures of norleucine and methionine. ................................ ................................ ............. 47 1 11 HIV 1PR nuclear Overhauser effect ( NOE ) values of liganded and unliganded HIV 1 PR ................................ ................................ ................................ ................................ .... 48 1 12 Flap conformations of HIV 1 PR captured by molecular dynamics (MD) simulations. ....... 49 1 13 Crystal structure of HIV 1PR Subtype F ................................ ................................ ............. 53 1 14 Crystal structure of Subtype C HIV 1PR ................................ ................................ ............ 54 1 15 Graphical description of the circulating recombinant form CRF01_A/ E ............................. 55 1 16 Points of inhibition within the HIV 1 viral life cycle. ................................ .......................... 56 1 17 Regions of amino acid conservation in protease inhib itor nave exposed HIV 1PR. .......... 63 1 18 Crystal structure of HIV 1PR multi drug resistant patient isolate MDR769. ...................... 64 1 19 Renin an giotensin system (RAS) and the sites of inhibition within the RAS. ..................... 67 1 20 Crystal structures of aspartic proteases renin, pepsin, and pepsin ogen ............................... 69 2 1 Example vector map highlighting necessary features of plasmid. ................................ ......... 80

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16 2 2 Structures of lactose and the lactose analog i sopropyl D 1 thiogalacto pyrano side ( IPTG ) ................................ ................................ ................................ ............................... 81 2 3 Internal structures of prok aryotic and eukaryotic cells ................................ ......................... 82 2 4 Structure of the detergent sodium dodecyl sulfate (SDS) ................................ ..................... 84 2 5 Anion exchange Q resin bound to a positively charged quaternary amine. ........................... 85 2 6 Structures of nitrilotriacetic acid and i minodiacetic acid. ................................ ...................... 85 2 7 Ell iptical polarized light. ................................ ................................ ................................ ....... 86 2 sheet, and rand om coil. ............................. 87 2 9 Site directed spin labeling (SDSL) scheme for (1 oxyl 2,2,5,5 tetramethyl pyrroline 3 methyl) methanethiosulfonate ( MTSL ) addtion to a thiol group of a CYS residue. ................................ ................................ ................................ ............................... 89 2 10 Structures of MTSL and other common nitroxide spin labels ................................ ............. 90 2 11 E lectron paramagnetic resonance (E PR ) spectral line shape s of HIV 1PR Subtype B si with various spin labels .. ................................ ................................ ................................ .... 91 2 12 Graphical representation of the 4/ 5 model for the MTSL label. ................................ ....... 92 2 13 Energy level diagram for a free electron in an applied magnetic field and corresponding spectr al line shape ................................ ................................ ..................... 94 2 14 Energy diagram for a system with a free electron undergoing hyperfine interacti on with the nucleus of nitrogen and a representative spectr al line shape ............................. 94 2 15 Dependence of EPR spectral line shape on motion. ................................ ............................. 95 2 16 Common spectral parameters H pp I LF I CF and I HF and second moment ......................... 98 2 17 The 4 pulse double electron electron resonance ( DEER ) sequence. ................................ ... 99 2 18 A bsorption spectra for a nitroxide spin label with positions of the low field transition ................................ ................................ ................................ .......... 100 2 19 Sample dipolar evolution curve showing locations of raw dipolar modulation, background subtraction, and background subtracted echo curve with fit. ...................... 102 2 20 Typical results of an echo dec ay experiment usin g a 2 pulse Hahn echo sequence for d etermination of T m ................................ ................................ ................................ ....... 103 2 21 Pictorial representation of the direct Fourier transfor m method of analysis ..................... 105

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17 2 22 Pictorial representation of the Monte Carlo analysis. ................................ ......................... 106 2 23 Selection of regularization parameter in T ikhonov regularization (T KR ) DEER an alysis method ................................ ................................ ................................ ................ 107 2 24 Example of zero time selection for dipolar modulated echo data. ................................ ..... 108 2 25 Self consistent analysis scheme. ................................ ................................ ......................... 110 2 26 Semi quantitative analysis of distance distribution profiles. ................................ .............. 111 2 27 Quantitative description of distance distribution profiles using Gaussian reconstruction pro cedure ................................ ................................ ................................ ......................... 112 2 28 Example of populat ion suppression error analysis ................................ ............................ 113 3 1 S odium d odecyl sulfate (S DS ) polyacrylamide gel electrophoresis (P AGE ) gel of self cleavage products for Subtype B with stabilizing mut ations and V6 and MDR769 w ith no stabilizing mutations. ................................ ................................ .......................... 115 3 2 Ribbon diag ram of HIV 1P R structure. ................................ ................................ ............... 116 3 3 Overlay of day 1 and day 47 CW EPR spect ra for Subtype B HIV 1 protease .................. 118 3 4 X band CW EPR spectra of 100 M HIV 1P R as a function of salt conce ntration ............. 119 3 5 V ector maps of pJ201:24237 and pJ201:24236 ................................ ................................ .. 121 3 6 Decomposition of ur ea; with heat and time, urea degrades into ammonium and cyanate. .. 128 3 7 Thermo brand 35 mL French pressure cell and Fisher Scientific brand tip sonicator. ......... 128 3 8 Typical anion exchange chromatogram for purification of HIV 1PR ................................ 130 3 9 Typical size exclusion chromat ogram of HIV 1PR on S 100 column ................................ 131 3 10 SDS PAGE gel showing purity of HIV 1PR at various steps in the purification .. ............ 132 3 11 Temperature control set up for CW EPR experime nts ................................ ...................... 135 3 12 CW EPR nitroxide spectral line shapes for HIV 1PR Subtype F si K55 MTSL ................. 137 3 13 CW EPR nitroxide spectral line s hapes for HIV 1PR CRF_01A/E si K55MTSL .............. 138 3 14 Circular dichroism spectra for spin la beled HIV 1PR, subtypes B F, CRF01_A/E ......... 141 3 15 Overlay of day 1 and day 30 CW EPR spectra for s ubtype F s HIV 1PR stored at 37 C, 25 C and 4 C, and CRF01 A/Es HIV 1PR stored at 37 C, 25 C, and 4 C. ............. 142

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18 3 16 Mole fraction of protease for samples stored at 37 C, 25 C, and 4 C. ................................ ................... 144 3 17 CW EPR spectra of HIV 1PR s ubtype F with tipranavir over the course of 30 days. ....... 145 3 18 CW EPR spectra of HIV 1PR CRF01_A/E with CA p2 over the course of 30 days. ....... 145 3 19 Mass spectrum for HIV 1PR CR F01_A/E K55MSL ................................ ........................ 149 3 20 Mass spectrum for HI V 1PR CRF01_A/E K55MSL ................................ ....................... 150 3 21 Mass spectrum for HIV 1PR s ubtyp e Fs K55MSL ................................ ........................... 151 3 22 Mass spectrum for HIV 1PR CRF01 A/E K55MSL. ................................ ........................ 15 2 3 23 Sites of autop roteolytic cleavage in HIV 1PR s ubty pe Fs K55MSL. ................................ 153 3 24 Sites of autoproteolytic cleavage in HIV 1PR CRF01 A/E K55MSL. .............................. 153 4 1 Ribbon diagrams showing HIV 1PR in the closed and semi open flap conformations.. .... 154 4 2 DEER results of subtype B HIV 1P R with and without Ritonavir ................................ ...... 156 4 3 Distance distributi on profiles of subtype B HIV 1PR with inhibitors. ................................ 159 4 4 Ribbon diagrams of HIV 1PR with amino acid differences relativ e to subtype B .............. 163 4 5 Circular d ichroism spectra for spin labeled HIV 1 PR. ................................ ........................ 166 4 6 Dipolar modulated echo curves used for zero time selection of Subtype B si C si ,F si CRF01_A/ E si V6 i and MDR769 i ................................ ................................ .................. 168 4 7 Background subtracted time domain echo data for Subtypes B, C, F, CRF01_A/E and MDR769 and V6, with fits generated by TKR ................................ ................................ 170 4 8 Data analys is for HIV 1PR subtype Bsi apo. ................................ ................................ ...... 171 4 9 Population validation process for HIV 1PR s ubtype Bsi apo. ................................ ............ 172 4 10 Data analysis for HIV 1PR Subtype Csi apo. ................................ ................................ .... 173 4 11 Population validation process for HIV 1 PR s ubtype Csi apo. ................................ .......... 174 4 12 Data analysi s for HIV 1PR Subtype Fsi apo. ................................ ................................ .... 175 4 13 Population validation process for HIV 1P R s ubtype Fsi apo.. ................................ ........... 176 4 14 Data an alysis for HIV 1PR CRF01_A/Esi apo. ................................ ................................ 177

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19 4 15 Population validation process for H IV 1P R CRF01_A/Esi apo. ................................ ...... 178 4 16 Data analysis for HI V 1PR patient isolate V6i apo. ................................ .......................... 179 4 17 Population valid ation process for HIV 1PR V6i apo.. ................................ ....................... 180 4 18 Data analysis for HIV 1PR patient isolate MDR769i apo. ................................ ............... 181 4 19 Population validation process for HIV 1P R MDR769i apo.. ................................ ............. 182 4 20 DEER results for apo HIV 1PR s ubtypes B, C, F, CRF01_A/E, V6, MDR769 ............... 184 4 21 Relative percentage of conformations of protease constructs in the apo form. .................. 184 4 22 R elative percentage of conformations for apo protease constructs ................................ ... 185 4 23 Distance distribution profile overlays of s ubtype B with ea ch other proteae construct .... 186 4 24 Derivative spectra and second derivative spectra of distance profiles for apo protease ... 187 4 25 Truncated dipolar modulated echo curves used for zero time selection of CRF01_A/E with inhibitors ................................ ................................ ................................ ................. 189 4 26 Data analysis fo r HIV 1PR CRF01_A/E with CA p2. ................................ ...................... 190 4 27 Population valid ation process for HIV 1PR CRF01_A/E with CA p2.. ............................ 191 4 28 Data analys is for HIV 1PR CRF01_A/E NFV. ................................ ................................ 192 4 29 Population validation process for HIV 1P R CRF01_A/E NFV. ................................ ....... 193 4 30 Data analysis for HIV 1PR CRF 01_A/E TPV. ................................ ................................ 194 4 31 Population validation process for HIV 1P R CRF01_A/E TPV.. ................................ ....... 195 4 32 Data analy sis for HIV 1PR CRF01_A/E LPV. ................................ ................................ 196 4 33 Population validation process for HIV 1 P R CRF01_A/E LPV. ................................ ....... 197 4 34 Data analy sis for HIV 1PR CRF01_A/E SQV. ................................ ................................ 198 4 35 Population validation process for HIV 1P R CRF01_A/E SQV.. ................................ ....... 199 4 36 Data analy sis for HIV 1PR CRF01_A/E ATV. ................................ ................................ 200 4 37 Population validation process for HIV 1P R CRF01_A/E ATV. ................................ ........ 201 4 38 Data analy sis for HIV 1PR CRF01_A/E DRV. ................................ ................................ 202 4 39 Population validation process for H IV 1P R CRF01_A/E DRV. ................................ ...... 203

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20 4 40 Data analy sis for HIV 1PR CRF01_A/E APV. ................................ ................................ 204 4 41 Po pulation validation process for HIV 1P R CRF01_A/E APV. ................................ ........ 205 4 42 Data analysis fo r HIV 1PR CRF01_A/Esi with RTV. ................................ ...................... 206 4 43 Popu lation validation process for HIV 1P R CRF01_A/Esi with RTV.. ............................ 207 4 44 Data analysis for HIV 1PR CRF01_A/Esi with IDV.. ................................ ....................... 208 4 45 Population validation proces s for HIV 1P R CRF01_A/E. ................................ ................ 209 4 46 Overlay of distance distribution profiles for apo HIV 1PR CRF01_A/Esi and with inhibitors. ................................ ................................ ................................ ......................... 210 4 47 Distance distribution profiles of HIV 1PR CRF01_A/Esi with inhibitors. ........................ 210 4 48 Population analysis for HIV 1PR CRF01_A /E. ................................ ................................ 210 4 49 Truncated dipolar modulated echo curves used for zero time selection of HIV 1PR CRF01_A/E with inhibitors ................................ ................................ ............................ 212 4 50 Data analysis for HIV 1PR s u btype Fsi with RTV. ................................ .......................... 213 4 51 Population validation process f or HIV 1PR Fsi with RTV. ................................ .............. 214 4 52 Data analysis for HIV 1PR s ubt ype Fsi with IDV. ................................ ............................ 215 4 53 Population validation process for HIV 1 P R s ubtype Fsi with IDV. ................................ .. 216 4 54 Data analysis for HIV 1PR s ubtype Fsi with LPV. ................................ ........................... 217 4 55 Population validation process for HIV 1P R Fsi with LPV. ................................ ............... 218 4 56 Data analysis for HIV 1PR s ub type Fsi with TPV.. ................................ ........................... 219 4 57 Population validation process for HIV 1P R Fsi with TPV.. ................................ .............. 220 4 58 Data analysis for HIV 1PR s ubty pe Fsi with SQV.. ................................ .......................... 221 4 59 Population validation process for HIV 1P R Fsi with SQV.. ................................ .............. 222 4 60 Data analysis for HIV 1PR s ubtype Fsi with DRV.. ................................ .......................... 223 4 61 Population validation proce ss for HIV 1PR s ubtype F with DRV. ................................ ... 224 4 62 Data analysis for HIV 1PR s ub type Fsi with NFV. ................................ .......................... 225 4 63 Population validation process for HIV 1P R Fsi with NFV ................................ ............... 226

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21 4 64 Data analysis for HIV 1PR s ubtyp e Fsi with ATV.. ................................ .......................... 227 4 65 Population validation process for HIV 1 P R Fsi with ATV. ................................ .............. 228 4 66 Data anal ysis for HIV 1PR Fsi with APV. ................................ ................................ ........ 229 4 67 Population validation process f or HIV 1P R Fsi with APV. ................................ .............. 230 4 68 Data analysis for HI V 1PR s ubtype Fsi with C A p2. ................................ ....................... 231 4 69 Population validation process for HIV 1 PR s ubtype Fsi with CA p2. .............................. 232 4 70 Population analysis for s ubtype Fs i ................................ ................................ ................... 233 5 1 Crystal structure of human renin. ................................ ................................ ......................... 236 5 2 Ribbon diagram showing x ray structure of oxidized thioredoxin. ................................ ...... 239 5 3 pJ2:G02057 storage vector in w hich prorenin DNA was obtained. ................................ ..... 241 5 4 pET 32a(+) vector map.. ................................ ................................ ................................ ...... 242 5 5 1% agarose gel used to confir m purity of pET32a_XaPR plasmid ................................ ..... 243 5 6 Domain diagram of the prorenin thioredoxin fusion construct. ................................ ........... 243 5 7 Pilot expression of prorenin thioredoxin fusion construct in BL21(DE3) strain Escherichia coli cells. ................................ ................................ ................................ ..... 249 5 8 Pilot expression of proreni n thioredoxin fusion construct in OrigamiB(DE3) strain E. coli cells. ................................ ................................ ................................ .......................... 249 5 9 Typical chromatogram from HiTrap TM Chelating HP affinity column. ............................... 250 5 10 Typical chromatogram from HiTrap TM Q HP a nion e xchange column. ........................... 251 5 11 SDS PAGE gel demonstrating purity of prorenin thioredoxin fusion construct following sequent ial chromatographic steps. ................................ ................................ ... 251 5 12 SDS PAGE gel demonstrating purity of prorenin thiore doxin fusion construct and prorenin after separation from thioredoxin.. ................................ ................................ .... 252 5 13 Circular dichroism spectrum of prorenin after liberation from thioredoxin. ...................... 253 5 14 4 (dimethylaminoazo)benzene 4 carboxylic acid ( DABCYL ) absorpti on overlaps with th e 5 ((2 aminoethyl)amino)naphthalene 1 sulfonic acid (E DANS ) fluorescence thereby quenching the fluorescence through fluorescence resonance energy transfer.. .. 254 5 15 Renin Substrate I Activity screening resuts ................................ ................................ ....... 255

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22 5 16 SDS PAGE gel showing purity of V28C mutant following anion exchange chromatography.. ................................ ................................ ................................ ............. 257 B 1 Map of pJ201:19864 with D215N Pro Yeast proteinase A insert (red). ............................. 271 D 1 Pilot expression of prorenin GM2 activator protein ( GM2AP ) thioredoxin fusion construct in OrigamiB( DE3) strain E. coli cells. ................................ ............................ 277 D 2 Ty pical chromatogram for GM2AP thioredoxin fusion construct using 5 mL Ni charged HiTrap HP affinity column. ................................ ................................ ............... 278 D 3 SDS PAGE gel showing in purity of GM2AP trx fusion construct following purification ................................ ................................ ................................ ...................... 278 D 4 Results o f a fluorescence resonance energy transfer ( FRET ) based functi onal assay for GM2AP function. ................................ ................................ ................................ ............. 279

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23 LIST OF AMINO ACIDS AND AMIN O ACID ABBREVIATIONS Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

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24 LIST OF HIV 1 PROTEASE I NHIBITORS AND ABBREV IATIONS APV Amprenavir ATV Atazanavir CA p2 Capsid p2 substrate mimic DRV Darunavir FPV Fosamprenavir IDV Indinavir LPV Lopinavir NFV Nelfinavir RTV Ritonavir SQV Saquinavir TPV Tipranavir

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25 LIST OF ABBREVIATIONS (+)mRNA plus sense messenger RNA ACE Angiotensin Converting Enzyme AIDS Acquired Immunodeficiency Syndrome AII Angiotensin II AmpR ampicillin resistance AP post acidification pellet ARB Angiotensin Receptor Blockers AR V antiretroviral drugs AS post acidification supernatant AT adenine thymine BEV b aculovirus BME Mercaptoethanol, 2 Mercaptoethanol bp base pair B.S. bachelor of science BSA bovine serum albumin CD circular dichroism CD4 Cluster of Differentiation 4 CHO C hinese h amster o vary CID collision induced dissociation CRF circulating recombinant form CW continuous wave DABCYL 4 (dimethylaminoazo)benzene 4 carboxylic acid dB decibel

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26 DEER double electron electron resonance DMSO dimethylsulfoxide DNA deoxyribonucleic acid Dr. doctor E. coli Escherichia coli EDANS 5 ((2 Aminoethyl)amino)naphthalene 1 sulfonic acid EI entry inhibitor E n v envelope EPR electron paramagnetic resonance ER endoplasmic reticulum ESEEM electron spin echo envelope modulation ESI electrospray ionization ESR electron spin resonance FDA Food and Drug Administration FI fusion inhibitors FIV feline immunodeficiency virus FRET fluorescence resonance energy transfer FWHM full width at half maximum G Gauss GHz gigahertz gp 120 glycoprotein 120 gp 41 glyc oprotein 41 HAART Highly Active Antiretroviral Therapy

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27 HEK h uman e mbryonic k idney HIV Human Immunodeficiency V irus HIV 1 Human immunodeficiency virus type 1 HIV 1PR HIV 1 protease HIV 2 Human immunodeficiency virus type 2 I AP 3 (2 Iodoac etamido) PROXYL I A SL 4 (2 Iodoacetamido) TEMPO IB inclusion body IDA iminodiacetic acid II integrase inhibitor IHRP In house research program IMAC immobilized metal ion affinity chromatography Int integrase IPTG Isopropyl D 1 thio galacto pyrano side ITC isothermal titration calorimetry kD kilodalton LB Luria Bertani media LP lysed cell pellet LS lysed cell supernatant LTR long terminal repeat M major group MALDI TOF matrix assisted laser desorption ionization t ime of flight MC Monte Carlo

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28 MCS multiple cloning site MD molecular dynamics MDR769 Multi drug resistant variant 769 mmHg m illimeters of mercury Mol. wt. molecular weight mS milli Siemens MSL 4 Maleimido TEMPO MTSL (1 oxyl 2,2,5, 5 tetramethyl 3 pyrroline 3 methyl)methanethiosulfonate MW molecular weight NHLBI National Heart, Lung, and Blood Institute NHMFL National High Magnetic Field Lab NIH National Institute of Health NK natural killer Nle norleucine NMR nuc lear magnetic resonance NNRTI non nucleoside reverse transcriptase inhibitor NOE Nuclear Overhauser Effect NRTI nucleoside reverse transcriptase inhibitor NSF National Science Foundation NTA nitrilotriacetic acid O outlier group OD opt ical density OI Opportunistic infection

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29 ORF open reading frame PDB Protein Data Bank PEG polyethylene glycol Ph.D. Doctor of Philosophy PI protease inhibitors PMPR pentamutated protease RAS Renin Angiotensin System RNA ribonuclei c acid RP resuspension pellet rpm rotations per minute RS resuspension supernatant RT reverse transcriptase SDS sodium dodecyl sulfate SDSL site directed spin labeling SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SIV Simian Immunodeficiency virus SIVcpz Simian Immunodeficiency virus, chimpanzee SIVsm Simian Immunodeficiency virus, sooty mangabey SNR signal to noise ratio SPR surface plasmon resonance SU surface TKR Tikhonov Regularization T m melting temperature

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30 T M phase memory time TM transmembrane tRNA transfer RNA trxA Thioredoxin A UF University of Florida UNAIDS Joint United Nations Programme on HIV/AIDS UV ultraviolet VMD visual molecular dynamics WHO World Healt h Organization WP wash pellet WS wash supernatant YPRA yeast proteinase A

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31 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Ph ilosophy AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF HIV 1 PROTEASE AND THE DEVELOPMENT OF A SOLUBLE EXPRESSION SYSTEM FOR PRORENIN By Jamie Laura Kear August 2010 Chair: Gail E. Fanucci Major: Chemistry All work performed for this dissertation dealt with, in general, the expression, purification, and biophysical charact erization of aspartic proteases, specifically human immunodeficiency virus type 1 ( HIV 1 ) p rotease (HIV 1PR) and the activatable renin zymogen called prorenin. Chapters 1 and 2 descri be the relevant biology and methodologies, including pulsed and continuous wave electron paramagnetic resonance spectroscopy. H IV 1PR is a viral aspartic protease that functions in regulating post translational processing of the viral polyproteins gag and gag pol The enzyme is a dimer comprised of 99 amino acid monomeric subunits. Accessibility of substrate to the active site is med hairpins called the flaps (one belonging to each monomer). The flaps have been shown to undergo a large conformation al change during substrate binding and cat alysis; molecular dynamics simulations have captured three distinct conformations of the flaps in HI V 1 protease, namely the closed semi open, and wide open conf ormations Reported in this work are results of continuous wave and pulsed electron paramagnetic resonance studies of HIV 1 protease. Continuous wave electron paramagnetc res onance (CW EPR ) spectroscopy though it does not report on the flap motions of the protease, was used to examine the autoproteolytic activity of the protease. The EPR spectral line shape is highly sensitive to mobility in the environment of the spin label thus it changes

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32 dramatically with changes in correlation time. Autoproteolysis affects the rate of global protein tumbling by decreasing rotational correlation time of the spin labeled protein as a smaller spin label ed peptide fragment is liberated As total correlation time decreases, the derivative EPR spectra decrease in breadth and resonance line shapes become sharper and increasingly narrow. T he appearance of a sharp component in the high field proportional to the amount of degraded protein in th e sample was monitored T he intensity of the high field line was quantitatively analyzed to give a term proportional to the amount of uncleaved peptide remaining in the sample The pulsed technique double electron electron resonance (DEER) was uti lized to examine the differential flap conf ormations an d flexi bility of various HIV 1PR constructs under various conditions. D EER experiments provided a means to determine distance profiles between two spin label ed sites in the flaps (sites K55C and K55C ), which were used to describe and quantify conformational sampling of protease constructs DEER echo curves were analyzed via Tikhonov Regularization methods and the resulting distance profiles were regenerated using a series of Gaussian shaped functions each representative of a distinct flap conformation. Distance profiles from spin label ed constructs of s ubtype s B, C, and F, CRF01_A/E, and drug resistant patient isolate s V6 and MDR769 without ligand were analyzed in order to identify what e ffect natu ral and drug induced polymorphisms have on the conformational ensemble of the protease The dipolar modulated echo data and resulting distance distribution profiles differed greatly among the apo protease constructs These results demonstrated that natur al and drug induced polymorphisms in the amino acid sequence of various subtypes and patient isolates alter the average flap conformations and flexibility of the flaps. A dditionally, in order to monitor differences in flap conformations upon inhibitor bin ding between Subtype B and CRF01_A/E protease s, constructs were analyzed upon addition of inhibitors and a non hydrolysable substrate

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33 mimic, CA p2. These studies yielded interesting results in that the conformational ensemble s of the protease differ drast ically with the various inhibitors. Renin, also known as angiotensinogenase, is an aspartic protease that plays a vital role in blood pressure regulation by catalyzing the first and rate limiting step in the activation pathway of its substrate angiote nsinogen. The protease cleaves angiotensinogen to form angiotensin I, which is then converted into angiotensin II by angiotensin I converting enzyme in a process known as the renin angiotensin cascade, which has an important effect on aldosterone release, vasoconstriction, electrolyte imbalance, congestive heart failure, and an increase in blood pressure leading to hypertension. M any hypertension drugs function by regulating blood pressure at various points in the renin angiotensin system. Prorenin is an inactive zymogen of renin that circulates through the plasma until it reaches the secretory granules, where the pro segment is cleaved and active renin is released. Currently, very little structural data on prorenin is available in the literature, likely because current methods for recombinant bacterial expression of multiply disulfide bonded aspartic proteases from Escherichia coli have been plagued by difficulty due to expression as inclusion bodies that require denaturation and refolding in order to ob tain properly folded, functional protein. Refolding, however, does not ensure that the protein will be both properly folded and active. A bacterial expression system to circumvent these difficulties was developed and work presented herein Thioredoxin fu sion methodology was employed in order to maintain protein solubility and avoid inclusion body formation. The resultant protein was shown to have secondary structure consistent with aspartic protease zymogens and a fluorescence resonance energy transfer ( FRET ) based assay was used to demonstrate pH dependent activation; however, the system was only minimally successful due to low yields and protein instability upon cleavage from the fusion partner thioredoxin

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34 CHAPTER 1 INTRODUCTION TO ASPA RTIC PROTEA SES Introduction t o Proteases Proteases are defined as enzymes that hydrolyze a polypeptide chain. Also called proteinases or proteolytic enzymes, proteases are a class of hydrolases that are necessary for life and are th us naturally occurring in all orga nisms Protein hydrolysis is defined by the lysis of a polypeptide chain by water, which i s broken down into a hydrogen cat ion and a hydroxide anion during the course of the reaction (McKee and McKee 2003) The general reaction scheme is given in Figure 1 1. Figure 1 1. Scheme of hydrolysis of a polypeptide chain. Note charge states will vary depending on pH. Proteases c an either exhibit limited proteoly sis, that is they target hydro lysis on specific peptide bonds, or unlimited proteolysis in which they carry out complete digestion of a peptide into its amino acid constituents Proteases can be classified as either exoproteases or endoproteases based upo n the region of the target protein or peptide in which proteolysis is carried out Exoproteases a re a subclass of proteases that exhibit hydrolytic activity only near the end of a peptide chain, while endoproteases can cleave near the center of the peptid e sequence. Proteases often contain a highly conserved catalytic triad which is involved in hydrolysis of the substrate. A catalytic triad refers to a series of three amino acids that function together and are directly involved in catalysis. Proteases a re active in a specific pH range, and based on the particular active pH range of the enzyme, they can be classified as acidic neutral, or basic proteases. Due to the varying nature of this class of enzyme, proteases are involved in a

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35 wide variety of diff erent physiological reactions (McKee and McKee 2003) Proteases are crucial to the peptide hydrolysi s process. Uncatalyzed peptide bond hydrolysis reactions a t 25 C and pH 7 are carried out with half times of approximately 500 years for the C terminal bond of acetylglycylglycine, 600 years for the internal peptide bond of acetylglycylglycine N methylamide, and 350 years for the dipepti de glycylglycine (Radzicka and Wolfenden 1996) Introduction to Aspartic Proteases The re exist six classes of proteases; namely, serine proteases, cysteine proteases, metalloproteases, threonine proteases, glutamic proteases, and aspartic proteases. Each class functions by providing a nucleophile, usually a water molecule or an amino acid, for attack on the peptide carboxyl group. All of the key proteins investigated throughout this dissertation are aspartic proteases. Aspartic proteases are a subclass of endoproteases that often play an important role in health and disease. Found in a range of different organisms, from mammals and f ungi to viruses and plants, aspartic proteases util ize aspartate residues to render catalytic activity. Generally speaking, aspartic proteases have two highly conserved aspartic acid residues in the active s ite of the enzyme and are optimally active at a cidic (or sometimes neutral) pH. Some aspartic proteases function as monomers while others function as dimers; however, all aspartic proteases, regardless of whether they function as a monomer or dimer, gener ally always have a tertiary structure composed of two similar lobes, thereby creating a two fold symmetry. Aspartic proteases carry out a wide variety of reactions to regulate physiological phenomenon, including but not limited to regulation of blood pres sure, protein catabolism, digestion, and lysosomal protein degradation, as illustrated by the examples given in Table 1 1.

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36 Table 1 1. Re presentati ve aspartic proteases by name, function, s ource, size and structure N ame Function Source Size (kDa) Structure Renin Blood pressure regulation Kid ney 37 Monomer HIV 1 protease Maturation of HIV Virus HIV 1 21 Dimer Pepsin Digestion Stomach 35 Monomer Cathepsin D Cel lular turnover Lysosome s 48 Dimer *kDa = kilodalton : 1 kDa is equal to approximately the weight of 1000 hydrogen atoms, or the weight of 1/16 th of the weight of 1000 Oxygen 16 atoms or 1.66 x 10 21 grams. General Mechanism of Catalysis by Aspartic Proteases Aspartic proteas es are a subclass of endoproteases that cleave specific dipeptide bonds within their target substrate. M ost often these dipeptide bonds are in close proximity to hydrophobic residues and a methylene group. The most widely accepted mechanism of catalysis by aspartic proteases shown in arrow pushing format in Figure 1 2 is based upon a general acid base catalysis mechanism that involves coordination of a water molecule which acts as a nuc leophile, between the two active site catalytic aspartate residues Figure 1 2. Generally accepted mechanism of catalysis by aspartic proteases. Figure generat ed in ChemDraw and modified from Brik et al. (Brik and Wong 2003)

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37 A unique characteristic of the two aspartate residues is that one has a relatively low pKa (usually between 1 and 3.5), while the other has a relatively high pKa (usually between 4 and 5.5) The deprotonated residue acts as the general base, accepting a proton from the coordinated H 2 O, rendering it available to make a nucleophilic attack on the carbonyl group of the cleavable peptide bond. The protonated aspartate residue acts as a general acid, donating a proton for formation and rearrangement of the tetrahedral oxyanion intermediate and protonation of the amide group. Introduc t ion to HIV 1 Protease HIV as a World Pan demic Much federally and privately funded medical research fo cuses on Acquired Immune D eficiency Syndrome (AIDS), which is a devastating world wide health crisis caus ed by the Human Immunodefi ci ency V irus (HIV). This disease of the human immune system is currently spreading at frightening rates though epidemiologic al data indicate that the spread of HIV appears to have peaked in 1996 when 3.5 million new infections occurred; i n 2008, the estimated number of new HIV infections was approximately 30% lower (2009) Nonetheless, the World Health Organization (WHO) has reported tha t the num ber of people living with HIV or AIDS a s of December 2008 was approximately 33 .4 million 31 .3 million of which were adults ( WHO 2009) This number is approximately 20% higher than the number in 2000, and roughly three times the number in 1990 ( WHO 2009) Approximately 2.7 million people were newly infected worldwide an d approximately 2 million people died from AIDS related deaths in 2008 alone ( WHO 2009) The Joint United Nations Programme on HIV/AIDS ( UNAIDS ) reported that a pproximately three quarters of those deaths t ook place in Sub Saharan Africa, where an estim ated 15 30% of the population was living with HIV or AIDS in 2007 This region accounted for approximately 72% of AIDS related deaths worldwide in 2008 ( WHO 2009) By

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38 comparison, the United States had approximat ely 0.5% 1% of its population living with HIV or AIDS in 2007 (Fig ure 1 3 ) Figure 1 3 Map showing percent of adult population living with HIV infection in 2007 according to country. Figure modified from UNAIDS. HIV infection is characterized by a su ppression of the immune system, and is said to have four stages. The first stage of HIV infection is the period following infection and is generally referred to as t he window. During this stage, a person will likely not test positive for HIV because most HIV tests look for antibodies to the virus rather than for the virus itself. The window phase is characterized as the period of time after which a person has become infected but has not yet developed antibodies to the virus. The second stage is called serconversion and is characteriz ed by production of antibodies; a person is highly infectious during this stag e. The third stage is the often symptom free stage and can last anywhere from a few months to more than ten years. Stage four is called AIDS. A person is said to have AID S whe n his/her CD4 (Cluster of Differentiation 4) cell count falls below 200 per cubic milli meter of blood (normal CD4 cell count is between 500 and 1500 per cubic millimeter of blood) and/ or the patient has one or more opportunistic infections (OIs ). OIs are typically normal infections from which a person with a healthy immune system could usually recover. Though there are many drugs aimed at slowing the course of the disease, there is currently no vaccine and no cure.

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39 Introduction to HIV 1 Viruses are smal l, non living infectious particles comprised of at least two main components, the viral genome composed of either deoxyribonucleic acid or ribonucleic acid (DNA or RNA) and a protein coat designed to house the genetic material and insert it into a host cel l. Additionally c ertain viruses also contain a n exterior lipid coat. There exist two distinct major strains of HIV called HIV 1 and HIV 2. E pidemiologic data suggest that HIV 1 originated from the simian immunodeficiency virus (SIV) called SIVcpz, of the chimpanzee, which is highly homologous with HIV 1. Similarly, a simian immunodeficiency virus strain called SIVsm from the sooty mangabey is highly homologous with HIV 2, thus it is likely that HIV 2 originated from that strain HIV 1 and HIV 2 poss ess si milar modes of transmission, but HIV 2 is more difficult to spread HIV 2 is characterized by a longer incubation period and lower infectivity than HIV 1 due to a lower viral density in the bloodstream In addition, HIV 2 has a rather isolated geog raphic pattern; it is pri marily found in the Western portions of Africa and i t is estimated that less than one hundred people are infected by HIV 2 in the U nited S tates. Figure 1 4. Cartoon representation of t he structure of the HIV 1 virus, showing t he locations of the host cell derived outer lipid membrane, the Env protein comprised of gp120 and pg41 glycoprotein subunits, the matrix, and the capsid, which houses two strands of viral RNA, plus viral proteins integrase, protease, and reverse transcrip tase Adapted from NIAID.

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40 HIV is roughly spherical in shape and has a diameter of approximately 1000 Angstroms The virus is encompassed by a viral envelope of lipid s d erived from a budding event from the human host cell. Also found in the viral envelope are various host cell proteins and numerous copies of the viral protein called Env elope ( Env ) which is c omprised of glycoproteins called glycoprotein 120 (gp120) and glycoprotein 41 (gp41). Within the viral envelope is the capsid, which surrounds two strands of HIV 1 RNA and several viral proteins, including Integrase (Int) and Reverse Transcriptase (RT) The structure of HIV 1 is shown in Figure 1 4. Because HIV is a virus, it depends on a host cell for reproduction. HIV is in the family of viruses called retroviruses, which are characterized by possessing a single stranded plus sense (+) mRNA genome w hich must be reverse transcribed to give a DNA intermediate. The enzyme responsible for this process is called reverse transcriptase and it lacks proofreading activity ; the mutation rate is estimated to be approximately 3.4 x 10 5 mutations/bp/replication or one every three rounds of replication (Mansky and Temin 1995) Most eukaryotic polymerases p ossess proofreading activity and as a result have very low mutation rate. For example human DNA polymerase replicates DNA with an estimated 5x10 11 mutations/bp/replication (Drake, Charlesworth et al. 1998) Some of these mutations will be silent (DNA mutations that do not change the amino acid sequence of the protein), but those that are not silent can result in proteins containing a polymorphism. Figure 1 5 Phylogenic tree for HIV 1 and HIV 2 groups subtypes and circulating recombi nant forms

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41 Table 1 2 Primary g eographic p revalence of s ubgroups of HIV 1 Group/Subtype/CRF Primary Geographic Prevalence Group M Subtype A East and Central Africa; Central Asia; Eastern Europe CRF01_ A / E Southeast Asia CRF01_A/G West Afri ca Subtype B North and South America ; Western Europe; East Asia; Oceania Subtype C India; Southern and Eastern Africa Subtype D East Africa Subtype F Africa; South America; Eastern Europe Subtype G West Africa Subtype H Central Africa Subtype J Cent ral America Subtype K Cameroon and Democratic Republic of Congo Group N Cameroon Group O West Africa Group P Cameroon HIV 1 is categorized into different groups, subtypes (or clades), and circulating recombinant forms (CRFs) Groups re fer to viral lineages, subtypes to taxonomic groups within a particular lineage, and CRFs to recombinant forms of the virus comp rised of different viral strains; Figure 1 5 shows a rough phylogenic tree for HIV 1 (Kantor, Shafer et al. 2005) Within HIV 1, there are four groups, namely group M (major), group O (outlier), and groups N and P. Groups N and P are found primarily in Cameroon and are very rare. Group O is slightly more common and is found primarily in west central Africa. Group M is by far the most common with at least nine distinct subtypes and several CRFs, which are recombinant forms of two or more subtypes. Within group M are subtypes A D, F H, J, and K, where e ach subtype exhibits a unique se t of naturally occurring polymorphisms. G enetic diversity can reach 1 5 to 20% within subtypes and 25 to 30% between subtypes The primary geographic prevalence of each of these subgroups is given in Table 1 2. Considering the natural frequency of mut ations within HIV 1, new genetic subtypes and CRFs

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42 will most certainly continue to develop as virus recombination and viral polymorphisms continue to occur. HIV 1 Viral Life Cycle The life cycle of HIV 1 is sim ilar to many other retroviruses, in that it i s characterized by a series of well defined steps, as shown in the cartoon representation of the viral life cycle in Figure 1 6 The HIV virus primar ily infects cells that have CD 4 cell surface receptors, including monocytes, macrophages dendritic cells, T helper cells, regulatory T cells natural killer (NK) cells, hematopoietic stromal cells, and microglial cells The gp120 s urface glyco protein recognizes and binds to the CD4 receptors, a conformational change in the viral protein gp41 takes place, and fusion of the virus to the host cell membrane occurs. T helper cells are a type of lymphocyte that plays an imperative role in the immune response. These cells exhibit no cytotoxic or phagocytic activity, but rather they are involved in activating and d irecting other cells involved in the immune response. A mature HIV 1 virion requires a CD4 cell surface receptor for binding and fusion events to occur. After the fusion event occurs, an uncoating event is triggered in which the RNA viral genome and mat ure viral proteins are released into the cytoplasm of the host. The viral RNA is then reverse transcribed resulting in a DNA intermediate which forms an integration complex of viral DNA and viral proteins and is imported into the cell nucleus. At this po int, the proviral DNA is integrated into the host cell chromosome and transcribed and translated via host cell machinery. The viral polyproteins subsequently accumulate on the outer membrane of the host cell, triggering a budding event in which non infect ious, immature viral particles are released from the host cell. In a final step, viral maturation takes place as HIV 1 protease cleaves the viral polyproteins gag and gag pol into their respective protein components.

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43 Figure 1 6 Cartoon represen tation of the HIV 1 l ife c ycle; s teps include r eceptor recognition and binding, fusion, u ncoating and r everse t ranscription, n uclear i mport and integration, transcription and t ranslation, p rotein a ssembly and b udding, and m aturation. Figure adapted from NIAIDS.

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44 The HIV 1 Viral Genome The HIV genome consists of one strand of RNA that encodes three major genes, including gag pol (or gag pol ), and env as well several other regulatory genes ( tat rev nef vif vpr and vpu ). The long terminal repeat (LT R) serves as a promoter of transcription. A cartoon representation of the HIV 1 viral genome is given as Figure 1 7 Figure 1 7 Cartoon representation of the HIV 1 viral genome. Figure modified from NIAIDS. The gag gene encodes for four proteins whi ch are important in building the core of the virus; these proteins include the capsid protein p24, the matri x protein p17, the nucleocapsid protein p9, and protein p6. The pol gene encodes for four proteins that are imperative for the life cycle of the vi rus, and include reverse transcriptase, HIV 1 p rotease, RNAse H, and Integrase. Reverse transcriptase is required to copy the viral RNA into DNA once inside a host cell. HIV 1 protease functions in regulating post translational processing of viral polypr oteins, which is required for viral maturation. RNAse H breaks down the viral genome following infection. Integrase is involved in integrating the reverse genome. The env gene encodes for only one protein, calle d gp160, which is cleaved to become gp120 and gp40 after new viral particles bud off from the host cell. Accessory genes encoded by HIV 1 include tat rev nef vif vpr and vpu tat encodes for a regulatory protein that increases the rate of transcripti on of HIV rev encodes for a regulatory protein which lead s to production of other viral proteins. nef encodes for a regul atory protein that accelerat es endocytosis of CD4 from the surface of infected cells. The vif vpr and vpu

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45 genes encode proteins th at appear to play a role in generating infectivity and pathologic effects. Vif also called p23, increases viral infectivity by antagonizing APOBECC3G, a protein that prevents spread of the infection by causing G to A hypermutation (Malim and Emerman 2008) Vpr also referred to as p15 has several functions including a role in the pre integration comple x (Malim and Emerman 2008) Vpu also referred to as p16 is an integral membrane protein that helps bind CD4 receptors (Malim and Emerman 2008) Structure and Function of HIV 1 Protease Figure 1 8 Structure of Subtype B HIV 1PR (PDB ID 2BPX); A) r ibbon dia gram, and B) space filling model of Subtype B HIV 1PR (top view), and C) ribbon diagram, and D) space filling model of Subtype B HIV 1PR (side view). Structures rendered with Chimera (Pettersen, Goddard et al. 2004) Human immunodeficiency virus type 1 (HIV 1) protease (HIV 1PR) (EC 3.4.23.16) is a member of the aspartic protease family (A02.001) (Barrett, Rawlings et al. 1998) The enzyme is a homodimer of two 99 amino acid monomers and is essential for the life cycle and maturation of the retrovirus HIV 1 because it functions in regulat ing post translational processing of the viral polyproteins gag and gag po l into an array of individual functional proteins that assemble

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46 into mature and infectious HIV particles Inhibition of HIV 1PR prevents viral maturation and lengthens the l ife span reduces viral load, and increases CD4 cell counts in HIV sufferers; as such, this enzyme is a primary target of AIDS antiviral therapy (Ashorn, McQuade et al. 1990) As of January 2010, more than 315 structures of HIV 1 Protease, both apo and in complex with various substrates and inhibitors were deposited in the Prot ein Data Bank (PDB), the first of which was reported in 1989 (Miller, Schneider et al. 1989) A ribbon diagram of the side and top view of HIV 1 pro tease, as well as a space filling model emphasizing the inaccessibility of the active site to a peptide substrate when the flaps are in the closed confor mation, are given in Figure 1 8 The nomenclature for the substrate amino acid cleavage positions is P4 P3 P2 P1/ where the slash represents the scissile bond. Table 1 3 provides a listing of the polyprotein processing sites of HIV 1 Protease. HIV 1PR has been crystallized with non hydrolyzable CA p2 bound and is shown in Figure 1 9 Table 1 3 HIV 1 P R polyprotein processing sites Loca tion HXB2 Consensus Cut Site Sequence P5 P4 P3 P2 gag polyprotein MA/CA VSQNY / PIVQN CA/p2 KARVL / AEAMS p2/NC TSAIM / MQRGN NC/p1 ERQA N / FLGKI P1/p6 gag RPGNF / LQSRP pol polyprotein NC/TFP ERQAN / FLREN TFP/p6 gag pol EDLAF / LQGKA P6 pol /PR TSFSF / PQITC PR/RTp51 CTLNF / PISPI RT/RTp66 GAETF / YVDGA RTp66/INT IRKVL / FLDGI Nef polyproteins Ne f AACAW / LEAQE

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47 Figure 1 9. HIV 1PR (silver ribbon) bound to the non hydrolyzable substrate mimic CA p2 (colored ball and stick) Structures rendered with VMD (Humphrey 1996) ; A) side and B) top views. The dissertation work describes experiments with a non hydrolyzable substrate mimic of the CA p2 cleavage site of the sequence Lys Ala Arg Val Leu Ala Glu Ala Met Ser; the substrate mimic has the sequence H Arg Val Leu r Phe Glu Ala Nle NH 2 (r = reduced). position been replaced by the methionine analog Norleucine (Nle), shown in Figure 1 10 A; methionine i s shown in Figu re 1 10 B for comparison. Norleucine is a non natural amino acid isomeric to leucine and isoleucine Figure 1 10 Structures of A) norleucine and B) methionine. Accessibility of substrate to the active site hairpins termed the flaps which must undergo a large conformational change from an open to a closed conformation during substrate binding and catalysis, as demonstrated by nuclear magnetic resonance ( NMR ) isothermal titration calorimetry ( ITC ) and molecular dyn amic (MD) simulations. NMR has been used to elucidate the structure of HIV 1PR in solution, both of the monomer (Ishima,

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48 Ghirlando et al. 2001; Ishima, Torchia et al. 2003; Louis, Ishima et al. 2003) and the dimer (Ishima, Freedberg et al. 1999; Freedberg, Ishima et al. 2002) Torchia et al. were able to provide evidence of millisecond microsecond timescale flap motion in unliganded protease and nanosecond to subnanosecond flap motion in ligand bound protease. Additionally, they were able to demonstrate via nuclear Overhauser effect (NOE) that the flap region undergoes an unordered to o rdered transition upon inhibitor binding (Figure 1 11). Figure 1 11. HIV 1 P R NOE values in black refer to the liganded form of HIV 1 PR and NOE values in gre y refer to the unliganded HIV 1 P R Isothermal Titration Calorimetry (ITC) has been used to exa mine many aspects of HIV 1 Protease, including stability and ligand binding. ITC is a technique that is used to determine the thermodynamic parameters of a system, including binding affinity and stoichiometry of a reaction, as well as changes in enthalpy and entropy. Freire et al. used the technique to determine that the flap region is the least stable part of the protein, indicating the presence of a major conformational change in that region (Todd, Semo et al. 19 98; Todd and Freire 1999; Todd, Luque et al. 2000; Velazquez Campoy, Todd et al. 2000)

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49 Molecular dynamics simulations have provided some of the most direct evidence of the necessary conformational changes within the flap region to facilitate ligand bind ing and catalysis. Molecular dynamics (MD) simulations performed by Simmerling et al. have indicated three distinct conformations of the flaps in HI V 1 protease, namely the closed semi open, and wide open conformations, as seen in Figure 1 12 (Hornak, Okur et al. 2006) Figure 1 12 Flap conformations of HIV 1 protease captured by molecular dynamics simulations; namely A ) closed in presence of inhibitor, B ) sem i open, and C ) wide open. Structures rendered with Chimera (Pettersen, Goddard et al. 2004) The closed conformation i s found primarily in the presence of an inhibitor and the semi open and wide open conformations are seen in the absence of inhibitor. This was a landmark study because prior to the work of Simmerling et al. no all atom MD simulations were able to capture flap reclosing, and the results were in agreement with previous NMR and X Ra y crystallographic evaluations of the flap ensembles. These MD s imulations have an advantage over NMR and X Ray in that X Ray tends to capture a single conformation and NMR reports on the average conformation.

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50 Conformational Sampling and the Conformati onal Ensemble of the HIV 1 Protease Flaps As noted above, the protease samples several distinct flap conformations. The sum of each of these conformations can be described as the conformational ensemble of the protease. The conformational sampling of the protease changes when inhibitors or substrate are added (Blackburn, Veloro et al. 2009) polymorphisms are incorporat e d into the protease (Kear, Blackburn et al. 2009) or the conditions of the protein solution are modified [B lackburn, unpublished] The double electron electron resonance ( DEER ) methodology described in later chapters allows us to examine the individual conformational populations that comprise the conformational ensemble, rather than evaluating the average of t he ensemble (as by NMR), or by capturing a single conformation of the protease (as by X Ray crystallography). Hydrophobic Sliding Mechanism As previously described, it is known that HIV 1PR must undergo a dramatic conformational change for substrates or inhibitors to gain access to the active site pocket. A mechanism, called the hydrophobic sliding mechanism, has been proposed by Schiffer et al. that explains how this conformational change takes place, in addition to providing an explanation as to how se condary mutations (those mutations outside the active site) in the protease confer drug resistance (Foulkes Murzycki, Scott et al. 2007) Analysis of molecular dynamics simulations have suggested that nineteen core hydrophobic residues facilitate the conformational changes by sliding past one another, exchanging v an der Waal contacts with little to no energy penalties. This type of motion would preserve hydrogen bonding that is likely imperative for maintaining structural integrity. Seven of the nineteen core hydrophobic residues described above are isoleucines w hich have numerous rotameric states that may facilitate the sliding mechanism. Additionally, this mechanism offers insight into the effect of mutations in these regions because

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51 changes in the hydrophobic residues would likely affect the conformational sam pling and flexibility of the protease (Foulkes Murzycki, Scot t et al. 2007) HIV 1 Protease Construct Terminology Numerous different HIV 1 protease constructs, including Subtypes B, C, F, CRF01_A/E, and patient isolates V6 and MDR769, will be discussed throughout this dissertation. For that reason, abbreviation s will be used to quickly identify which construct is being discussed. Each of these abbreviations is given in Table 1 4, and a description of the construct is also included. Before proceeding, a brief introduction to the abbreviations and nomenclature u sed throughout this dissertation is necessary. Amino acid substitution code (e.g. D25N) is given by amino acid residue to be substituted out, followed by the residue number, followed by the amino acid to be substituted in (e.g. D25N the aspartic acid residue at position number 25 was mutated to an asparagine residue). 1 protease undergoes autoproteolysis under the high concentrations required for biophysical s tudies. In Subtype B, the cleavage sites have been identified as occurring between positions 6 and 7, 33 and 34, and 63 and 64 (Mildner, Rothrock et al. 1994) ; thus, the stabilizing mutations Q7K, L33I and L63I were introduced to avoid self proteolysis. The subsc inactive protease in which a D25N mutation was engineered into the sequence. This mutation has been shown by X Ray and NMR not to perturb the structure of the protease. In addition, all constructs possess mutations C67A and C95A to fa cilitate site directed spin labeling and to avoid non specific disulfide bonding.

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52 Table 1 4. HIV 1 P R construct abbreviations and descriptions Construct abbreviation Construct Details A/E s CRF01_A/E; K55C, C67A, C95A, Q7K, L33I, L63I A /E si CRF01_A/E; K55C, C67A, C95A, Q7K, L33I, L63I, D25N B s Subtype B; K55C, C67A, C95A, Q7K, L33I, L63I B si Subtype B; K55C, C67A, C95A, Q7K, L33I, L63I, D25N C s Subtype C; K55C, C67A, C95A, Q7K, L33I, L63I C si Subtype C; K55C, C67A, C95A, Q 7K, L33I, L63I, D25N F s Subtype F; K55C, C67A, C95A, Q7K, L33I, L63I F si Subtype F; K55C, C67A, C95A, Q7K, L33I, L63I, D25N V6 Patient isolate V6; K55C, C67A, C95A V6 i Patient isolate V6; K55C, C67A, C95A, D25N MDR769 Patient isolate MDR769; K55C, C67A, C95A MDR769 i Patient isolate MDR769; K55C, C67A, C95A, D25N HIV 1 Subtype B Protease Subtype B is the dominant HIV subtype in the United States and Europe. For this reason, a substantial proportion of HIV 1PR research, and most drug develo pment, clinical trials, and observational studies have been performed on Subtype B virus. However, only about 10% of HIV worldwide is Subtype B (2009) Top and side views of a ribbon diagram as well as a space filling model of the crystal structure (PDB ID 2BPX) of Subtype B HIV 1PR were shown in Figure 1 8 The Los Alamos HIV database contains four primary sequences of the Subtype B viral genome namely HXB2 (Wong Staal 1985) BK132 (Hierholzer, Montano et al. 2002) 671 (Geels, Cornelissen et al. 2003) and 1058 (Bernardin, Kong et al. 2005) HXB2 ( Acc ession Number K03455) is the primary reference an d is a specific clone from the French isolate LAI The HXB2 sequence was utilized for all Subtype B work throughout the dissertation (with the exception of several necessary mutations described in detail later). BK132 (Acc ession Number AY173951) was isol ated in Thailand in 1990. 671 ( Acc ession Number AY423387) was isolated in the Netherlands in 2000, and 1058 ( Acc ession Number AY331295) was isolated from the United States in 1998.

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53 HIV 1 Subtype F Protease The subtype F protease is generally differentiate d from the LAI consensus sequence of Subtype B by the following polymorphisms: I15V, E35D, S37N, R41K, and R57K None of these polymorphisms are primary, i.e. occur in the substrate binding pocket but rather, are located on the hinge region and on the o uter wall of the protease Two crystal structures of Subtype F protease have been reported in the Protein Data Bank ( PDB ID 2P3C and 2P3D). 2P3C shown in Figure 1 13 is a structural complex between the protease and the TL 3 inhib itor TL 3 is a protea se inhibitor developed for the feline immunodeficiency virus (FIV), but has been shown to have broad based activity in that it functions to inhibit replication of the human, simian, and feline immunodeficiency viruses (Buhler, Lin et al. 2001) Figure 1 13 Crystal structure of HIV 1PR s ubtype F (PDB ID 2P3C) ; A) side and B) top v iews Structure was rendered with VMD (Humphrey 1996) There are eight primary sequences for the Subtype F viral genome in the Los Alamos HIV database. These includ e 93BR020 1 (Gao, Robertson et al. 1998) VI850 (Laukkanen, Carr et al. 2000) FIN9363 (Laukkanen, Carr et al. 2000) MP411 (Triques, Bourgeois et al. 2000) MP255 (Triques, Bourgeois et al. 2000) MP257 (Tr iques, Bourgeois et al. 2000) 0016BBY (Kijak, Sanders Bue ll et al. 2004) and CM53657 (Carr, Torimiro et al. 2001) The 93BR020 1 (Accession number AF005494) was isolated in Brazil in 1993 ; VI850 (Accession number AF077336) wa s isolated in 1993 in Belgium; FIN9363 (Accession number AF075703) was

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54 isolated in Finland in 1993; MP411 was isolated in France in 1996; MP2555 and MP257 were isolated in Camer oon in 1995 ; AY371158 w as isolated in Cameroon in 2002, and AF377956 was isolated in Cameroon in 1997. HIV 1 Subtype C Protease Subtype C is the most prevalent subtype of HIV worldwide, accounting for approximately 48% of all cases. This subtype is fou nd in Sub Saharan Africa, as well as Asia and India. Subtype C generally differs from Subtype B by the following polymorphisms: T12S, I15V, L19I, M36I, S37A, H69K, N88D, L89M, and I93L. Three crystal structures of Subtype C have been reported in the lite rature, one in the apo form, one in complex with NFV (shown in Figure 1 14), and one in complex with IDV. Figure 1 14. Crystal structure of Subtype C (PDB ID 2R5Q) in complex with NFV (not shown in figure). Structure was rendered with VMD (Humphrey 1996) There are many full length genomes reported in the HIV database, some of which are ETH2220 (Accession number U46016 ) is o lated from Ethiopia in 1986 (Salminen, Johansson et al. 1996) 92BR025.8 (Accession number U52953 ) isolated from Brazil in 1992 (Gao, Robertson et al. 1996) IN21068 (Accession number AF067155 ) isolated f rom India in 1995 (Lole, B ollinger et al. 1999) and SK164B1 (Accession number AY772699 ) isolated from South Africa in 2004 (Kiepiela, Leslie et al. 2004)

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55 HIV 1 Protease Recombinant Form CRF01_A/E Most commonly, CRF01_A/E differs from Subtype B protease by the following polymorphisms: I13V, E35D, M36I, S37N, R41K, H69K, and L89M. The primary sequence of CRF01_A/E in the Los Alamos HIV database is CM240 (Accession number U54771) and was isolated in Thailand in 1990. CRF01_AE one of many reported circulating recombinant forms. This particular CRF is becoming highly prevalent in Asia, but originated from Central Africa. Circulating recombinant forms, as the name implies, are recombinant forms of the virus. CRF01_A/E is a putative subtype A/E recombinant. Figure 1 15 shows a diagram of what regions of the geno me recombined f rom what subtype The CRF01_A/E protease gene recombined entirely from Subtype A making the protease sequences the same. Figure 1 15 Graphical description of the circulating recombinant form CRF01_A/E, portions of DNA from several subtypes recombined to give the CRF01_A/E sequence. Portions in red are from Subtype A, those in yellow are from a parent subtype E, and those in white are from an unclassified parent source. Interestingly, no full length genome has been found for a pure subtype E However, because CRF01_A/E is often referred to as Subtype E, and the E designatio n is the common name for the env region of the genome, the current name will not be changed so as to avoid confusion In the future, regions of recombinants for which there is no full length parental strain will be considered unclassifi ed (U), thus a reco mbinant form of Subtype A and an unclassified region would be called CRF01_A / U.

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56 T herapeutic Approaches to HIV 1 Infection and Inhibition of HIV 1 Protease Figure 1 1 6 Points of inhibition within the HIV 1 viral life cycle.

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57 There are several points wit hin the HIV 1 viral life cycle which may be targeted for inhibition in an attempt to treat HIV 1 infection, including the fusion, reverse transcription, integration, and maturation step s, as illustrated in Figure 1 16 There is still no cure for HIV or AI DS, but rather just drugs that simply function to suppress the virus. As of January 2010, there were 31 antiretroviral drugs (ARVs) approved by the U.S. Food and Drug Administration (FDA) to treat HIV infection, grouped into s everal classes based upon poi nt of inhibition within the viral life cycle, given in Table 1 5. These include reverse transcriptase inhibitors of two classes, nucleoside (NRTIs) and non nucleoside (NNRTIs), fusion inhibitors, entry inhibitors, and integrase inhibitors. Patients with HIV/AIDS undergo a treatment known as Highly Active Antiretroviral Therapy, or HAART, which is a combined treatment of one or more inhibitors of the HIV viral life cycle. Table 1 5. FDA approved drugs for the treatment of HIV/AIDS. P Is NNRTIs N RTIs EIs IIs Saquinavir Delavirdine Zidovudine Enfuvirtide Raltegravir Ritonavir Efavirenz Emtricitabine Celsentri Indinavir Nevirapine Lamivudine Nelfinavir Stavudine Amprenavir Abacavir Lopinavir Didanosine Atazanavir Tenofovir Tipranavir Fosamprenavir Darunavir Protease inhibitors target the active site of the HIV 1PR with the objective of binding to the active site tightly enough to prevent binding by the substrate via a mechanism called co mpetitive inhibition. HIV 1 protease inhibi tors were first developed in 1989 by researchers at Hoffmann La R oche Inc. Abbott Laboratories and Merck & Co., Inc. HIV 1 protease inhibitors are u se d in the treatment of patients with HIV and AIDS and can l ower viral load in these patients. They function by specifically binding to the active site by mimicking the tetrahedral

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58 intermediate of the substrate. Currently there are ten FDA approved HIV 1 protease inhibitors used in the treatment of HIV 1 infectio n, including Ritonavir (with trade name Norvir), Indinavir (Crixivan), Lopinavir (Kaletra and Aluvia), Darunavir (Prezista), Saquinavir (Invirase and Fortovase), Tipranavir (Aptivus), Atazanavir (Reyataz), Amprenavir (Agenerase), Fosamprenavir (Lexiva and Telzir), and Nelfinavir (Viracept). Details about the protease i nhibitors, including structural information, are given in Table 1 6. Table 1 6 FDA a pproved protease inhibitors for HIV 1 HAART treatment Inhibitor (Market Name) Abbreviatio n Structure Formula Mol. wt. Solubility Amprenavir ( Agenerase ) APV C 25 H 35 N 3 O 6 S 505.63 DMSO CH 2 OH EtOH CH 2 Cl 2 MetCl Atazanavir (Rey ataz) A TV C 38 H 52 N 6 O 7 802.9 4 5mg/mL H 2 O DMSO EtOH Darunavir ( Prezista ) DRV C 27 H 37 N 3 O 7 S 5 93.73 0 .15mg/mL H 2 O Indinavir (Crixivan) IDV C 3 6 H 47 N 5 O 4 711.88 H 2 O CH 3 OH

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59 Table 1 6. Continued. Inhibitor (Market Name) Abbreviation Structure Formula Mol. wt. Solubility Ritonavir (Norvir) R TV C 37 H 48 N 6 O 5 S 2 720.96 DMSO Chloroform Toluene Saquinavir ( Invirase and Fortovase ) S QV C 38 H 50 N 6 O 5 67 5.10 DMSO CH 3 OH Tipranavir (Aptivus) TPV C 31 H 33 F 3 N 2 O 5 S 602.7 EtOAc Lopinavir (Kaletra and Aluvia) LPV C 37 H 48 N 4 O 5 628.81 DMSO CH 3 OH EtOH CH 2 Cl 2 MetCl Nelfinivir (Viracept) N FV C 32 H 45 N 3 O 4 S 567.78 4.5mg/mL H 2 O

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60 In December 1995, Saquinavir was the first HIV 1 protease inhibitor (and sixth antiretroviral) to gain FDA approval. The drug was manufactured by Hoffman La Roche and was given the brand name Invirase. In 1997 the drug was reapproved under the brand name Fortovase after being reformulated for higher bioavailability. Finally, in 2006 Fortovase was discontinued and Saquinavi r is now given in the form of Invirase coupled with Ritonavir. Unlike most HIV protease inhibitors, Saquinavir inhibits both the protease from HIV 1 and HIV 2 (Collier, Coombs et al. 1996; Noble and Faulds 1996; Vel la and Floridia 1998) Ritonavir, manufactured by Abbott Labs and given the brand name Norvir, was next to receive FDA approval in March 1996. Ritonavir is now most commonly used as a booster of other protease inhibitors, as it also inhibits cytochrome P450 3A4, an enzyme found in the liver which is known to break down protease inhibitors. Thus, when low doses are given in tandem other HIV protease inhibitors, bioavailability of those PIs is increased substantially (Zeldin and Petruschke 2004; Ortiz, Dejesus et al. 2008) Indinavir, manufactured by Merck under the name Crixivan, also received FDA approval in March 1996. When fir st introduced into the market, Indinavir was much more effective in the treatment of HIV/AIDS than any other retroviral known at that time (2008; Wolters Kluwer Health 2008; Hou 2009) Nelfinavir mesylate was next to be approved by the F DA for treatment of HIV in March 1997, making it the fourth HIV protease inhibitor and the twelfth antiretroviral. Nelfinavir, marketed under the name Viracept, was originally manufactured by Agouron Pharmaceuticals, which is now a subsidiary of Pfizer. Like Saquinavir, Nelfinavir also inhibits proteases from both HIV 1 and HIV 2 (Pai and Nahata 1999; Bardsley Elliot 2000; Zhang, Wu et al. 2001; Gills, Lopiccolo et al. 2007)

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61 The fifth HIV protease inhibitor, Ampre navir gained FDA approval in April 1999. The drug was manufactured by GlaxoSmithKline under the brand name Agenerase. Amprenavir is no longer being manufactured. However, the pro drug of Amprenavir called Fosamprenavir also manufactured by GlaxoSmith Kline, obtained FDA approval in October 2003 under the name Lexiva. By taking the pro drug of Amprenavir rather than Amprenavir itself, the body is forced to metabolize Fosamprenavir releasing Amprenavir and thereby increasing the amount of time that the drug is available to the body (Bulgheroni, Citterio et al. 2004) Lopinavir was the next HIV protease inhibitor to receive FDA approval which was granted in September 2000. Lopinavir is marketed and manufactured by Abbott labs as both Kaletra and Aluvia The drug is given with a sub therapeutic dose of Ri tonavir to inhibit CP450 3A4 (Bulgheroni, Citterio et al. 2004; Ortiz, Dejesus et al. 2008) Atazanavir, manufactured by Bristol Myers under the name Reyataz received its FDA approval for the treatment of HIV/AIDS in June 2003. In October 2006 the FDA approved a second formulation designed to lower the number of pills required by the therapy. Atazanavir is also used in co mbination with Ritonavir to boost bioavailability (C lemente, Coman et al. 2006) Tipranavir disodium is manufactured by Boehringer Ingelheim under the name Aptivus and was approved for treatment of HIV/AIDS by the FDA in June 2005. Aptivus is a member of the 4 hydroxy 5,6 dihydro 2 pyrone sulfonamide cl ass, with activity as a non peptide protease inhibitor (Cheonis 2004) It is administered with Ritonavir for improved bioavailabil ity. Notably, Tipranavir has been shown to inhibit proteases that have gained drug resistance to other protease inhibitors, and it has been suggested that resistance to Tipranavir requires numerous drug pressure induced mutations. Unfortunately, however the use of Tipranavir is associated

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62 with an increase in more severe side effects when compared to other protease inhibitors (Larder, Hertogs et al. 2000; Bulgheroni, Citterio et al. 2004; Cheonis 2004; Kandula 2005 ) The most recent HIV protease inhibitor to gain FDA approval is called Darunavir, which was approved for use in June 2006. The drug, marketed as Prezista, was developed and manufactured by Tibotec. Darunavir is a second generation protease inhibitor developed to overcome drug resistance problems associated with other protease inhibitors. It has been shown to be effective on proteases from numerous strains of HIV 1, as well as proteases from previously PI exposed patients who harbor multiple drug res istant proteases. However, the drug costs approximately $9000 for a one year supply (Ortiz, Dejesus et al. 2008) The FDA approved protease inhibitors have shown great promise in the treatment of HIV/AIDS by substantially decreasing mature viral load. Each of these inhibitors were designed with respect to the LAI sequence of Subtype B, and most clinical trials were carried out using Subtype B. As discussed above, however, the high mutation rate of HIV 1 Protease, as with any retrovirus, can render the clinical efficacy of certain i nhibitors with certain subtypes (Rose, Craik et al. 1998; Wlodawer and Vondrasek 1998; Velazquez Campoy, Vega et al. 2002; Clemente, Coman et al. 2006; Coman, Robbins et al. 2007; Sanches, Krauchenco et al. 2007; Ban daranayake, Prabu Jeyabalan et al. 2008; Coman, Robbins et al. 2008) F or this reason it is imperative to understand how other non B subtypes compare to Subtype B in structure, flexibility, dynamics, and kinetics. Drug Resistance in Protease Inhibitor Exposed Patient Isolates of HIV 1 Protease Drug resistance is a major problem concerning the efficacy of HIV protease inhibitors. Because of the high error rate in the production of the DNA intermediate, a high number of natural polymorphisms can become incorporated into the viral genome resulting in numerous

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63 variants even within a single patient. P atients that have been exposed to protease inhibitors often develop further drug induced polymorphisms. Figure 1 17 Regions of amino acid conservation in A) protease inhibitor nave and B) protease inhibitor exposed HIV 1PR. A comparison of HIV 1PR sequences from the Stanford HIV 1 Database of both protease inhibitor nave and exposed patients shows that there are several regions of conservation in the nave protease that lose conservation after prolonged exposure to protease inhibitors. These regions include the flap region, the active site, and the dimerization region (Figure 1 17 ). The active site is required for binding and catalysis of substrate and the flaps are necessary for regulation of accessibility of substrate to the active site. The dimerization domain is required to stabilize the dimer, as the monomer has essentially no activity. Many patterns of drug induced mutations that render resi stance have accumulated, and are recorded in 1 Database. The mutations occurring in the HIV 1 Protease are classified as either primary or secondary mutations. Primary mutations are found in or around the active site and provide the protease with some level of drug resistance Secondary mutations are found outside the active site, often on the periphery of the protease. Secondary mutations,

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64 though they are not found in the active site, provide some level of drug resistance, often in combina tion with p rimary mutations or other secon dary mutations. Multi drug Resistant Patient Isolate MDR769 The multi drug resistant patient isolate called MDR769 was isolated from a patient previously treated with Indinavir (IDV), nelfinavir (NFV), Saquinavir (SQV), and Amprenavir (APV). The protease most often differs from Subtype B by the following polymorphisms: L 10I, M36 V, M46L, I54V, I62V A71V, V82A, I84V, and L90M and each of these polymorphisms has been reported to confer drug resistance to protease i nhibitors (Logsdon 2004) One c rystal structure has been reported in the PDB (1T W7), and i s shown in Figure 1 18 Figure 1 18 Crystal structure of HIV 1PR multi drug resistant patie nt isolate MDR769 (PDB ID 1TW7); A) side and B) side views. Structure rendered with VMD (Humphrey 1996) The 1.8 resolution crystal structure shows an expanded active site cavity and flaps that stay open wider with respect to Subtype B pro tease. Polymorphisms at positions 82 and 84 exchanged V and I residues for smaller A and V residues, respectively, thus lending more space to the active site cavity. An MDR769 crystal s tructure of the protease in com plex with Lopinavir has shown differe nt binding interactions between the inhibitor and the protease, when compared with Subtype B. Addition ally, s urface plasmon resonance (SPR) measurements point to higher k off rates between inhibitor and protease (Logsdon 2004)

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65 Drug Resistant Patient Isolate V6 The drug resistant patient isolate called V6 was isolated from a pediatric patient previously treated with Ritonavir (RTV). V6 generally differs from Subtype B by the following polymorphisms: K20R, V32I, M36I, A71V, V82A, L90M V32I and V82A are primary mutations associat ed with Ritonavir resistance (found in or around the active site), while the rest are non active site polymorphisms. The I84V polymorphisms has been reported to have a 10 fold decrease in catalytic efficiency when compared to protease inhibitor nave Subt ype B (Clemente, Moose et al. 2004) Introduction to Prorenin Hypertension and Its Impact on Soci ety Hypertension is a medical term that describes the condition of having high blood pressure. Blood pressure readings are given as systolic pressure/diastolic pressure, both in units o f millimeters of mercury (mmHg), where s ystolic pressure is the press ure that i s created when the heart beats and diastolic pressure is the pressure inside the blood vessels when the heart is at rest. There are several classifications of hyperte nsion, as shown in Table 1 7 (Chobanian 2003) The American Heart Association reported that in 2008 the percentage of non institu tionalized adults over the age of 20 living with some degree of hypertension was approximately 32%, and approximately 24 ,000 deaths were attributed to hyperten sion in the United States alone (Clemente, Moose et al. 2004) The National Heart, Lung, and Blood Institute (NHLBI) estimated in 2002 that hypertension cost the Unite d States approximately 47 billion dollars (Clemente, Hemrajani et al. 2003) Although a person with hypertension can often experience little or no symptoms, possible symptoms can include headaches, blurred vision, nose bleeds, and dizziness

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66 Table 1 7 Cla ssifications of hypertension and respective systolic and diastolic pressure ranges (Chobanian 2003) Classification Systolic Pressure (mmHg) Diastolic Pressure (mmHg) Normal 90 119 60 79 Pre hypertension 120 139 80 89 Stage I Hypertension 140 159 90 99 Stage II Hypertension Hypertension can be caused by a variety of factors, both genetic and lifestyle induced Approximately 30% of hypertension cases can be contributed to genetic factors, although the genes that contribute to hypert ension have yet to be identified. Abnormalities in the arteries or in the adrenal hormone glands are known to cause hypertension. Additional factors such as ob esity high salt intake, smoking, pregnancy can cause an elevation in blood pressure leading to hypertension (Clemente, Moose et al. 2004) There exist many differen t types of FDA approved treatments for hypertension, including alpha blockers, beta blockers, angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), diuretics, re nin inhibitors, and vasodilators. Renin inhibitors are a relat ively new class of protease inhibitors that function on the juxtaglomerular cells to inhibit the production of renin. Renin, Prorenin and the Renin Angiotensin System The renin angiotensin system (RAS) is considered one of the key modulators of blood v olume, blood pressure, and cardiac and vascular function. As such, many hypertension drugs function by regulating blood pressure at vario us points in the RAS (Danser 2003) Renin also known as angiotensinogenase, is an aspartic protease that plays a vital role in blood pressure regulation by catalyzing the first and rate limiting step in the activation pathway of its substrate angiotensinogen. It cleaves an giotensinogen to form the decapeptide angiotensin I, which is then converted into angiotensin II by angiotensin I converting enzyme. A schematic of this pathway is shown i n Figure 1 19

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67 Figure 1 19 Renin angiotensin system and the s ites of inhibition within th e RAS ; 1) adrenergic antagonists; 2 ) ACE inhibitors; 3 ) renin inhibitors; and 4 ) angiotensin II (AII) receptor antagonists. This significant cascade has an effect on aldosterone release, vasoconstriction, electrolyte imbalance, congestive heart fa ilure, and an increase in blood pre ssure leading to hypertension (Danser 2003; Morris 2003; Marathias, Agroyannis et al. 2004; Schweda and Kurtz 2004; Berecek, Reaves et al. 2005) Because renin is involved in the control of blood pressure through the formation of the vasoactive peptide angiotensin II, the proteins renin, and perhaps prorenin, represent potential therapeutic targets in the regulation and control of hypertension (Danser 2003; Morris 2003) Biosynthesis and Intercellular Processing Human renin in synthesized via two separate pathways, the first is a constitutive pathway for pror enin secretion and the second is a regulated pathway for secretion of mature renin. Renin is synthesized in the granular cells of the juxtaglomerular apparatus in the kidney and secreted in response to several factors, including a decrease in arterial blo od pressure or a decrease in sodium chloride (NaCl) levels. Prorenin is an inactive zymogen of renin, me aning that it has a pro segment on its N terminal end that functions as the activation modulator Prorenin circulates through the plasma until it reac hes the secretory granules, where the pro segment is cleaved and active renin is released (Danser 2003) Interestingly prorenin is found in much higher

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68 conc entrations in the bloodstream than renin, representing a likely enzymatic contr ol mechanism and it is unclear if other biological or physiological roles of prorenin exist. Table 1 8 Pro peptide sequences of several aspartic protease zymogens. Aspar tic Protease Pro Peptide Amino Acid Sequence Human Prorenin LPTDTTTFK R IFLK R MPSI R ESLKERGVDMARLGPEWSQPMKR Mouse Prorenin FSLPTGTTFE R IPLK K MPSV R EILEERGVDMTRLSAEWKVFTKR Rat Prorenin SFSLPTDTASFG R ILLK K MPSV R EILEERGVDMTRISAEWGERKK Bovine Prochymosin AEIT R IPLY K GKSL R KALKEHGLLEDFLQKQQYGISSKYSGF Human Procathepsin D LV R IPLH K FTSI R ETMSEVGGSVEDLIAKGPVSKYSQAVPAVTE Human Pepsinogen C AW K VPLK K FKSI R ETMKEKGLLGEFLRTHKYDPAWKYRFGDL Human Pepsinogen A IMY K VPLI R KKSL R RTLSERGLLKDFLLKKHNLNPARKYFPQWEALPTL Porcine Pepsinogen A LV K VPLV R KKSL R QNLIKDGKLKDFLKTHKHNPASKYFPEAAAL In vitro, conversion of prorenin into active renin appears to be a two step process involving the generation of an interm ediary form of activated prorenin, where the active site is exposed but the pro segment has not yet been proteolytical ly cleaved from the enzyme The first step is thought to be an acid induced activation that occurs as the result of a conformational chan ge in the pro segment. In the inactive form, (based upon structural homology to pepsinogen) the pro segment is expected to be folded over the active site and in tertiary contact with other regions of the protein. At low pH, it is believed that a series of residues become protonated, thus breaking three salt bridges that are implicated in holding the pro segment over the active site and resulting in an unstructured conformation of the pro segment that is no longer in contact with the active site. This confor mer is referred to as the acid activated form of prorenin. This first step has been shown via enzymatic assays to be reversible via a pH switch back to neutral conditions. The second step in the activation of prorenin to renin is the proteolytic removal of the pro segment resulting in active renin (Danser 2003) The three amino acids thought to partake in salt bridge formation are shown to be conserved in n umerous aspartic protease zymoge ns (Table 1 8 ) leading to the hypothesis that perhaps most aspartic protease zymogens function in a similar fashion (Derkx, Schalekamp et al. 1987)

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69 Structure and Function of Prorenin Pepsinogen, the inactive zym ogen of pepsin, is a well studied member of the aspartic protease family. Though no crystal structure exists for prorenin, the crystal structures for pepsin and renin are very similar, also suggesting that the mechanisms of activation are similar. The cr ystal structure of pepsinogen illustrates how the pro segment physically blocks the active site of the enzyme. Figure 1 20. Crystal structures of aspartic proteases A) renin, B) pepsin, C) an overlay of the renin (orange) and pepsin (purple) for compari son of structural similarity, and D) an overlay of renin (orange) and pepsinogen (blue, with green pro segment) for comparison of structural similarity and prediction for positioning of renin pro segment; crystal structure of pepsinogen shows the pro segme nt of pepsinogen (green) physically blocking the active site of the protease. In the case of pepsinogen, it is generally accepted, though there is no direct structural evidence, that acid liberates the pro segment from the active site of the enzyme, matu re pepsin is then generated through an autocatalytic process where the pro segment is cleaved at residue 66

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70 (in preprorenin numbering), leaving a 340 amino acid renin of approximately 37 kDa. Although the pro segment of prorenin is not autocatalytically r emoved like that of pepsinogen, prorenin is thought to behave in a similar manner. Figure 1 20 demonstrates the high level of similarity between the crystal structures of renin and pep sin. Renin is shown Figure 1 20 A pepsin is shown in Figure 1 20 B, and an overlay of the two is shown in Figure 1 20 C. Shown in Figure 1 20 D is an overlay of renin and pepsinogen, the inactive zymogen of pepsin. This provides a model for what the crystal structure of prorenin might look like. Understanding the structur e a nd conformational changes that take place in the activation of prorenin could lead to potentially new avenues for rational drug desi gn in controlling hypertension. Difficulties in Expression and Purification of Prorenin Currently, very little structural da ta on prorenin is available in the literature. This is likely related to the fact that current methods for recombinant bacterial expression of aspartic proteases from Escherichia coli ( E. coli) have been plagued by difficulty. Most aspartic proteases have multiple disulfide bonds; hence these proteins, when cloned using the E. coli system, are usually expressed as inclusion bodies. Inclusion body proteins require that they be denatured and then refolded in order to obtain properly folded, functional prote in. Refolding, however, does not ensure that the protein will be refolded correctly and active ( Nishimori Kawaguchi et al. 1982; Imai, Cho et al. 1986; Kaytes, Theriault et al. 1986; Masuda, Nakano et al. 1986; Lin Wong et al. 1989; Yamauchi, Nagahama et al. 1990; Chen, Koelsch et al. 1991) Prorenin is known to be notoriously difficult to refold thus protein is generally obtained from renal isolation or via expression and secretion from insect cells ; both techn iques are costly and time consuming. Thus, part of my Ph.D. research focused on developing a new method for expression and purif ication of prorenin and the results of that study are reported in Chapter 5 of this dissertation. Prorenin was successfully c loned into a bacterial expression vector, over

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71 expressed in E. coli BL21(DE3) and Origami(DE3) cells as a fusion construct with thioredoxin, and purified in small yield. A CD spectrum was obtained that suggests that the protein was properly folded, and S DS PAGE gel electrophoresis confirmed that the size of the purified protein corresponded with the prorenin thioredoxin fusion construct. An activity assay confirmed that the fusion construct possessed some level of catalytic activity. Numerous CYS mutant s were generated for spin labeling and EPR studies of the motion of the pro segment. However, to date, expression levels remain low. An addition problem that remains involves stability of the zymogen following removal of the thioredoxin fusion tag. Scope of the Dissertation This first chapter of the dissertation, entitled Introduction to Aspartic Proteases served as an introduction to the biological aspects of the research included in this dissertation. As the title of the work implies, the researc h in this dissertation focuses on the aspartic proteases HIV 1 pro tease and prorenin. HIV was introduced as the causative agent of AIDS. HIV and AIDS have become a world epidemic, and a large proportion of medical research focuses on the virus. HAART tr eatment and other therapeutic approaches to treatment are described, followed by a detailed description of the function and structure of HIV 1 protease Conformational ensembles and conformational sampling is described with respect to flap motion and dyna mics. A summar y of previous NMR, ITC, MD and X ray data suggesting that the flaps of HIV 1 protease must undergo a large conformational change to facilitate binding and catalysis of substrate is given Natural and drug pressure selected polymorphisms are introduced and a description of groups, subtypes, circulating recombinant forms, and drug resistant patient isolates are discussed. Next, a discussion of the aspartic protease renin and it s zymogen prorenin is discussed. Renin is an integral part of the renin angiotensin system (RAS), which is a modulator of blood volume, blood pressure, and cardiac and vascular function; therefore, many hypertension drugs function

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72 by regulating blood pressure at vario us points in the RAS The structure of renin and pro renin are discussed and compared to pepsinogen, a well studied zymogen of the aspartic protease pepsin. The problems associated with recombinant bacterial expression of aspartic proteases with pro segments due to the necessity of protein refolding from in clusion bodies are discussed, and were related to the fact that very little structural data on prorenin is available in the literature. Current methods used to obtain prorenin for biophysical studies are described, and most commonly include expression and purification from human embryonic kidney ( HEK ) or Chinese hamster ovary ( CHO ) cells, the baculovirus ( BEV ) system, and isolation from human kidney cells. Project goals, including developing a novel method for recombinant bacterial expression and purifica tion without the need for refolding are described. Chapter 2, entitled Background for Techniques and Methodologies begins by providing a summary of the types of techniques and methodologies performed during the course of this work. The first major secti on of chapter two serves to discuss the theory of recombinant protein expression from E coli and subsequent purification of the target protein. Characteristics of proteins that can be exploited for the purposes of purification, including size, charge, an d binding affinity are detailed, along with the methods that can be utilized based upon each particular characteristic. Circular dichroism spectroscopy was utilized to ensure proper secondary structure of each individual recombinant protein, thus the theo ry and instrumentation of the technique are examined. A large proportion of the work presented in this dissertation deals with site directed spin labeling in conjunction with electron paramagnetic resonance spectroscopy. This chapter details the theory of site directed spin labeling as well as the procedures used in the spin labeling reaction and different types and characteristics of spin labels. Next, the principles of continuous wave and pulsed EPR are discussed, including a condensed mathematical

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73 b ackground, discussion of protein requirements, and detailed discussion of data analysis. Detailed data and error analyses have been developed for the DEER data presented in this dissertation, and those aspects are discussed in great detail. Chapter 3 is entitled Continuous Wave Electron Paramagnetic Resonance Studies of HIV 1 Protease The first part of this chapter gives results of CW EPR studies of subtype F and CRF01_A/E with each of 9 FDA approved protease inhibitors and the non hydrolyzable substrat e mimic CA p2 As described in detail, the CW EPR line shapes do not report on the true motions of the protease flaps as previously determined by other methodologies, thus providing a reason to expand these studies to pulsed EPR ( discussed in Chapter 4 ) The second part of Chapter 3 summarizes the results of a study that utilized site directed spin labeling and electron paramagnetic resonance spectroscopy to monitor the autoproteolysis of active HIV 1 Protease by s ite d irected spin labeling and electron p aramagnetic r esonance s pectroscopy Chapter 4 is entitled Pulsed Electron Paramagnetic Resonance Studies of HIV 1 Protease The first part of this chapter details the results of study designed to compare the apo proteases from each of the si x constructs namely Subtypes B, C, F, CRF01_A/E, and Multi Drug Resistant Patient Isolates V6 and MDR769 Dipolar modulated echo data was used to extract information regarding the conformational ensembles of the protease flaps. Interestingly, the distance profiles for each of th ese constructs differ substantia l l y from one to the next. Because the structures of these proteases are not extremely different, these results are of great importance. This work summarizes a publication in the Journal of the American Chemic al Society entitled 1 p rotease v arian ts c onfers a ltered f lap c onformations and f lexibility, by Kear et al (Kear, Blackburn et al. 2009) The next part of this chapter examines the

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74 results from DEER studies of CRF01_A/E w ith each of 9 FDA approved protease inhibitors and the non hydrolyzable substrate mimic CA p2 Chapter 5 is entitled Soluble Expression and Purification of Multiply Disulfide Bonded Prorenin fr om Escherichia coli. The work described in this c hapter details the setup and study of an innovative system for expression and purification of prorenin. Thioredoxin fusion methodology is utilized in an attempt to circumvent inclusion body formation in t he production of soluble, properly folded protein. Also included are the results of cysteine mutagenesis in creating several DNA constructs for potential electron paramagnetic resonance studies to examine the conformational change of the pro segment assoc iated with activation of the zymogen to the active protease. Chapter 6, entitled Conclusions and Future Directions provides a brief summary of the conclusions drawn from the data presented within this dissertation. Following these conclusions is a sum mary of several future studies that must be done to provide a thorough interpretation of the data discussed herein. Our early spectroscopic results on HIV 1PR are both exciting and promising, but there is a multitude of work still to be done.

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75 CH APTER 2 BACKGROUND FOR TECHN IQUES AND METHODOLOG IES Introduction Several techniques were utilized herein including protein expression and purification circular dichroism (CD), site directed spin labeling continuous wave (CW) electron paramagnetic resona nce spectroscopy (EPR), and the pulsed technique Double Electron Electron Resonance (DEER) also known as pulsed electron double resonance (PELDOR). General overviews to these methods are given in the following sections. Additionally, each research chapt er has a detailed materials and methods section for all experiments contained within. Setting up a Protein Expression System for E scherichia coli T he first step in the characterization or study of a protein is to either isolate the protein from natural sou rces or to over express the target protein from a recombinant source such as Escherichia coli (E. coli) Recombinant expression involves incorporation of the DNA (or gene) encoding the protein of choice into a double stranded DNA vector which may then be transformed into a bacterial cell for transcription and translation by cellular machinery. After over expression p roteins can be separated from unwanted cellular components and other non target proteins based upon numerous physical characteristics inclu ding size, charge or isoelectric point, and binding affinity to various solid phases. Numerous considerations must be made when setting up a bacterial expressio n system for a target protein, including how to obtain the DNA, what type of bacterial cells to use, and what expression vector to use. These are all questions that must be carefully deliberated prior to setting up a protein expression system and each of these considerations will be discussed in detail in the following pages. The first considera tion when set ting up a protein expression system in E. coli regard s the DNA sequence to be used as a template for the target protein. If your laboratory does not have

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76 access to a DNA synthesizer, numerous companies such as DNA2.0 ( https://www.dna20.com ) for example, are available to synthesize the gene of choice at a fai r cost. Synthetic genes have the added advantage that they can be optimized to enhance expression focusing on characteristics required by the express ion system and host. Synthetic genes can be optimized for codon bias and are free of unwanted regulatory elements such as pause or stop loops, which may hinder protein expression from a non native host. If the target protein is not a natural E. coli prote in, codon optimization may need to be considered Because there are 64 possible codons and only 20 naturally occurring amino acids, most amino acids are en coded for by numerous codons but most organisms prefere ntially synthesize transfer RNAs ( tRNAs ) I t is therefore imperative to compare the codon usage of E. coli with that of the organism from which the target protein is derived, and synthesize the gene accordingly Without codon optimization, bacterial expression levels of eukaryotic proteins may be v ery poor. Table 2 1 details differential codon usage in Homo sapiens and E. coli cells. Table 2 1. Differential codon usage in Homo sapiens and E. coli cells Codon Amino acid H. sapiens Codon Usage E. coli codon usage TTT Phenylalanine 0.4 3 0.57 TTC Phenylalanine 0.57 0.43 TTA Leucine 0.06 0.15 TTG Leucine 0.12 0.12 CTT Leucine 0.12 0.12 CTC Leucine 0.20 0.10 CTA Leucine 0.07 0.05 CTG Leucine 0.43 0.46 ATT Isoleucine 0.35 0.58 ATC Isole ucine 0.52 0.35 ATA Isoleucine 0.14 0.07 ATG Methionine 1.00 1.00 GTT Valine 0.17 0.25 GTC Valine 0.25 0.18 GTA Valine 0.10 0.17 GTG Valine 0.48 0.40 TCT Serine 0.18 0.11 TCC Serine 0.23 0.11

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77 Table 2 1. Continued. Codon Amino acid H. sapiens Codon Usage E. coli codon usage TCA Serine 0.15 0.15 TCG Serine 0.06 0.16 CCT Proline 0.29 0.17 CCC Proline 0.33 0.13 CCA Proline 0.27 0.14 CCG Proline 0.11 0.55 ACT Threonine 0.23 0.16 ACC Threonine 0.38 0.47 ACA Threonine 0.27 0.13 ACG Threonine 0.12 0.24 GCT Alanine 0.28 0.11 GCC Alanine 0.40 0.31 GCA Alanine 0.22 0.21 GCG Alanine 0.10 0.38 TAT Tyro sine 0.42 0.53 TAC Tyrosine 0.58 0.47 TAA **stop** 0.22 0.64 TAG **stop** 0.17 0.00 CAT Histidine 0.41 0.55 CAC Histidine 0.59 0.45 CAA Glutamine 0.27 0.30 CAG Glutamine 0.73 0.70 AAT Asparagine 0.44 0.4 7 AAC Asparagine 0.56 0.53 AAA Lysine 0.40 0.73 AAG Lysine 0.60 0.27 GAT Aspartate 0.44 0.65 GAC Aspartate 0.56 0.35 GAA Glutamate 0.41 0.70 GAG Glutamate 0.59 0.30 TGT Cysteine 0.42 0.42 TGC Cysteine 0. 58 0.58 TGA **stop** 0.61 0.36 TGG Tryptophan 1.00 1.00 CGT Arginine 0.09 0.36 CGC Arginine 0.19 0.44 CGA Arginine 0.10 0.07 CGG Arginine 0.19 0.07 AGT Serine 0.14 0.14 AGC Serine 0.25 0.33 AGA Argin ine 0.21 0.02 AGG Arginine 0.22 0.03 GGT Glycine 0.18 0.29 GGC Glycine 0.33 0.46

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78 Table 2 1. Continued. Codon Amino acid H. sapiens codon usage E. coli codon usage GGA Glycine 0.26 0.13 GGG Glycine 0.23 0.12 For the purposes of sub cloning the gene of choice into an expression vector, the gene must be flanked by two different restriction sites which are typically pali n dromic Restriction sites are sequences in the DNA that are recognized by restric tion enzymes, which are endonucleases found in bacteria. An example of a common restriction enzyme is BamHI, which where the apostrophe denotes the site of cleavage. Bam HI cleaves after the first G on each strand of a double stranded DNA sequence containing the restriction site, leaving an overhang on each strand (GATCC). Overhangs created by restriction sites are single stranded unpaired regions which can be ligated to DNA sequences containing complementary overh angs. Over 600 different restriction enzymes are commercially available (Roberts, Vincze et al. 2007) Restriction sites should be chosen based upon the sites available in the expression vector. It is important, however, to ensure that the chosen restriction sites are not found naturally in the gene. An expression vector is generally a double stranded circular DNA plasmid which is a n on chromosomal piece of DNA which can replicate in dependently from the chromosome. Plas mids used as tools for gene expression are often referred to as ve ctors. For this purpose, the gene of interest is inserted into the vector by a process called ligation; and the modified vector is subsequently inserted, or transformed, into a host cell. Expression vectors must possess four necessary components, the fi rst of which is called the multiple cloning site (MCS), or polylinker region. The MCS contains numerous restriction sites for insertion of the gene of i nterest into the expression vector The second necessary component of an expression vector is an antib iotic resistance gene which is used for selection of transformed

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79 host cells. The bla gene, which encodes TEM1 lactamase is one of the most common ampicillin resistance ( AmpR) markers used in molecular biology The third essential component of an expression vect or is the origin of replication, or site within the vector at which replicat ion is initiated. Structure of the origin of replication varies, but all origins of replication have high A denine T hymine (AT) content. A protein complex called the pre replication complex binds the origin of replication, unwinds the DNA, and begins the replication complex. A c ommon example of the origin of repli cation in expression vectors is called ori Large plasmids may require more than one origin of replication in order for timely replication to occur (Baker and Wickner 1992) The final necessary component of an ex pression vector is the promoter, or the region of the gene in which RNA transcription begin s. Certain promoters are constitutive meaning that they function all the time. It is advantageous, however, for promoters on expression vectors to be inducible, not constitutive A common type of inducible pr omoter is the T7 promoter, which is known as a strong promoter. A n example of an expression vector map with each of the four required features is shown in Figu re 2 1 A myriad of commercial vectors are available, each with particular purposes and characte ristics. C ertain vectors can be used to tag a protein for ease of purification or for identification via Western Blotting C ommon tag s for this purpose are the 6 His tag and 10 His tags in which 6 or 10 successive His residues are attached to either the N terminal or C terminal end of the protein to provide a means for purification via nickel affinity chromatography. The vectors used for the research in this dissertation are called pET vectors, which is named for p lasmid for e xpression by the T 7 RNA pol vectors, each with different characteristics; those chosen for use herein are called pET32a(+) and pET23b. Specific vectors will be described in more detail in future chapters.

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80 Figure 2 1. Example vector ma p highlighting necessary features of plasmid, including inducible promoter, origin of replication antibiotic resistance gene, and the multiple cloning site. Another important consideration when setting up a protein expression system for E. c oli is the ch oice of E. coli strain The prototype expression strain is ca lled BL21(DE3), with genotype F ompT gal dcm lon hsdS B (r B m B T7 gene 1 ind1 sam7 nin5]). BL21(DE3) contains the T7 expression cassette, which is a vector carrying a promoter sequence, an open reading frame (ORF) corresponding to the T7 RNA polymerase, the LacI gene, and a polyadenylation site The lacI gene encodes a repressor that binds the promoter when lactose (or often a lactose analog or derivative) is not present and transcription occurs only at basal levels. Isopropyl D 1 thiogalacto pyrano side ( IPTG ) is a common lactose analog (Fig ure 2 2) used in molecular biology. When added, IPTG binds to the repressor changing its shape and preventing it from binding to the promoter such that T7 RNA polymerase transcription can proceed. IPTG has the advantage over lactose that it cannot be me tabolized by E. coli and thus its concentration and concurrent rate of expression, remains constant Standard E. coli cells do not produce T7 RNA polymerase ; thus plasmids controlled by a T7 promoter have repressed expression until induction with IPTG.

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81 Figure 2 2. Structures of A) lactose and B) the lactose analog IPTG. Other strains of E. coli cells discussed within the scope of this dissertation are the Origami B (DE3) (taxon identifier 469008) and BL21(DE3)pLysS strain s. BL21(DE3)pLysS cells are ba sed upon the BL21(DE3) strain, but also contains the pLysS plasmid, which carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target genes but does not interfere with level s of expression post induction with IPTG This type of cell is necessary in order to produce proteins that are toxic to the E. coli cell (such as active HIV 1 Protease). B L21(DE3)pLys S cells are of the genotype BL21(DE3)pLysS F omp T, hsd S B (r B m B ), dcm gal (DE3), pLysS, Cm r Finally, Ori gami B (DE3) cells of the genotype F ompT hsdSB(rB mB ) gal dcm lacY1 aphC (DE3) gor522 Tn10 trxB (KanR, TetR), are E. coli strain K 12 derivatives that have mutations in both the thioredoxin reductase ( trxB ) and glutathione reductase ( gor ) genes. Muta tions in these genes function to enhance disulfide bond formation in the cytoplasm. Inclusion Body Isolation and Protein Refolding Eukaryotic cells utilize a process called secretion to synthesize proteins and release them to the external environment. Pr oteins are synthesized on ribosomes on the rough endoplasmic reticulum (ER) (Figure 2 3) whi ch has a pH of approximately 7 As the proteins are translated,

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82 they are translocated to the endoplasmic reticulum ( ER ) lumen for post translational modificati ons such as glycosylation, and chaperone proteins assist in proper protein folding. Properly folded proteins are packaged in vesicles and trafficked to the Golgi apparatus (pH 6.5) for further post translational modifications, then the proteins are packaged into secretory vesicles (pH 5.0 6.0) which fuse with the cell membrane thus releasing the properly folded, mature proteins into the exterior environment (Anderson 2006) Figure 2 3 Internal structures of A) prokaryotic and B) eukaryotic cells demonstrating the drastic differences between the two and the lack of compartmentalization in the prokaryotic cell as opposed to the h igh level of structure within the eukaryotic cell ; 1) nucleolus, 2) nucleus, 3) ribosome, 4) vesicle, 5) rou gh endoplasmic reticulum (ER), 6) Golgi apparatus, 7) c ytoskeleton, 8) smooth ER, 9) mitochondria, 10) vacuole, 11) cytoplasm, 12) lysosome, 13 ) cen trioles Figures obtained from Wikipedia and were free for copy and redistribution. Conversely, prokaryotic cells have a reducing, non compartmentalized internal environment. As a result, over expression of non bacterial proteins in a prokaryotic syste m often results in misfolded and aggregated protein Oftentimes when this occurs, the proteins accumulate in what are called inclusion bodies. Inclusion bodies are isolated as an insoluble component following cell lysis and washed with a series of wash b uffers designed to further purify the inclusion bodies away from non protein cellular components and certain non target proteins. The inclusion bodies are then solubilized by a denaturing solution such as urea, then

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83 further purified and subsequently refol ded. Though sometimes protein denaturation is irreversible, protein r efolding can sometimes be carried out by slow, drop wise addition to a solution that promotes folding. This solution is often a buffer, a weak detergent, or a dilute acid or base, and v aries from protein to protein. Common Methods of Protein Separation Introduction Protein purification is defined as the separation of a target protein from all non target proteins and other cellular components. Proteins possess many characteristics which can be exploited for the purposes of purification, including size, charge, and affinity to various ligands and solid supports. Described in the following sections are some of these techniques For a more comprehensive discussion, readers are referred to many great review articles and textbooks available for reference (Scopes 1994; Rosenberg 2005) Several steps, chromatographic or otherwise, often need to be applied in turn to successfully purify the protein of interest. The idea l purification scheme varies from protein to protein, as the physical characteristics of each protein differ. Various purification schemes, including buffer compositions and ionic strengths, protein concentrations, and type and order of chromatographic steps are often evaluated before an ideal scheme can be determined. Separation Based Upon Size Proteins can be separat ed based upon size using several techniques. Size exclusion chromatography requires a long, narrow column tightly packed with a porous resin. As the protein sample is introduced onto the column and moves down, smaller proteins will move through the pores in the resin, while larger proteins bypass the pores and move between the resin beads, meaning the pathlength of larger proteins is smaller than that of smaller proteins, thus the elution time is smaller. Hence, proteins will elute from the column with d e creasing size.

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84 A second protein separation method based upon size is SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) This method is most often used to determine the level of purity of a sample, but can be used to isolate a partic ular protein. A protein mixture is exposed to sodium dodecyl sulfate ( SDS ) which is a detergent that applies a negative charge to (Figure 2 4) most often causes denaturation of the secondary and tertiary structures of the proteins (with th e exception of disulfide bonds); however, depending on the inherent structure of the protein, boiling of the sample can also be required to ensure that the proteins will be separated based solely up on size with little to no influence by structure or charge. An electric field is applied to the SDS PAGE gel, causing the negatively charged proteins to move through the polyacrylamide gel matrix with a speed dependent upon the size of the protein as a r esult of the amount of resistance encountered, resulting in separation based upon size. Figure 2 4. Structure of the detergent sodium dodecyl sulfate. Separation Based Upon Charge or Isoelectric Point Ion exchange chromatography can be carried out by via two different techniques, namely anion exchange or cation exchange. Anion exchange chromatography involves binding a negatively charged protein to a positively charged stationary phase within a column. Conversely, cation exchange chromatography invo lves binding a positively charged protein to a negativ ely charged stationary phase within a column. The rest of this section will provide details on anion exchange chromatography. To ensure that the protein of interest carries a net negative charge, the mobile phase must be at a pH higher than the isoelectric point (pI) of the protein of

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85 interest and have a low ionic strength Typical resin used for anion exchange chromatography is called Q resin, which is bound to a quaternary amine that carries a posit i ve charge as shown in Figure 2 5 As the protein sample is applied to the anion exchange column, proteins with a net negative charge will bind via an ionic bond and neutral or positively charged proteins will come out in the flow through and wash Often a gradient of increasing ionic strength can then be applied to remove the proteins bound within the column. Ion exchange chromatography is generally a quite effective method of protein purification and thus is often used as a primary purification step. Figure 2 5 Anion exchange Q resin bound to a positively charged quaternary amine. Separation Based Upon Binding Affinity Affinity chromatography is an effective method of protein purification that separates proteins based upon specific interaction s or affinities to various compounds retained via a solid phase. A common example is immobilized metal ion affinity chromatography (IMA C), where the stationary phase con tains a resin with a chiral chelating group such as nitrilotriacetic acid (NTA) or imi nodiacetic acid (IDA) that is subsequently charged with a metal such as nickel, copper, or cobalt (Figure 2 6). Figure 2 6. Structures of A) nitrilotriacetic acid and B) iminodiacetic acid

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86 P roteins that were expressed wi th an N terminal or C terminal His tag have an affinity for these metals and will be retained within the column and proteins that have no natural affinity for the chelated metal ions will be removed with the flow through or wash Finally, proteins bound within the colum n can be releas ed using a pH gradient, or a competitive binder or metal ion ligand, such as imidazole which can be applied to the column over an increasing gradient of concentration. Circular Dichroism Spectroscopy Circular dichroism (CD) spectroscopy is a technique use d to observe differences in absorption of left and right handed circular polarized light by optically act ive materials as a function of wavelength Circularly polarized light is, by definition, chiral, and thus it interacts differently with chiral molecul es. Circularly polarized light is defined by a n angle of ( Figure 2 7) where E R and E L are the magnitudes of the electric field vectors of right and left circularly polarized light, respectively. As a result, after passage through the sample, elliptical polarized light is measured to provide wavelength dependent differential absorption Figure 2 7. Elliptical polarized light (purple) is composed of unequal contributions of right (blue) and left (red) circular polarized light. Figure adapted from Wikipedia. When being used to identify secondar y structural elements in proteins CD spectroscopy is carried out in the far UV regions of approximately 180 260 nm where the chromophore is the peptide bond. Left and right handed circularly polarized light will be absorbed to different e xtents by the chiral amino acids, thus a CD signal will arise when the peptide bond is in a

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87 sheet. CD spectroscopy in the far UV will yield different spectra for different types of secondary structure in peptides and prote ins ( Figure 2 8) ; thus, analysis of CD spectra can yield useful information regarding structure of a protein. Often, the raw data reported by the CD instrument is in the units of millidegrees ( ) The units in which CD results are typically reported are m ean residue ellipticity [ ] (deg cm 2 dmol 1 residue 1 ), as shown in Equation 2 1, (2 1) where [ ] is the mean residue ellipticity, is the ellipticity, M r is the protein molecular weight, c is the protein concentration (in mg/mL ), l is the cuvette path length and N A is the number of amino acids in the protein. Figure 2 8 Sample circular dichroism spectra for sheet and random coil To accurately measure the secondary structura l ensemble of a protein sample, several factors must be considered in sample preparation. T he protein sample needs to be as pure as possible since non target protein, particulate matter, or misfolded protein will contribute to the CD signal. Ideal protei n concentrations are typically in the range of 0.5 mg/mL. Buffers,

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88 detergents, or other additives should not absorb in the region of interest (260 180 nm). Buffers should typically be at or below 5 mM, or as low as possible while maintaining the stabil ity of the protein. CD spectroscopy only requires small amounts of protein is non destructive to the sample, and does not require difficult or extensive data processing. In addition, even small changes in overall secondary structure can be monitored very accurately. The drawback, however, is that even though secondary structure can be determined, it cannot be determined what regions of the peptide or protein contains what elements of the secondary structure, thus CD cannot be used to definitively determi ne i f a protein is properly folded and should only be used as evidence of the contrary. CD gives far less specific structural information than other techniques such as NMR or X ray crystallography. With respect to proteins, CD provides little information on membrane proteins, as lipids and other membrane structures cause light scattering and are thus unreliable. Sit e D irected Spin L abeling Introduction Site directed spin labeling (SDSL) is a technique utilized to inco rporate a paramagnetic tag into a protein or other biological macromolecule at a specific site, often an engineered cysteine residue, to facilitate electron paramagnetic resonance (EPR) studies (EPR will be discussed in detail in the following section) (Griffith and McConnell 1965; Stone, Buckman et al. 196 5) SDSL in conjunction with EPR is a well suited technique for studying the conformations and conformational changes of proteins. A unique, reactive CYS residue is incorporated into a protein using a technique called site directed mutagenesis, in whi ch the DNA is manipulated such that a codon for CYS is positioned at a chosen point within the sequence. Upon protein expression, the modified DNA sequence results in a CYS substituted protein construct which can then be modified with a spin label. Spin

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89 labels are incorporated into a protein via a thiol based chemistry reaction, of which a general reaction scheme for incorporation of (1 oxyl 2,2,5,5 tetramethyl 3 pyrroline 3 methyl)methanethiosulfonate (MTSL) is shown in Figure 2 9. The spin label ed sid e chain can easily be accommodated by most sites within proteins ( especially when that site is aqueous exposed ) and has been shown t o be no more perturbing than most other single amino acid substitution s The resulting modified CYS residue is often refer red to as R1. Figure 2 9. S ite directed spin labeling scheme showing the reaction of MTSL with a free thiol group of an engineered CYS residue. Conformational changes can be easily monitored with conventional EPR spectrometers as the EPR nitroxid e spectral line shape is sensitive to local secondary structural elements, local dynamics, and conformational changes. The EPR spectra of R1 are very sensitive to changes in secondary structure, which provides an accurate detection of con formational chang es in proteins (Hubbell and Altenbach 1994; McHaourab, Lietzow et al. 1996; Hubbell, Gross et al. 1998; McHaourab, Kalai et al. 1999; Hubbell, Cafiso et al. 2000; Columbus, Kalai et al. 2001; Columbus and Hubbell 20 02; Fanucci and Cafiso 2006) Three types of motion can be detected using the SDSL/EPR technique: the intrinsic motion of the spin label, the backbone flexibility in the region of the R1 label, and the overall t umbling of the molecule in solution Exper imental parameters such as temperature, viscosity, or modified spin label side chains, can be adjusted such that individual modes of moti on can be studied more directly (Hubbell and Altenbach 1994; McHaourab, Lietzow et al. 1996; Hubbell, Gross et al. 1998; McHaourab, Kalai et al. 1999; Hubbell, Cafiso et al. 2000; Columbus, Kalai et al. 2001; Columbus and Hubbell 2002; Fanucci and Cafiso 2006)

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90 Choice of Spin Label Dr. Luis Galiano while a graduate student, e xamined the a ffect of the four different spin labels, namely (1 Oxyl 2,2,5,5 Tetramethyl Pyrroline 3 Methyl) Methane thiosulfonate (MTSL), 4 Maleimido TEMPO (MSL), 3 (2 Iodoacetamido) PROXYL ( IAP), and 4 (2 Iodoacetamido) TEMPO (IASL) the structures of which are shown in Figure 2 10 on the CW EPR nitroxide spec tral line shape of HIV 1PR Subtype Bsi Figure 2 10. Structures of A) M T SL, B) MSL, C) I ASL, and D) IAP. From the structures of each of the spin labels, it is clear that each one contains very different structure, with respect to size and mobility of head group, rotable bonds, and degrees of freedom. Each of these spin labels are nitroxide based where the nitroxide head group is either a five or six membered ring with four methyl groups to help defer collision induced reactions with the radical. E ach of the spin labels have a flexible linker, or group of atoms that link the C of the CYS residue, of between four and six bonds. MSL clearly has the bulkiest head group, leading to its more restricted, anisotropic EPR line shape. On the other extreme, IASL has the most degrees of freedom with respect to rotable bonds, leading to its highly mobile, isotropic line shape. Additionally, however, one must consider reversibility of attachment to the protein backbone. MTSL, for example, m akes a S S bond with the free thiol of

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91 a CYS residue. Under certain conditions, the disulfide bond linking the spin label to the protein may be reduced, liberating free spin label that could complicate EPR line shape analysis. On the other hand, MSL make s a non reducible C S bond with a free thiol of a CYS residue. T he changes inferred upon the line shapes are due to the differences in inherent spin label mobility. That data was reported in his 2008 dissertation and is shown in Figure 2 11 as derivative spectra (Galiano 2008) All things considered, two different spin labels were ultimately chosen for use in this work, namely MTSL and MSL. Figure 2 11. Effect of choice of spin label on the derivative EPR line shape of H IV 1PR Subtype B si with A) MTSL, B) MSL, C) IASL, and D) IAP. All spectra were collected at X band frequency with 100 Gauss sweep width. Spin Label Conformations and the 4/ 5 M odel for MTSL The intrinsic motions and conformations of the spin label are of important consideration, as they contribute to the mobility of the EPR nitroxide spectral line shape. Here, we will talk specifically about the 4/ 5 model for the MTSL label, which was developed in the laboratory of

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92 Dr. Wayne Hubbell at UCLA and is based upon the spin label positioned in a solvent exposed helix in T4 Lysozyme (Langen, Oh et al. 2000) As discussed previously, MTSL has a linker region with five theoretically rotable bonds. However, Hubbell et al. and limiting rotational degrees of freedom to the fourth and fifth rotable bonds, labe led 4 and 5 in the Hubbell model. Of course, this should only be taken as a model since each individual protein has the potential to alter the intrinsic mobility of the label. Figure 2 12 graphically illustrates the theory behind the 4/ 5 model. Figure 2 12. Graphical representation of the 4/ 5 model for the MTSL label, which was developed in the laboratory of Wayne Hubbell at UCLA and is based upon the spin Continuou s Wave Electron Paramagnetic Resonance Spectroscopy Introduction Electron paramagnetic resonance (EPR) spectroscopy also known as electron spin resonance (ESR) spectroscopy is the study of absorption of microwave radiation by a paramagnetic species in th e presence of a n external magnet ic field The unpaired electron has a net dipole due to a magnetic moment that arises predominantly from spin angular momentum of quantum number m = In the simplest case of the free electron s magnetic moment is degenerate, meaning th at the energy of the two spin st ates (m s = + and m s = ) is equal.

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93 However, when a magnetic field is applied, the electron can align itself either parallel or antiparallel to the field in what is known as the Zeeman Effect The Zeeman Equation ( 2 2) describes the diffe rence between the energy levels E and E where g is the spectroscopic g factor (equal to approximately 2 for most samples), e is the Bohr magneton, and B is the strength of the applied magnetic field. The Bohr magneton (9.274x10 24 JT 1 ), is a proportionality c onstant defined in Equation 2 3, where e is the electric charge is the Plank constant divided by 2 (1.054 10 34 Js), m e is the mass of the electron (9.109 10 31 kg) (Weil, Bolton et al. 1972; Poole 1983) Energy is absorbed, i.e. resonance occurs, when the applied energy is equal to the difference in energy levels E and E ; this is achiev ed by maintaining a constant frequency and sweeping the magnetic field. The energy diagram describing this graphically i s given as Figure 2 13A, while F igure 2 13B shows a typical derivative absorption spectrum for a free electron in solution (Weil, Bolton et al. 1972; Poole 1983) e B (2 2 ) e = e m e (2 3 ) When EPR is performed in conjunction with SDSL, the free electron ( m s = ) on the nitroxide spin label couples with the nuclear spin from nitrogen ( m I = 1) via the hyperfin e interaction. As such, both levels E and E split into three Hyperfine energy levels (based upon the 2I + 1 splitting rule), providing the system three allowed energy transitions. The energy diagram and corresponding derivative a bsorption spectrum is given in Figure 2 14 (Weil, Bolton et al. 1972; Poole 19 83)

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94 Figure 2 13. A ) Energy level diagram for a free electron in an applied magnetic field and B ) c orresponding derivative of absorption spectrum. Figure 2 14 A) Energy diagram for a system with a free electron ( m s = ) undergoing hyperfine int eraction with the nucleus of nitrogen ( m I = 1), and B) r epresentative derivative EPR spectrum for a nitroxide spin label. All EPR work described in this dissertation was collected at X band, for which resonance occurs at a magnetic field of approximate ly 3480 Gauss and 9.75 GHz. A list of fields for resonance at varying microwave frequencies commonly available in EPR is given in Table 2 2.

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95 Table 2 2. Common microwave bands and frequencies used in CW EPR. Band Frequency (GHz) B r esonance (Gauss) S 3.0 1070 X 9.75 3480 Q 34.0 12000 W 94.0 34000 Nitroxide S pectral L ine S hapes The EPR spectral line shape is highly sensitive to motion in th e environment of the s pin label and thus changes dramatically with changes in correlation time as shown in Figure 2 15 (Hubbell, Gross et a l. 1998; Hubbell, Cafiso et al. 2000; Fanucci and Cafiso 2006) For sites capable of u ndergoing fast isotropic motion with a low rotational correlation time, the spectra show sharp, narrow peaks (top). As motion becomes more restricted and correlation ti me increases, resonance peaks become increasingly less sharp, and significant line broadening occurs, as can be seen in the intermediate, slow (middle) and rigid spectra (bottom). Figure 2 15. Dependence of EPR spectral line shape on motion. Correlat ion time is defined by the level of a convolution of mobility, and can be broken R ), the rate at which the protein is tumbling in solution. This rate is modulated by the bulkiness of the protein, as well as experimental facto rs such as temperature, viscosity, and the presence of solutes. The

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96 i ), which can is defined by torsional oscillations within the spin label, as well as bulkiness of t he spin label head group and the degrees of freedom separating the nitroxide moiety and the carbon of the CYS residue to which the nitroxide moiety is attached as discussed in previous sections. The intrinsic spin label mobility can be experimentally d etermined to a certain extent by repeating experiments with nitroxide spin labels of varying size and flexibility. The third and final class of mobility as seen by the EPR spectral line shape is defined by local dynamics and backbone B ). Th is type of mobility is modulated by changes in secondary and tertiary structure, and well as conformational changes within the protein (McHaourab, Lietzow et al. 1996; Columbus, Kalai et al. 2001; Columbus and Hubbell 2002) Protein Requirements for CW EPR T h e CW EPR methodology requires nanomole quantities of spin label ed protein. T ypical sample sizes are 3 10 microliters of 50 200 M protein for a loop ga p resonator used at X band frequencies These values are true for all work reported within. CW EPR D at a A nalysis CW EPR line shapes can be analyzed both qualitatively and quantitatively to provide information regarding the motional ensemble of the spin labeled protein. The overall breadth and width of EPR spectral features are narrowed by molecular motion s, and as such, the line shape can be analyzed in terms of empirical parameters. Most commonly analyzed are the peak to peak width of the central resonance line, called H pp the second moment (), and the normalized resonance line intensities (I LF I CF I HF ) These parameters have been shown to correlate with protein secondary structure and can be used to characterize pr otein conformation and dynamics.

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97 is defin ed as the dist ance in Gauss between the minimum and maximum of the central resonance line of the EPR spectrum. An EPR spectrum resulting from fast, isotropic motion of a highly mobile spin label with short correlation time contains sharp, narrow resonance lines As motion becomes more restricted and /or anisotropic and the correlation time increases the EPR line shape becomes increasingly broad ened Thus, the value of increases as the overall motion of the system decreases and /or anisotropy increases. For all work this allows for a more precise calculation since all s pectra are collected over 1024 points. Another empirical parameter that is frequently analyzed is called s caled mobility Scaled mobility can be calculated by normalizing using spectral line widths of the most mobile and immobile proteins in order t o compare from data collected on different instruments, and is given in Equation 2 4 i m exp and experimental EPR spectra, respectively (Hubbell, Cafiso et al. 2000) (2 4 ) Resonance line peak intensities can be compared (when EPR spectra are area normalized with respect to the number of spins ) as a way to compare mobility of two or more spin labeled samples. Most commonly, either the high field resonance intensity (I HF ) or the low field/ center field resonance intensity ratio (I LF /I C F ) are used. As motion becomes more restric ted and anisotropic, the EPR line shape becomes increasingly broad and the intensity of each of the high ( 1) center (0) and low (+1) field resonances decrease. A s motion in the system increases, the high field resonance intensity and the ratio of the l ow field/center field resonance will increase. Calculation of s econd moment is more complex than previously described spectral parameters.

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98 (2 5) A precise baseline correction is necessary, and asymmetric spectra require a correcti on factor to account for the asymmetry. The n th moment is given by Equation 2 5 where H 0 is the center field, H j H j 1 is the step size, H j is the field value for any point j and y j is the intensity at point j. Figure 2 16 shows a graphical representa tion of the determination of these parameters. Figure 2 16. Pictorial representation of the determination of common spectral parameters A) H pp B) I LF I CF and I HF and C) second moment (). Pulsed Electron Paramagnetic Resonance Spectroscopy Introduction Pulsed EPR was pioneered by W. B. Mims in the 1960s and has since become a valuable tool used in a wide variety of applications. The specific pulsed technique utilized in this research is called pulsed electron double resonance (PELDOR), or double electron electron resonance (DEER). DEER experiments use two different microwave frequencies to measure the strength of the coupling betwee n two electron spins; in our case, to measure the distance between two nitroxide spin labels. This technique can be describe d by a simplified Ha miltonian, given in Equation 2 6 comprised of three sets of terms: the Zeeman terms for the spin subsets A and B, and the dipolar coupling term that relates the two, where A S z A is the Zeeman term for the spin

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99 subset A, B S z B is the Zeeman term for the spin subset B, and ee Sz A z B is proportional to the dipolar coupling between spins. AB = A S z A + B S z B + ee Sz A z B (2 6 ) All pulsed EPR work reported in this dissertation made use of the common 4 pulse DEER sequence at X band frequencies. The pulse sequence is shown in Figure 2 17 The echo sequence followed by a r efocusing 180 pulse ( ) This channel is applied at a frequency (1) which corresponds to the low field resonance that lays approximately 26 Gauss or 72 MHz below the central resonance, shown in Figure 2 18. Figure 2 17. The 4 pulse DEER sequence. (1) is comprised of a Hahn echo sequence followed by a refocusing at the low field resonance approximately 26 Gauss or 72 MHz below the central ( 2 ) at the location of the center resonance. In the experiment, the Hahn Echo sequence is used to produce an echo for the spins in resonance with the microwave frequency (1) the magnetization in the XY 1 1 this sequence will give rise to an echo lined up 2 ho. Table 3 describes the standard 4 pulse table used for DEER experiments described within.

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100 Figure 2 18. Absorption spectra for a nitroxide spin label with positions of the low field ransition marked as approximately 26 Gauss or 72 Table 2 3. Standard pulse table used for DEER experimen ts. +x Pulse Acquisition Trigger Position 200 600 Position Pulse Length 16 32 Pulse Length 16 Pos. Display 8 Pos. Display The frequency (2) is applied to the central resonance located at approximately 3460 Gauss in the X band EPR spectrum (corresponding to B spins) At the pump frequency (corresponding to A spins) spins. the refocused echo is monitored. The set of spins affected by the probe frequency experiences a different magnetic The change in the magnetic environment experienced by the spins at (1) affects how the spi The overall effect is a modulation in the intensity of the refocused echo as a function of the strength of the coupling between spins. The strength of the coupling is proportional to the inverse cube of the dista nce between the spins ( proportional to 1/r 3 ). Note, ap plying the pump pulse ( (1) ) to the central resonance line results in an incr eased signal: noise ratio (SNR) because it is the

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101 (2 ) ) is applied to the low field transition because it is the next most populated region of the spectrum. Theoretically, the technique is useful to extract distances in the range of 1.5 8 nm (15 80 ), but in practice this distance is closer to 1.5 5 nm (15 50 ). DEER experiments were configured with the parameters listed in Table 2 4. Table 2 4. Standard pulsed EPR parameters used in this study. Parameter Value Shot repetition time 4000 Sweep width 160 200 G Number of scans variable Shots/point 100 Center Field ~3460 G Low Field ~3432 G Frequency ~9.6 GHz Pulsed Attenuation 0.1 Video Bandwidth 25 MHz 5 Modulation Amplitude ~1 G Time Constant 0.082 0.164 sec Receiver Phase 100 Figure 2 19 shows typical dipolar modulated echo data from a system containing two separate nitroxide spin labels. The raw dipolar modulated echo cur ve, designated V(t) (Equation 2 7), is shown as the solid black line. The background, designated B(t), is plotted as a solid green line. Given the concentration of protein used for DEER experiments, A spins will come in contact with B spins from separate proteins causing a random distribution of intra protein spin spin interactions, giving rise to a backgrou nd signal in the form of an exponential decay. The background corrected dipolar evolution data, designated F(t), is plotted as a solid blue line. It is quite imperative to select the correct background for subtraction; though the most probable distance o f the distance distribution profile is not likely to change from improper background subtraction, the high level of detail obtainable from DEER distance measurements can be lost. V(t) = F(t)B(t) (2 7)

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102 After background subtraction of the dipolar evolution curve, the dipole dipole interaction can be written as the functi on V(t), as shown in Equation 2 Equation 2 9. (2 8) (2 9) Figure 2 19. Sample dipolar evolution curve showing locations of raw dipolar modulation, background subtraction, and background subtracted echo curve with fi t. Phase M emory T ime T m For DEER experiments, T m limits the length of the dipolar evolution curve, which can affect data analysis particularly for longer distances. T 2 is the transverse relaxation time and describes how quickly the magnetizat ion in the x y plane dissipates; T m is a broader term that encompasses more of the processes that affect the refocusing of spins into an echo, including but not limited to local spin concentrations and dipolar interactions with nuclear spins. The use of glycerol and deuterated materials are common and effective methods of extending the T m of samples with solvent exposed labeling sites. Additionally, glycerol functions as a glassing agent

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103 and cryoprotectant. Glycerol is the most common glassing agent/cryoprotectant used in pulsed EPR studies; others include polyethylene glycol (PEG), sucrose, and ethylene glycol. A glassing agent is an additive that increases the glass transition temperature of the sample which helps avoid protein aggregation during the freezing pro cess. The protein samples examined in this work were placed in a deuterated buffer with 30% d8 glycerol. T m is also highly dependent on experimental temperature; a ll DEER experiments here were d one at 65 K, where T m is substantially extended. To measure T m a simple echo decay experiment is performed using a 2 pulse Hahn ec h o sequence given as /2 with typical parameters given in Table 2 5 Table 2 5. Typical parameters used for the echo decay experiment for determination of T m Parameter Value (ns) /2 16 32 200 8 Figure 2 2 0 Typical results of an echo decay experiment using a 2 pulse Hahn echo sequence ( /2 ) for determination of T m (black). The data is fit to a n exponential decay function of the form in Equation 2 8 (red). Spacing between /2 and cho intensity is measured as a function of igure 2 20 is an example of typical echo

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104 decay data (black), which is fit to an exponential decay function (red) of the form in Equation 2 10, where T m is the rate of decay A is initial intensity, B is the time offset, C is a variable that describes the exponential function stretching, and D is the value of y as t approaches infinity. Modulations seen in the data are the result of proton modulation via the electron spin en velope echo modulation (ESEEM) effect. (2 10) Protein Requirements for Pulsed EPR Experiments T he pulsed EPR methodology with the ELEX S YS E580 with MD 4 or MD 5 dielectric resonator requires nanomole quantities of spin lab eled protein. Typical sample sizes are 100 L of 150 300 M protein. These values are accurate for all work repor te d within, but may vary from instrument to instrument Analysis of DEER Data DEER data must be analyzed very carefully and extensively to provide the most accurate and useful information regarding distances between two spin labels in a protein system, and there are numerous aspects that are important to consider in data analysis steps. For a comprehensive review on DEER data collect ion and analysis, the reader is referred to a number of excellent sources by Gunnar Jeschke, Jack Freed, and others (Jeschke 2002; Jeschke 2002; Jeschke 2004; Chiang 2005; Jeschke, Chechik et al. 2006; Jeschke and Polyhach 2007) The dipolar evolution curve is the result o f the coupling between the A spins and the B spins; however, converting this information into a distance profile is complex due to the ill posedness of the problem (meaning that there is not a single uniq ue answer). There are several methods used to analyze DEER data, the most common are the direct Fourier transform, Monte Carlo, and Tikhonov regularization.

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105 Direct Fourier t ransform The simplest metho d used to analyze DEER data is direct Fourier transfo rm of the time domain data to give a frequency spectrum where the Pake pattern splitting is proportional to 1/r3 (where r is the interspin distance). However, this method does not provide a detailed a nalysis or distribution profile. Instead, this method only reports on a most probable distance between spins. A graphical representation of this analysis method is shown in Figure 2 21. Figure 2 21 Pictor i al representation of the direct Fourier transform method of analysis, A) time do main spectrum is con verted to a B) frequency domain spectrum, where the singularities in the Pake pattern are proportional to 1/r 3 where r is C) the most probable distance between two spins. Curve fitting and Monte Carlo analysis In order to gain more information than the di rect Fourier transform method provides, i.e. a distance distribution profile a more complex analysis method is required. A common technique, referred to as curve fitting, can be employed to solve the inverse problem of generating a distance profile base d on known information about the system, and optimize it until the theoretical and experimental dipolar evolution curves match. In Monte Carlo (MC) analysis, the scheme for which is shown in Figure 2 22, the distance profile has a pre determined form (e.g ., Gaussian) (Fajer 2006 ) The MC analysis has the advantage of being a fairly easy and expansive

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106 approach to finding a suitable answer; however, it has the severe limitations that it is both dependent on an original model and a pre determined form. Figure 2 22. Pictorial representation of the Monte Carlo analysis. Tikhonov r egularization A third approach, and the one used for all DEER analysis described in this dissertation, is called Tikhonov regularization (TKR) (Tikhonov 1943; Hansen 1998; Chiang, Borbat et al. 2005) TKR us es the function in E quation 2 11 to find the best answer to the ill posed problem by balanc ing the quality of fit with the smoothness of the solution by varying the regularization parameter (often referred to as ). The first term represents the quality of the fit and the second term represents the smoothness of the solution; P is probability of the spin spin distance, K and L are operators, S is the experimental data vector. Following the TKR process, the log of ( ) is plotted against the log of ( ) to produce an L curve which is analyzed in order to determine the optimal regularization parameter. Each individual point on the plot is given by Equation 2 12, an d ( ) and ( ) are given in Equations 2 13 and 2 14. (2 11) (2.12) (2.13)

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107 (2.14) Figure 2 23. Selection of regularizat ion parame ter in TKR DEER analysis method; A) r aw and b ackground subtracted dipolar evolution curve and B) typical L curve showing C) undersmoothed and D) resulting distance profile, E) optimal and F) corresponding distance profile, and G) oversmoothed with H) corresponding distance profile.

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108 When the regularization parameter is small, G(P) is dominated by the quality of the fit with experimental data and a s the regularization parameter is increased, G(P) is driven by a larger contribution from smoo thness of the fit as illustrated in Figure 2 23 A quality L curve to the optimal regularization parameter. By selecting this value of the optimal dista nce distribution profile can be determined. Zero time s election Figure 2 24 Example of zero time selection for dipolar modulated echo data; A ) s election of zero time, and B ) results of incorrect zero time selection In order to obtain the most accur ate distance profile, it is imperative that the correct zero time be determined. DEER data is collected with a small amount of negative time, as shown in Figure 2 24A. If the incorrect zero time is selected, the distance distribution will be shifted slig htly towards either smaller or greater distances, as demonstrated in Figure 2 24B. In order to do this, truncated dipolar modulated echo curve in the region of 300 300 ns is plotted in Origin8.0 and fit to a GaussAmp function of the form shown in Equati on 2 15 where y 0 is the y offset, w is the width, A is the amplitude, and x c is the center of the Gaussian shaped function, and thus designated as the zero time.

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109 (2 15) Self c onsistent a nalysis As previously described, the typical dipolar modulated echo curve is affected by the dipolar interactions between spin labels on different proteins. To accurately analyze the DEER data with respect to the lowly populated states in a conformational ensemble, it is absolutely imperative to ensure that the appro priate level of background is subtracted. In efforts to obtain information about the shape and location of minor populations within the distance profile, and to ensure that the correct background subtraction was selected, our research group developed a te chnique termed self consistent analysis that facilitates this need the scheme for which is shown in Figure 2 25 In short, the self consistent analysis consists of an initial determination and subtraction of background via an approximate Pake transforma tion using DeerAnalysis2008 software, following by TKR analysis, resulting in a distance distribution profile. The distance distribution profile is then regenerated, using the DeerSim program, with a series of Gaussian shaped curves representing individua l flap conformations. The sum of the Gaussian shaped curves is used to generate a theoretical dipolar modulated echo curve, which is free of any contributions from background. The theoretical and experimental dipolar modulations are compared. If the the oretical and experimental dipolar modulations are not exactly the same, then the background subtraction is incorrect, a new background subtraction is selected, and this process is repeateduntil both the distance profile and the dipolar modulated echo curve are accurately reproduced.

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110 Figure 2 25 Self consistent analysis scheme. Interpretation of distance distribution profiles There are several ways to describe and/ or interpret the distance distribution profiles for a biological system. One way is a simple, semi quantitative description in which the profile is characterized with respect to most probable distance av e rage distance, and/or full width at half

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111 max (FWHM) as shown in Figure 2 2 6 Here, the most probable distance is defined as the most i ntense point in the distance profile, and the full width at half max is indicative of flexibility or range of motion within the system A sharp, narrow profile can be related to low flexibility or small range of motion, whereas a broad profile suggests hi gh flexibility and range of motion. However, a more thorough quantitative description can be made using a procedure developed in our lab called Gaussian reconstruction, where in individual sub populations are identified and used to regenerate the distance distribution profiles. Figure 2 2 6 Semi q ua nt itative analysis of distance distribution profiles, including most probable distance and full width and half max. Gaussian reconstruction process The Gaussian reconstruction process facilitates a high ly quantitative interpretation of the distance profiles of a biological system. Here the distance profile is regenerated using a series of Gaussian shaped sub populations, where the sum of the sub populations defines the conformational ensemble of the sy stem. An example of this type of analysis is given in Figure 2 27. Each of the sub populations can be analyzed for most probable distance, FWHM, and the relative percentage of the conformational ensemble. Figure 2 27A shows an example of a distance dist ribution profile generated using DeerAnalysis2008 software (red), and a regenerated distance distribution profile (blue dashed) created using the sum of the Gaussian shaped sub

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112 populations shown in Figure 2 27B. The two small populations centered at appro ximately 15 and 22 were suppressed due to a negligible effect on the dipolar modulation; this process will be discussed in more detail in the following section. Figure 2 27. Quantitative description of distance dist ribution profiles using Gaussian reconstruction procedure ; A) distance distribution profile generated using DeerAnalysis2008 software (red) and regenerated distance distribution profile (blue dashed), and B) series of Gaussian shaped sub populations used t o regenerate the distance distribution profile. E rror analysis by population suppression and validation When DEER data analysis is performed using our Gaussian reconstruction process, a method of error analysis called population suppression is performe d. Population suppression is utilized in order to determine if each of the sub populations is necessary to properly regenerate the experimental dipolar modulated echo data. The principle behind this analysis technique is to individually remove, or suppre ss, each of the sub populations one at a time and also linearly, then regenerate a theoretical dipolar modulated echo curve that is then overlaid with the experimental background subtracted dipolar modulated echo data. If the theoretical curve overlays w ith the experimental curve within the noise of the signal, the suppressed population can be labeled as either questionable or unnecessary. If, however, the theoretical echo data no

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113 longer overlays well with the experimental data, the suppressed population is known to contribute to the overall experimental outcome. An example of the population suppression error analysis technique is given in Figure 2 28. Figure 2 28. Example of population suppression error analysis, A) distanc e profile (red) overlaid with regenerated distance profile (blue dashed), B) series of Gaussian shaped sub populations used to regenerate the distance distribution profile, C E) theoretical dipolar modulation with various sub populations suppressed (blue) overlaid with experimental dipolar modulation. In each case, it can be seen that when the given population is suppressed the theoretical echo curve no longer overlays with the experimental echo curve data, therefore each of the sub populations was validat ed as being a real contributor to the experimental data.

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114 CHAPTER 3 CONTINUOUS WAVE ELEC TRON PARAMAGNETIC RE SONANCE STUDIES OF H IV 1 PROTEASE Introduction As described in previous chapters, human immunodeficiency virus type 1 (HIV 1) protease (HI V 1PR) is the viral enzyme responsible for virus maturation. Several different HIV 1PR constructs will be discussed in chapter 3; namely A/E s A/E si F s F si B s and B si Refer to Chapter 1 for more detailed discussion on construct nomenclature and am ino acid substitution code. In summary, a (Q7K, L33I, L63I) ( D25N ). Amino acid substitution code (e.g. D25N) is given by amino acid residue to be substituted out, followed by the residue number, followed by the amino acid to be substituted in (e.g. D25N the aspartic acid residue at position number 25 was mutated to an asparagine residue ). Unpublished work performed by graduate student A. Mike Veloro in our lab on the drug resistant patient isolate s V6 and MDR769 (without the three stabilizing mutations Q7K, L33I, and L63I) has resulted in substantial autoproteolysis of the active proteases during the course of the purification. Thus, most of our structural work to date has been focused on protease constructs that have incorporated the Q7K, L33I, and L63I substitutions. Figure 3 1 shows a 16.5% tris tricine SDS PAGE gel of self cleavage products for Subtype B, here called PMPR (pentamutated protease) with stabilizing mutations Q7K, L33I and L63I (PMPR) and V6 and MDR769 (MDR) with no stabilizing mutations. Aliquots were taken from freshly prepared stock solutions immediately after purification As seen in the SDS PAGE gel, stabilized Subt ype B has enhanced stabilization from self cleavage while b oth V6 and MDR769 undergo rapid degradation during the course of the purification The self cleavage products of V6 and MDR769 are not known.

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115 Figure 3 1 16.5% tris tricine SDS PAGE gel of self cleavage products for Subtype B with stabilizing mutations Q7K, L33I and L63I (PMPR) and V6 and MDR769 (MDR) with no stabilizing mutations. Aliquots taken from freshly prepared stock solutions immediately after purification. B ecause of the rapid degra dation of the protease during the course of the purification, e ach of the Subtype B, F, and CRF01_A/E constructs examined were stabilized against autoproteolysis by incorporating amino acid substitutions Q7K, L33I and L63I These sites have been reported to slow the rate of autoproteolysis more than 100 fold. Edman degradation sequencing was used to identify t hree primary sites of proteolytic cleavage for Subtype B protease, and include the peptide bonds located between amino acid res idues at positions L5 W6, L33 E34 and L63 I 64 In an attempt to slow the rate of autoproteolysis by rendering the primary site of cleavage less labile, Rose et al. engineered a Q7K substitution into an HIV 1 PR construct, which reduced autoproteolysis in the protein more than 100 fold (Rose, Craik et al. 1998) Subsequently, Mildner et al. introduced the additional substitutions L33I and L63I to produce a triply substituted construct which was shown to retain the specificity and kinetic properties of the wild type enzyme but was hi ghly stabilized against autoproteolysis (Mildner, Rothrock et al. 1994) When studying inactive protease, these mutations are not necessary. However, because much of the research in our lab deals with active protease, where purification practically requires the s tabilizing mutations, all constructs examined (with the exception of the drug resistant

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116 constructs discussed in later chapters due to the location of certain drug induced polymorphisms ) have incorporated Q7K/ L33I / L63I mutations to allow for comparison betw een active and inactive protease. Fi gure 3 2 shows a crystal structure of HIV 1PR highlighting the D25 active sites residues, the K55 site chosen for modification as a reporter site, and stabilizing mutation s at positions Q7, L33, and L63. Figure 3 2 Ribbon diagram of HIV 1 P R structure PDB ID 2BPX Site s of K55 reporter site D25 active site residues and s ites of Q7K, L33I and L63I stabilizing substitutions are labeled. Structure rendered in VMD (Humphrey 1996) Within this chapter are re sults from continuous wave electron paramagnetic resonance ( CW EPR ) studies on spin labeled HIV 1PR constructs The materials and methods section con tains detailed information about all aspects of the experiments described within chapter 3, including cloning of the HIV 1PR gene, site directed mutagenesis, expression and purification of the protein and details of constructs examined, spin labeling circ ular dichroism, sample preparation and storage, CW EPR and analysis of EPR results, as well mass spectrometry experiments and analysis. The next section of the chapter gives the results of a study on the effect of inhibitors on the CW EPR nitroxide spect ral line shape of HIV 1PR Subtype F si and CRF01_A/E si The normalized spectra of the apo constructs were compared to those of the protease in the presence of nine different FDA approved protease inhibitors used in the treatment of HIV, and a non hyd rolyza ble substrate mimic CA p2 Because the EPR nitroxide spectral line shape is sensitive

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117 to motion, it is feasible that upon substrate binding and flap closure the line shape would appear more motionally restricted. The final section of the chapter gives r esults from a study regarding autoproteolysis in the active Subtype F s and CRF01_A/E s constructs. The EPR nitroxide spectral line shape is highly sensitive to mobility in the environment of the spin label, thus changes dramatically with changes in correla tion time. Contributions to correlation time come from three modes of motion, including global protein tumb R i ), and backbone B ). Autoproteolysis affects the rate of global protein tumbling by decreasing rotational correlation time of the spin labeled protein. As total correlation time decreases, the der ivative EPR spectra decrease in breadth and resonance line shapes become sharper and increasingly narrow. Here, we monitored the development of a sharp component in the high field that is proportional to the amount of degraded protein in the sample, and t he intensity of the high field line (I HF ) was quantitatively analyzed. Dr. Luis Galiano performed a similar experiment with Subtype B s in which autoproteolysis was monitored for approximately 50 days using CW EPR (Galiano 20 08) Minimal cleavage occurred in the protease sample, as evidenced by the small change in the high field intensity of the EPR line shape (Figure 3 3 ). Subtype B, because it is the most prevalent subtype in the United States and Europe is the most stud ied protease subtype. It is important, however, not to neglect the structural and dynamic changes that subtype polymorphisms can incur on the protease. Thus, this study was performed in part to compare the rates and locations of the autoproteolysis of Su btype F s and CRF01_A/E s to Subtype B s In addition, storage conditions and a ffect of the inhibitor were examined. Subtype F and CRF01_A/E protease samples were

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118 prepared and stored at 37 C, 25 C, or 4 C, and in the presence of a protease inhibitor or n on hydrolyzable substrate mimic. Figure 3 3 Overlay of day 1 (black) and day 47 (grey) area normalized X band 100 Gauss CW EPR spectra for s ubtype B HIV 1 protease labeled with MSL at position 55 and stored at 25 C. Also performed by Galiano were e xperiments to determine the effect of salt concentration on the CW EPR spectral line shape of H I V 1PR (Galiano 2008) While developing the p urification scheme, it was observed that HIV 1 PR was very sensitive to salt concent ration To study the effect of salt concentration on the EPR spectrum of HIV 1PR, 100 Gauss X band CW EPR spectra were collected for 100 M HIV 1PR, labeled with MTSL at the K55 C and K55 C positions, in 2 mM NaOAc with increasing concentrations of salt (0, 50, 500, and 2500 mM), and r esults are shown in Figure 3 4. It is known that the enzymatic activity of HIV 1PR is dependent upon ionic strength, with greater activity observed in high salt concentrations near 2 M (Szeltner and Polgar 1996) However, the EPR spectral line shapes from 100 M Subtype B HIV 1 PR labeled with MTSL show significant broadening when the salt concentration is increased above 50 mM NaCl These changes in spectral line shapes can be indicative of protein aggregation which was confirmed

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119 upon inspection of the capilla ry tube which revealed precipitated protein above 500 mM NaCl. From th ese findings, buffers for all EPR experiments are prepared at low ionic strength with a maximum of 50 mM NaCl (Galiano 2008) Figure 3 4 Area norma lized 100G X band CW EPR spectra of 100 M HIV 1 P R as a function of salt concentration; A) 2 mM NaOAc + 0 mM NaCl, B) 2 mM NaOAc + 50 mM NaCl C) 2 mM NaOAc + 500 mM NaCl, and D) 2 mM NaOAc + 2500 mM NaCl. Materials and Methods Materials The chemicals, reagents, and supplies were obtained from Fisher Scientific (Pittsburg, Pennsylvania) and used as received, with a few noted exceptions pET23 DNA was purchased from Novagen (Gibbstown, New Jersey). AG 501 X 8 (D) resin, 20 50 mesh was purchased from BioR ad ( Hercules, California ) HiTrap Q HP Anion Exchange column HiPrep 16/60 Sephacryl S 200 h igh r esolution s ize e xclusion column was purchased from GE Biosciences (formerly Amersham Pittsburg, Pennsylvania) HIV 1 Protease DNA was synthesized and subseque ntly purchased from DNA2.0 ( Menlo Park, California) 4 m aleimido 2,2,6,6 tetramethyl 1 piperidinyloxy (4 m aleimido TEMPO, MSL) was purchased from Sigma Aldrich (St. Louis, MO). (1 o xyl 2,2,5,5 tetramethyl 3 pyrroline 3 methyl) methanethiosulfonate spin label ( MTSL ) was purchased from Toronto Research Chemicals, Inc (North York, Ontario,

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120 Canada). The QuikChange site directed mutagenesis kit was purchased from Stratagene (La Jolla, California). 0.60 I.D. x 0.84 O.D. capillary tubes (Cat # CV6084) were p urchased from Fiber Optic Center ( New Bedford, M assachusetts) BL21*(DE3) pLysS E. coli cells were purchased from Invitrogen (Carlsbad, California). Ritonavir, Indinavir T ipranavir Darunavir Amprenavir Atazanavir, N elfinivir, Saquinavir and Lopinavir w ere generously received from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH ( Bethesda, Maryland) (NIH) The non hydrolyzable substrate mimic CA p2 (H Arg Val Leu r Phe Glu Ala Nle NH 2 (R V L r F E A Nle NH 2 r = reduced) was synthesized and purchased from the University of Florida Protein Chemistry Core Facility (Gainesville, Florida) Metho ds Cloning of HIV 1 p rotease The Escherichia coli ( E. coli ) codon and expres sion optimized genes for HIV 1 protease CRF01_A / E si K55C and Subtype F si K45C ( sequences are given in Tables 3 1 and 3 2 respectively ) were purchased from DNA 2.0 and received in pJ2 01 : 24237 and pJ201:24236 vector s (Figure s 3 5A and 3 5B ) Plasmids containing the CRF01_A/E si K55C and Subtype F si K45C were cleaved using the restriction enzymes NdeI and BamHI and subsequently ligated into the pET23a vector (Figure 3 5C ) that had bee n previously digested using the same enzymes. Standard restriction digestion and ligation procedures were utilized. The ligated vector was then transformed into XL1 blue cells, then isolated and purified using the Qiagen mini prep k it and checked by sequ encing. These constructs will now be referred to as pET23a_F si K45C and pET23_A/E si K55C.

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121 Figure 3 5 Vector maps of A) pJ201:24237 with HIV 1P R CRF01_A/ E insert (red), B) pJ201:24236 with HIV 1P R s ubtype F insert (red) (vector maps in A) and B) were created by DNA 2.0 and received with DNA constructs), and (C) pET23 vector (vector map adapted from Novagen). Site d irected mutagenesis of HIV 1 protease c onstructs To obtain each of the constructs describe d within this chapter, including protease const ructs pET23a_F si K55C, pET23_F s K55C, and pET23_ A/E s K55C, several rounds of site directed mutagenesis were performed. P rimer sequences melting temperatures (T m ) and molecular weights are given in Tables 3 1 and 3 2 for Subtype F and CRF01_A/E, respective ly T hermal cycling p arameters are given in Table 3 3 and the DNA and amino acid sequences of all constructs discussed within this chapter are given in Table s 3 4 3 8 A B C

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122 Table 3 1. PCR p rimers utilized to introduce mutations to HIV 1 PR CRF01_A/E Mut ation Primer (5` 3`) Tm ( o C) m.w. %GC C55K Forward TGGCGGCATCGGCGGCTTTATCAAA 72.0C 12,611.2 56.0% Reverse CAATCTCGATAATGATCTGGTCGTA 71.6C 12,440.4 40.0% N25D Forward GTGGTCAACTGAAAGAAGCGCTGC 70.9C 14,9 62.7 54.1% Reverse TCAATGACCGTATCATCCGCACCGGT 70.1C 14,310.3 53.8% T74C Forward GGTAAGAAGGCAATTGGCTGCGTCTT 69.4C 12,094.8 50.0% Reverse CCATTCTTCCGTTAACCGACGCAGAA 69.1C 11,881.7 50.0% Table 3 2. PCR p rimers utilized t o introduce mutations to HIV 1 PR s ubtype F Mutation Primer (5` 3`) Tm ( o C) m.w. %GC C45K Forward GACATGAATCTGCCGGGTAAGTGG 68.1 C 13,718.9 54.2% Reverse TTTGATAAGACCAATACCGCCAATC 67.8 C 14,901.7 40.0% K5 5C Forward ATGATTGGCGGTATTGGTGGTTTCAT 67.2 C 13,319.7 42.3% Reverse ATGATAATCTGATCGTATTGCTTGAC 67.5 C 15,008.8 34.6% N25D Forward CCAATTGAAGGAGGCCCTGCTGGAT 71.6 C 11,769.6 56.0% Reverse AATCACGGTATCGTCCGCACCGGTA 72.3 C 12,202.9 56.0% T74C Forward GGCCACAAAGCGATCGGTTGTGTTC 70.3 C 11,710.6 56.0% Reverse CCGGTGTTTCGCTAGCCAACACAAG 69.8 C 11,648.6 56.0% Table 3 3 Thermal c ycling parameters for HIV 1 p rotease site directed mutagenesis reactions Segment Cycles Temperature Time 1 1 95 o C 30 seconds 2 18 95 o C 30 seconds 55 o C 1 minute 68 o C 6 minutes Table 3 4 E. coli codon optimized HI V 1 P R s ubtype F si K45C DNA and amino acid sequences 1 ccg cag att acc ctg tgg aag cgt ccg ctg P Q I T L W K R P L 11 gtc acg atc aaa gtt ggc ggc caa ttg aag V T I K V G G Q L K 21 gag gcc ctg ctg aac ac c ggt gcg gac gat E A L L N T G A D D

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123 Table 3 4. Continued. 31 acc gtg att gag gac atg aat ctg ccg ggt T V I E D M N L P G 41 a ag tgg aaa ccg tgc atg att ggc ggt att K W K P C M I G G I 51 ggt ggt ttc atc aaa gtc aag caa tac gat G G F I K V K Q Y D 61 cag att atc atc gaa atc gct ggc cac aaa Q I I I E I A G H K 71 gcg atc ggt act gtt ctg gtt ggc cca acc A I G T V L V G P T 81 ccg gtg aat atc att ggt cgc aac ttg ctg P V N I I G R N L L 91 acg cag att ggt gca acg ctg aac ttc T Q I G A T L N F Table 3 5 E coli codon optimized HIV 1 PR s ubtype F si K55C DNA and amino acid sequences 1 ccg cag att acc ctg tgg aag cgt ccg ctg P Q I T L W K R P L 11 gtc acg atc aaa gtt ggc ggc caa ttg aag V T I K V G G Q L K 21 gag gcc ctg ctg aac acc ggt gcg gac gat E A L L N T G A D D 31 acc gtg att gag gac atg aat ctg ccg ggt T V I E D M N L P G 41 aag tgg aaa ccg tgc atg att ggc ggt att K W K P K M I G G I 51 ggt ggt ttc atc aaa gtc aag caa t ac gat G G F I C V K Q Y D 61 cag att atc atc gaa atc gct ggc cac aaa Q I I I E I A G H K 71 gcg atc ggt act gtt ctg gtt ggc cca acc A I G T V L V G P T 81 ccg gtg aat atc att ggt cgc aa c ttg ctg P V N I I G R N L L 91 acg cag att ggt gca acg ctg aac ttc T Q I G A T L N F Table 3 6 E coli codon optimized HIV 1 PR s ubtype F s K55C DNA and amino acid sequences 1 ccg cag att acc ctg tg g aag cgt ccg ctg P Q I T L W K R P L 11 gtc acg atc aaa gtt ggc ggc caa ttg aag V T I K V G G Q L K 21 gag gcc ctg ctg aac acc ggt gcg gac gat E A L L D T G A D D 31 acc gtg att gag gac atg aat ctg ccg ggt T V I E D M N L P G

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124 Table 3 6. Continued. 41 aag tgg aaa ccg tgc atg att ggc ggt att K W K P K M I G G I 51 ggt ggt ttc atc aaa gtc aag caa tac gat G G F I C V K Q Y D 61 cag att atc atc gaa atc gct ggc cac aaa Q I I I E I A G H K 71 gcg atc ggt act gtt ctg gtt ggc cca acc A I G T V L V G P T 81 ccg gtg aat atc att ggt cgc aac ttg ctg P V N I I G R N L L 91 acg cag att ggt gca acg ctg aac ttc T Q I G A T L N F Table 3 7 E coli codon optimized HIV 1 PR A/E si K55C DNA and amino acid sequences 1 ccg cag atc acg ctg tgg aaa cgt cca ctg P Q I T L W K R P L 11 gtt acc gtt aag att ggt ggt caa ctg aaa V T V K I G G Q L K 21 gaa gcg ctg ctg aac acc ggt gcg gat gat E A L L N T G A D D 31 acg gtc att gag gac atc aat ctg ccg ggt T V I E D I N L P G 41 aag tgg aaa ccg aaa atg att ggc ggc atc K W K P K M I G G I 51 ggc ggc ttt atc tgc gtg cgc caa tac gac G G F I C V R Q Y D 61 cag atc att atc gag att gct ggt aag aag Q I I I E I A G K K 71 gca att ggc acc gtc ttg gtt ggt ccg acc A I G T V L V G P T 81 ccg gtg aat atc atc ggt cgt aac atg ctg P V N I I G R N M L 91 act cag att ggt gcc acg ctg aac ttc T Q I G A T L N F Table 3 8 E coli codon optimized HIV 1 PR A/E s K55C DNA and amino acid sequences 1 ccg cag atc acg ctg tgg aaa cgt cca ctg P Q I T L W K R P L 11 gtt acc gtt aag att gg t ggt caa ctg aaa V T V K I G G Q L K 21 gaa gcg ctg ctg aac acc ggt gcg gat gat E A L L N T G A D D 31 a cg gtc att gag gac atc aat ctg c cg ggt T V I E D I N L P G 41 aag tgg aaa ccg aaa atg att ggc ggc atc K W K P K M I G G I

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125 Table 3 8. Continued 51 ggc ggc ttt atc tgc gtg cgc caa tac gac G G F I C V R Q Y D 61 c ag atc att atc gag att gct ggt aag aag Q I I I E I A G K K 71 g ca att ggc acc gtc ttg gtt ggt ccg acc A I G T V L V G P T 81 ccg gtg aat atc atc ggt cgt aac atg ctg P V N I I G R N M L 91 act cag att ggt gcc acg ctg aac ttc T Q I G A T L N F Expression of HIV 1 p rotease c onstructs Modified pET 23 a vectors (pET23a_F si K45C and pET23_A/E si K55C, pET23a_F si K55C, pET23_F s K55C, and pET23_ A/E s K55C were transformed separately into E. coli strain BL21 (DE3) pLysS via s tandard heat shock methodology. The transformed cells were inoculated in 5 mL sterile Luria Bertani (LB) media ( Table 3 9 ) and grown at 37 C with shaking at 250 rpm to an optical density (OD 600 ) of approximately 0.60 then transferred to 1 L sterile LB m edia and grown to an OD 600 of approximately 1.0 with shaking at approximately 200 RPM Cells were then induced using 1 mM isopropyl beta D thiogalactopyranoside (IPTG ) The cultur e was then incubated with shaking at 250 rpm for 5 6 hours at 37C Cells were harvested by centrifugation for 15 minutes at 8500 g using a Sorvall RC6 floor model centrifuge with SLA 3000 rotor a t 4 C and s upernatant was discarded. Table 3 9 Luria Bertani m edia (1L) Component Amount Yeast extract 5 g NaCl 10 g Tryptone 10 g Ampicillin 1 mL (100 M) (opt.) Sterile H 2 O Volume to 1 L Details of p rotease c onstructs Several d ifferent HIV 1PR constructs were designed and prepared ; namely A/E s (CRF01_A/E; K5 5C, C67A, C95A, Q7K, L33I, L63I), A/E si (CRF01_A/E; K55C, C67A, C95A,

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126 Q7K, L33I, L63I, D25N), F s (Subtype F; K55C, C67A, C95A, Q7K, L33I, L63I), F si (Subtype F; K55C, C67A, C95A, Q7K, L33I, L63I, D25N), and B s (Subtype B; K55 C, C67A, C95A, Q7K, L33I, L63I) T he D25N amino acid substitution was incorporated into some of the constructs in order to render i nactivity on the protease. The D25N substitution has been shown by X Ray and NMR not to perturb the structure of the protease. All construct s contained th ree stabilizing mutations that help provide protection from autocatalyti c cleavage: Q7K, L33I, and L63I. Additionally, each construct had both naturally occurring cysteine residues (C67 and C95) substituted to alanine residues These mutations all ow for site specific labeling and protect ion against intramolecular disulfide cross linking. Site s K55 solvent exposed site s in the flap region, were substituted with CYS residues for site directed spin labeling Active c onstructs were labeled with 4 Maleimido TEMPO (MSL) and inactive constructs were labeled with ( 1 oxyl 2,2,5,5 tetramethyl 3 pyrroline 3 methyl)methanethiosulfonate ( MTSL ). Structures of the spin labels and spin labeled s ide chains are given in Figure 2 10 HIV 1 p rotease p ur i fication b uffers For compositions of all buffers used in HIV 1PR purification, see Table 3 10 All buffers containing urea were made fresh prior to each round of protein purification. With heat and time, urea degrades to give products that can carbamylate free c ysteines (Figure 3 6), thus limiting spin labeling efficiency (Stark 1965; Stark 1965; Stark 1965; Lippincott and Apostol 1999) Additionally, all urea buffers contain glycylglycine (diGly) to function as an ion sc avenger for urea decomposition products and were ion exchanged using a mixed ion bed resin (AG501 X8) by adding 5 g resin/100 mL urea.

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127 Table 3 10 HIV 1 P R p urification b uffers Buffer (Volume) Compo nent Re suspension Buffer 20 mM Tris HCl 1 mM EDTA 10 M BME added fresh *Adjust pH to 7.5 and filter using a 0.22 m membrane. Store at 25 o C. Wash Buffer 1 25 mM Tris HCl 2.5 mM EDTA 0.5 M NaCl 1 mM Gly Gly 50 M BME added fresh *Adjust pH to 7.0 and filter using a 0.22 m membrane. Store at 4 o C. Wash Buffer 2 25 mM Tris HCl 2.5 mM EDTA 0.5 M NaCl 1 mM Gly Gly 50 M BME added fresh 1 M urea *Adjus t pH to 7.0 and filter using a 0.22 m membrane. Must be fresh! Prepared an hour before use. Wash Buffer 3 25 mM Tris HCl 1.0 mM EDTA 0.5 M NaCl 1 mM Gly Gly 50 M BME added fresh *Adjust pH to 7.0 and filter using a 0.22 m membrane. Store at 4 o C. Inclusion Body Resuspension Buffer (250 mL) 25 mM Tris HCl 2.5 mM EDTA 0.5 M NaCl 1 mM Gly Gly 50 M BME added fresh 9 M urea *Adjust pH d epending on isoelectric point of construct ; pH should be slightly less than pI of protein to give a good yield. *F ilter using a 0.22 m membrane. Must be fresh! Prepared an hour before use. Spin labeling Buffer (1 L) 10 mM Tris HCl Adjust pH to 6.9 and filter using a 0.22 m membrane. Size Exclusion Column buffer (1 L) 50 mM NaOAc Adjust pH to 5.0 sonicate to remove air, and filter using a 0.22 m membrane. Abbreviations: Ethylenediaminetetraacetic acid mercaptoethanol ( BME), sodium acetate (NaOAc)

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128 Figure 3 6 Decomposition of urea; w ith heat and time, urea degrades into ammonium and cyanate. HIV 1 p rotease p urification Prior to protein purification, 250 mL of 9 M and 150 mL of 1 M urea buffers were freshly prepared in separate containers and 1 2 g AG 501 X8 (D) resin ( 20 50 mesh ) was added to each of the urea buffers. These solutions were placed on a heated (30 C) stir plate and mixed by stir bar for approximately 2 hours to dissolve the urea and the resin was re moved by filtration. All buffer exchange steps described in the following sections were carried out using a 5 mL HiTrap Desalting column from GE Healthcare (packed with Sephadex G25), which is first washed successively with 3 4 column volumes (15 20 m L) of nanopure water (nH 2 O), 1 M NaCl, nH 2 O, 0.5 M NaOH, nH 2 O, and then equilibrated in the desired buffer. Figure 3 7. A) Thermo brand 35 mL French pressure cell and B) Fisher Scientific brand tip sonicator. Pelleted cells from 1 L growth were r esuspended in 30 mL resuspension buffer ( Table 3 10 Mercaptoethanol ( B ME) was added to the reaction and mixed well by

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129 swirling. The approximate weight of the wet pellet was generally 5 6 grams, but varied from purification to purificati on. In order to lyse the cells and release the cellular contents, t he sample was sonic ated for 2 minutes using a Fisher brand tip sonicator (Figure 3 7 ) at approximately 25 watts output power Sonication was always performed in cycles of 5 sec onds on fol lowed by 5 sec onds off to avoid shearing the proteins present in the lysate. The sample was then passed 3 times through a 35 mL French pressure cell (Thermo Scientific, Waltham, Massachusetts) operating at approximately 1200 pounds per square inch ( psi ) Next, the lysed cells were centrifuged for 30 minutes at 18500 x g and 4 C using the Eppendorf 5810R centrifuge with F34 6 38 rotor t o collect cell debris and protease containing inclusion bodies, and the supernatant was discarded. The inclusion body containing pelle t was resuspended, homogenized (using a 50 mL Dounce Tissue Homogenizer) and sonicated in 40 mL fresh wash buffer #1 (Table 3 10 ) then centrifuged for 30 minutes at 18500 x g and 4 C, and the supernatant was discarded. This process was r epeating with 40 mL wash buffer #2 (Table 3 10) and 40 mL wash buffer #3 (Table 3 10) Each of these steps functioned to isolate and wash the inclusion bodies, removing non target proteins and remaining cellular components. In order to solubilize the inc lusion bodies, t he pellet was then resuspended, homog enized and sonicated in 30 mL inclusion body resuspension buffer containing 9 M urea, then centrifuged at 18500 x g and 4 C for 30 minutes. The supernatant, which contained solubilized target proteins in unfolded, monomeric form was collected. Because purification proceeds by anion exchange chromatography, the pH of the inclusion body resuspension buffer needs must be adjusted according to the specific isoelectric point (pI) of the protein being purif ied. Th e of the F si K55C, F s K55C, A/E si K55C, and A/E s K55C constructs are 8.95, 8.95 9.53 and 9.53

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130 respectively; and, the respective pH values of the buffer utilized for anion exchange chromatography are 8.5, 8.5, 9.08, and 9.08 A 5 mL HiTrap Q HP a nion e xchange column was equilibrated with inclusion body resuspension buffer on an Akta Prime liquid chromatography system, and the supernatant containing solubilized HIV 1PR monomers was ap plied to the column at a rate of 5 mL/min F raction collection was started immediately and flow through was collected in 4 mL fractions, (specific fraction numbers depend on the results of the chromatogram) Figure 3 8 shows a typical chromatogram for the ani on exchange step chromatography step Figure 3 8. Typical anion exchange chromatogram (blue: UV 280 red: conductivity, green: constant buffer composi tion) for purification of HIV 1 P R via 5 mL anion exchange Q column. Black square highlights peak of eluted protein. Fractions 1 8 were selec ted and pooled for further purification, refolding, and spin labeling Fractions containing HIV 1PR were collected and pooled into a clean 50 mL Eppendorf tube (approximately 32 mL from 8 separate 4 mL fractions), then acidified to pH 5 (typically with ap C to allow some contaminants to precipitate, after which time the protease sample was decanted into a 50 mL polypropylene Eppendorf centrifuge tube and centrifuged for 30 minutes at 12000 rpm and 6 C to separate precipitated contaminants. 300 mL 10 mM formic acid solution was prepared in a clean beaker and coo led on ice to approximately 0 C. HIV 1PR was refolded by doing a 10

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131 fold stepwise dilution on ice using a peristaltic pu mp for approximately 2 hours, and the pH was subsequently adjusted to approximately 3.8 (typically by adding about 1 mL 2.5 M sodium acetate). The solution temperature was brought to approximately 30 C and the pH was adjusted to 5 (typically by adding ab out 3 mL 2.5 M sodium acetate). Figure 3 9. T ypical size exclusion chromatogram of HIV 1PR on S 100 column. chromatogram (blue: UV, red: conductivity, green: constant buffer composition). Box encircles UV peak corresponding to HIV 1PR. After approxim ately 20 minutes of wait time the solution was moved to balanced centrifuge tubes and centrifuged for 20 minutes at 18500 x g and 23 C to remove contaminants that precipitated during refolding The sample was concentrated to OD 280 = 0.5 using an Amicon 100 mL equipped with a Millipore 10,000 Da MW cut off polyethersulfone membrane If further purification was required, the protease sample was buffer e xchanged into 50 mM NaOAc, pH 5, and 5 mL of the concentrated protein sample was loaded on to a HiPrep 1 6/60 Sephacryl S 200 equilibrated with the same buffer and run at 0.5 mL/min and 2 mL fractions containing HIV 1PR were collected (fraction numbers vary according to chromatogram). A typical size exclusion chromatog ram is shown in Figure 3 9 The result is >95 % pure HIV 1 Protease.

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132 Aliquots were collected after each step in the purification protocol and run on a Biorad Criterion pre cast 16.5% tris tricine peptide SDS PAGE gel to illustrate the purity of the protein sample after each step of the purifica tion protocol, as shown in Figure 3 10. Note, though many typical protein purifications utilize protease inhibitors to prevent cellular proteases from acting upon the target protein, no protease inhibitors were added to the lysate at any time because the target protein is a protease. Figure 3 1 0 Purification of HIV 1PR Biorad Criterion pre cast 16.5% tris tricine SDS PAGE gel Lane 1: Promega b road r ange p rotein m arkers (MW) 2: total cell pellet (TC) 3: lysed cell supernatant (LS) 4: lysed cell pellet (LP) 5: wash buffer 1 supernatant (WS) 6: wash buffer 1 pellet (WP) 7: wash buffer 2 supernatant (WS) 8: wash buffer 2 pellet (WP) 9: wash buffer 3 supernatant (WS) 10: wash buffer 2 pellet (WP) 11: s olubilized washed i nclusion bodies (IB) 1 2 19 : Q column flow through fraction s, 20: a cidification supernatant (AS) 21: a cidification pellet (AP) 22: p ost refolding supernatant (RS) 23: p ost refo lding pellet (RP) 24: s ize exclusion fraction s (SF) 25: empty Spin l abeling When spin labe ling was needed, the purified and refolded protein sample was buffer exchanged into 10 mM Tris HCl, pH 6.9 (further details of spin labeling discussed in next section) after the final purification step. Approximately 1 mg of spin label ( IASL, IAP, MTSL or MSL) was dissolved in 1 00 L ethanol, and added to approximately 40 mL of HIV 1 PR in 10

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133 mM Tris HCl, pH 6.9. The spin labeling reaction was carried out in the dark (via wrapping reaction tube in aluminum foil) at room temperature for approximately 4 6 ho urs followed by 6 8 hours at 4 C for inactive (D25N) constructs, and at 4 C for approximately 8 12 hours for active (D25) constructs. At this time, t he sample solution was centrifuged at 12000 rpm for 20 minutes at 4 C to remove solid impurities and ag gregated proteins. The sample was then buffer exchanged into 2 mM NaOAc, pH 5 and concentrated to OD 280 =1.25. Circular d ichroism s pectroscopy To ensure that the spin labeled HIV 1 PR constructs contained proper secondary structure, circular dichrois m (CD) experiments were performed. All measurements were collected on an Aviv 400 spectrometer using Hellma CD cuvettes with 1 cm pathlength with samples in 2 mM NaOAc buffer, pH 5.0 at approximately 30 M protein concentration Protein concentration was determined by absorption at 280 nm, using an extinction coefficient of 12490 M 1 cm 1 for each construct Typica l parameters used for circular d ichroism experiments are summarized in Table 3 1 1 For each data set, 3 5 scans were taken and averaged to give a final result. Background scans of all buffers were collected and subtracted from the final averaged spectra. Table 3 11 Standard parameters used for c ircular d ichroism experiments Parameter Value Experiment type Wavelength Bandwidth 1 nm Temperature 25 o C Wavelength Start 260 nm Wavelength End 190 nm Wavelength Step 0.5 nm Averaging time 3.000 sec Settling time 1.0 sec Multi scan wait 1.0 se c Scans 3

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134 S ample preparation for EPR data collection The first set of experiments reported in the results section of this chapter was performed in order to analyze the effect of inhibitors and substrate on the EPR nitroxide spectral line shape s. Samples were prepared by adding 1 L of 0.08 mM inhibitor to 9 L of 125 M MTSL labeled HIV 1PR (a final molar ratio of approximately 4:1 inhibitor:enzyme) For inhibitors not dissolved in dimethylsulfoxide ( DMSO ) (i.e. indinavir and tipranavir ), 1 L DMSO was included in the 10 L sample (1 L inhibitor, 1 L DMSO, 8 L HIV 1PR) to make al l samples isovisc ous with one another (10% v/v DMSO) Samples were loaded into capillary tubes and allowed to equilibrate to a controlled temperature, as describe d above. The next set of experiments was performed in order to analyze the autoproteolysis of active HIV 1PR under various conditions. MSL labeled s amples were utilized here for reasons to be discussed later. A po (non substrate/inhibitor bound) HIV 1 PR Subtype F s and CRF01_A/E s were prepared at 150 M protein concentration in 2 mM NaOAc, pH 5.0 Protein concentration was measured by absorption at 280 nm with an extinction coefficient of 12490 M 1 cm 1 for each construct Extinction coefficients were calculated using the program ProtParam at the EXPA SY server ( http://www.expasy.ch/tools ) Three identical 10 L samples were prepared and loaded into c apillary tubes and both ends were sealed. T he tubes were then stored in three conditions, one at 37 C, anoth er at 25 C, and the third at 4 C for a total of 97 days Samples of HIV 1PR Subtype F containing 0.08 mM Tipranavir and CRF01_A/E containing 0.08 mM CA p2 substrate mimic (1 L TPV or CA p2 in 10 L total sample volume) were also prepared in sealed capi llary tubes and stored at 25 C.

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135 CW EPR m easurements C W EPR data were collected on a modified Brker ER200 spectrometer with an ER023 M signal channel, an ER032 M field control unit, and a loop gap resonator ( Molecular Specialties, Milwaukee, WI). A qu artz dewar (Wilmad Labglass) surrounded the loop gap resonator for temperature controlled experiments. Nitrogen gas was passed through a copper coil submerged in a recirculating 25 C water bath (Thermo Scientific) containing 40% ethylene glycol. This se tup is shown in Figure 3 1 1 Figure 3 11. Temperature control set up ; A) t hermocouple thermometer, B) quartz dewar surrounding the loop gap resonator, C) copper coil submerged in a recirculating water bath containing 40% ethylene glycol, with nitrogen pas sing through the line into the D) back of the quartz dewar. Samples were removed from storage conditions and allowed to equilibrate in the loop gas resonator with quartz dewar for at least 15 minutes prior to sample collection. CW EPR spectra were coll ected with 1 G auss modulation amplitude and 100 Gauss sweep width. Additional pp Each

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136 spectrum contains 1024 points with a center field of approximately 3450 Gauss. Sp ectra were collected and averaged from between 1 20 scans with a frequency of 9.6 9.7 GHz. A complete listing of other typical parameters is shown in Table 3 1 2 Table 3 1 2 Standard CW EPR parameters used in this study. Parameter Value Number of points 1024 Center field ~3450 G Number of scans 5 25 Sweep width 20 100 G Acquisition time 40.63 sec Frequency ~9.6 GHz Power 20 dB 20 mW Receiver Gain 1x10 3 5x10 5 Modulation Amplitude ~1 G Time Constant 0.082 0.164 sec Receiver Phase 100 Mass spectrometry e xperiments After a total of 100 days of storage at 37 C, apo samples were removed from the sealed capillary tubes used for EPR analysis and prepared for analysis via mass spe ctrometry (MS) All sample preparation and MS experiments were performed by Dr. Laura Busenlehner at the University of Alabama. Samples were diluted to 6 pmol/ L in 50% HPLC grade acetonitrile in HPLC grade water with 0.1% formic acid. Samples were in troduced to the Br ker HCTultra Discovery ion trap mass spectrometer by direct infusion at a flow rate of 2 L/min. The electrospray ionization source maintained a nebulizer gas pressure of 10 psi, a dry gas flow of 5 L/min, and a drying temperature of 250 C. The positive mode mass range swept was 50 2000 m / z and data dependent collision induced dissociation (CID) fragmentation of peptides was collected. MS spectra were deconvolved for charge using DataAnalysis (Br ker Daltonics) with an abundance cutoff of 10%, a molecular weight tolerance of 0.01% and an envelope cutoff of 75%. For peptides with a monoisotopic mass of less than 3000 Da, CID spectra were collected

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137 and the fragmentation pattern analyzed de novo using MassXpert2 ( Rusconi 2009) and MS Product (Protein Prospector, U niversity of C alifornia S an F rancisco, California) http://prospector.ucsf.edu/prospector/cgi bin/msform.cgi?form=msp roduct ). The MSL spin label was included as a permanent modification on cysteine 55 of mass 354.28 m / z Results and Discussion A ffect of Inhibitors on CW EPR Line S hape s of HIV 1PR Subtype F and CRF01_A/E CW EPR spectra were obtained for several HIV 1 PR Subtype F s i and CRF01_A/E s i with nine separate FDA approved protease inhibitors, with the non hydrolyzable substrate mimic CA p2, and in the apo form (free of inhibitors and substrate). Most of the inhibitors used here are dissolved in DMSO, which cha nges the viscosity of the sample and thus affects the EPR spectral line shape. As a result, each of the samples were made isoviscous to one another by ensuring that each one has the same percent DMSO (10% v/v). Figure 3 1 2 100 Gauss CW EPR nitroxid e spectra l line shapes for HIV 1PR s ubtype F si K55MTSL, collected at X band frequency, same data given in two views: (A) waterfall and (B) overlay.

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138 Table 3 13 CW EPR data analysis for s ubtype F si K55MTSL

low/center fiel d high/center field intensity (normalized) i ntensity (normalized) HIV 1PR ( 2 mM NaOAc ) 1.85 188 0.64 0.18 HIV 1PR ( 10% DMSO ) 1.93 196 0.60 0.17 CA p2 2.01 195 0.58 0.16 RTV 1.97 194 0.58 0.16 APV 2.05 193 0.56 0.15 ATV 2.01 205 0.56 0.16 DRV 2.03 187 0.56 0.16 IDV 1.95 195 0.62 0.18 LPV 2.05 192 0.59 0.17 NFV 1.91 192 0.58 0.16 SQV 2.05 189 0.59 0.16 TPV 1.97 189 0.59 0.16 Shown in Figure 3 1 2 are the spectra for Subtype F si K55MTSL, and Table 3 13 summarizes the spectral parameters described by those data. Shown in Figure 3 13 are the spectra for CRF01_A/E si K55MTSL, and Table 3 14 summarizes the spectral parameters described by those data. Figure 3 1 3 100 Gauss CW EPR nitroxide spectral line shapes for HIV 1PR CRF_01A/E si K55MTSL collected at X band frequency shown in (A) waterfall and (B) overlay.

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139 Table 3 1 4 CW EPR data analysis for A/Esi K55 MTSL

low/center field high/center field intensity (normalized) intensity (normalized) HIV 1PR ( 2 mM NaOAc ) 1.93 192 0.60 0.17 HIV 1PR ( 10% DMSO ) 2.01 196 0.64 0.17 CA p2 1.94 189 0.59 0.16 RTV 1.97 186 0.60 0.17 APV 1.94 193 0.59 0.15 ATV 1.93 189 0.58 0.16 DRV 1.97 190 0.57 0.17 IDV 1.95 195 0.61 0.18 LPV 2.03 194 0.60 0.17 NFV 1.99 192 0.58 0.17 SQV 2.05 195 0.58 0.18 TPV 1.97 196 0.57 0.16 Dat a from a number of biophysical techniques, including NMR and ITC, have suggested that the flaps of HIV 1 PR become locked down into the closed conformation in the presence of various inhibitors and that minor backbone fluctuations are the major source of p rotein motion (Todd, Semo et al. 1998; Ishima, Freedberg et al. 1999; Todd and Freire 1999; Todd, Luque et al. 2000; Velazquez Campoy, Todd et al. 2000; Freedberg, Ishima et al. 2002; Hornak, Okur et al. 2006) Thu s, it might be expected that changes would be reported in the CW EPR line shape for apo and ligand bound samples; however, this is not the case. Because the change in the EPR nitroxide spectral line shape with and without inhibitor/substrate is so minor, it is likely that the CW EPR is not reporting on the true motion of the flaps in these particular protein systems, but rather the internal motion of the spin label itself. As such, pulsed EPR methodology was employed, and results from those studies will b e discussed in Chapter 4. Monitoring the Autoproteolysis of HIV 1 Protease by SD S L EPR and Mass Spectrometry It is well known that HIV 1PR undergoes self proteolysis, particularly at the high concentrations necessary for many types of spectroscopic analysi s, and that the sites in which proteolytic cleavage occurs most frequently in Subtype B are positions W6 Q7, L33 E34 and L63 I64 (Tomasselli, Mildner et al. 1995; Szeltner and Polgar 1996) In addition, it is known

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140 that the amino acid substitut ions, Q7K, L33I and L63I reduce autoproteolysis in Subtype B protease without affecting protein fold ing activity, or kinetic prop erties, but rather remains similar to the wild type (Tomasselli, Mildner et al. 1995) In agreement with the literature, the constructs investigated here have the amino acid substitutions Q7K, L33I and L63I in order to slow the self proteolysis process. Note, aut oproteolysis is slowed, not completely removed. Protease inhibitors used in treatment of HIV 1 are generally designed with respect to S ubtype B (Wlodawer and Vondrasek 1998) Subtype B is the dominant HIV 1 subtype in Western Europe and the United States, though it is estimated that only about 12% of HIV infection worldwide is due to that strain. Additionally, various reports show that key difference s occur in structure and dynamics of the protease among the subtypes and CRFs as a result of the assorted sequences that define ea ch subtype. For example, we recently showed, with site directed spin labeling, that subtype polymorphisms alter the conformations and flexibility of the apo protease flaps (Kear, Blackburn et al. 2009) demonstrating the need to understand the key variation s between subtypes that may affect the e fficacy of inhibitors In addition to these general implications, this work was also specifically appl icable to the studies performed in our lab. Due to the autoproteolytic ability of HIV 1PR, our early EPR investigations have all been conducted with inactive (D25N) protease constructs. (Galiano, Bonora et al. 2007; Blackburn, Veloro et al. 2009; Galiano, Ding et al. 2009; Kear, Blackburn et al. 2009) In order to continue investigations on active protease, we need to understand how conditions during sample preparation and storage affect the autoproteolytic process. CW EPR was used to monitor the autoproteolysis of HIV 1 PR Subtype F s and CRF01_A/E s The EPR nitroxide spectral line shape is highly sensitive to mobility in the environment of the spin label, thus changes dramatically with variations in correlation t ime.

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141 Contributions to correlation time come from three modes of mo tion, including global protein R i B ). Autoproteolysis affects the rate of global protein tumbling by decreasing rotational correlation time of the spin labeled protein. As the pr otease undergoes self cleav age a peptide fragment containing the spin label is generated, which gives rise to an isotropic nitroxide line shape component that is easily discernable in the high field resonance line in the EPR spectrum. By m onitoring the i ntensity of th is spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions Data was collected on samples that were stored at 4 C, 25 C, and 37 C, as well as on samples that contain ed eithe r 0.08mM Tipranavir or 0.08mM of the substrate mimic CA p2 Circular dichroism experiments were performed prior to carrying out the EPR experiments to ensure that the spin labeled HIV 1 PR constructs co ntained proper secondary structur e. Measurements wer e collected on an Aviv 400 spectrometer using Hellma CD cuvettes with 1 cm pathlength. Samples were prepared at approximately 30 M protein concentration in 2 mM NaOAc, pH 5.0. Figure 3 14 Circular d ichroism spectra for spin labeled HIV 1 PR, subtype B (black), subtype F (red), CRF01_A/E ( blue).

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142 Figure 3 15 Overlay of day 1 (black) and day 30 (grey) area normalized 100 Gauss X Band CW EPR spectra for (A) s ubtype F s HIV 1PR stored at 37 C, 25 C and 4 C, and (B) CRF01 A/E s HIV 1PR stored at 37 C, 25 C, and 4 C. Figure 3 15 shows overlays of spectra collected immediately after purification and labeling (black) and after 30 days (grey) for HIV 1PR Subtype Fs and CRF01_A/Es at all three storage conditions (37 C, 25 C, or 4 C). LabVIEW soft ware which was provided by Drs. Christian Altenbach and Wayne Hubbell (University of California Los Angeles), was used for baseline correction and dou ble integral area normalization, allowing for easy comparison of various spectral parameters. Subsequent ly, the peak to peak intensity of the high field line (I HF ) was measured for each spectrum and converted to mole fraction of proteolyzed, unfolded protein ( b ) using Equation 3 1 and then plotted with respect to time, as shown in Figure 3 16. For these ca lculations, t = 100 is presumed equal to I and I 0 was calculated from an EPR spectrum

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143 collected immediately after purification, spin labeling, and sample preparation was complete. The value obtained is proportional to the amount of uncleaved protease re maining in the sample. The intensity of the high field line (I HF ), as expected, increased substantially over time and with increasing storage temperature (3 1) Figure s 3 17 and 3 18 give an indication of t he stability to self proteolysis of HIV 1PR at these concentrations. The prompt addition of a substrate to the enzyme solution greatly reduced the rate of autoproteolysis of the samples, as evidenced by the small change in I HF over the course of the 30 da ys when the protease samples were stored a t 25 C over the course of 30 days. These results seem to indicate that s ub type F prot ease, with tipranavir, undergoes negligible self proteolysis, and the CRF01_A/E sample underwent very little. As previously st ated, most of our early EPR investigations have all been conducted with inactive (D25N) protease constructs due to the instability of the autoproteolytic activity of the protease (Galiano, Bonora et al. 2007; Blackbu rn, Veloro et al. 2009; Galiano, Ding et al. 2009; Kear, Blackburn et al. 2009) Thus, in order to continue investigations on active protease, experiments were performed to determine how preparation and storage affect the autoproteolytic process. From t his work, it seems likely that SDSL EPR studies could be successfully applied to active HIV 1PR if the protein is purified quickly and the addition of inhibitor or substrate is swift following the refolding process. This SDSL EPR methodology has shown to be very useful in monitoring the slow autoproteolysis or degradation of a protein sample. Fitting the plots with an exponential growth function allowed for generating a term that is proportional to the amount of uncleaved protein in the sample. This type of methodology has the advantage over SDS PAGE in that it does not deplete the sample.

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144 Figure 3 16 Mole fraction of degraded protein ( b) vs. time (days) for CRF01_A/E and s ubtype F protease for samples stored at 37 C 25 C and 4 C. Function fitting was done in Origin 8.0 with the 2 component exponential growth function ( ExpGro2 ) of the form given in Equation 3 2, under the assump tion that there are two pseudo first order processes occurring in the autoproteolytic process, a fast component likely representative of the initial conversion of intact, properly folded protease to cleaved, unfolded protease, and a slow component likely r epresentative of further degradation of the protease. In this E quation, y 0 is equal to the y offset, A 1 and A 2 are amplitudes, and t 1 and t 2 are growth constants y = y 0 + A 1 e x/t1 + A 2 e x/t2 (3 2) The degradation constant t1 (in days) was ex tracted for each sample as a measure of the rate of conversion of intact, properly folded protease to unfolded/cleaved protease. For subtype F, the degradation constants t1 are 12.1 1.2, 24 3 and 34 6 days, for samples stored at 37 C, 25 C, and 4 C; respectively. For CRF01_A/E, the degradation constants t1 are 8.2 0.9, 38 7, and 54 10 days, for samples stored at 4 C, 25 C, and 37 C, respectively. The values of t1

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145 generally indicate that autoproteolysis in the Subtype F protease const ruct proceeded more quickly than did the autoproteolysis of the CRF01_A/E protease construct. Figure 3 17 Area normalized 100 G X Band EPR spectra of HIV 1PR s ubtype F in the presence of 0.08 mM tipranavir (FDA approved protease inhibitor) over the c ourse of 30 days, where A) the data are stacked such that day 1 spectrum is at the bottom and day 30 is at the top and B) overlaid with inset showing magnified high field line. Figure 3 18 Area normalized 100 G X Band EPR spectra of HIV 1PR CRF01_A/ E in the presence of 0.08 mM non hydrolyzable substrate mimic CA p2 over the course of 30 days, where A) the data are stacked such that day 1 spectrum is at the bottom and day 30 is at the top and B) overlaid with inset showing magnified high field line. After a total of 100 days of EPR data collection, apo samples were removed from the sealed capillary tubes and prepared for analysis via mass spectrometry in order to identify sites of auto proteolytic cleavage Mass spectra are reported in Figures 3 19 3 22. The identities of smaller peptide fragments were confirmed by MSMS (data not shown), but longer peptides could

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146 only be identified via deconvolution (too large for MSMS). Sites of autoproteolysis in subtype Fs and CRF01_A/Es were identified by mass spectrometry. MALDI TOF and electrospray (ESI) mass spectrometry of the subtype F and CRF 01_A/E HIV 1PR constructs indicated that both proteins have complete MSL spin labeling and were of the correct masses. In order to identify the specific sites of aut oproteolysis, electrospray mass spectrometry was used to sequence peptide fragments via collision induced dissociation (CID), more commonly known as tandem MS. Because of the incorporation of the MSL label at position K55C, the modification was included in the de novo analysis. The substitution of amino acids between the subtypes clearly influences the cleavage sites. For Subtype F, four peptides which contain the MSL label were identified conclusively by tandem MS (peptide residues 31 63, 41 55, 53 61 and 53 63). In contrast, only one peptide from the CRF 01_A/E autoproteolysis sample was identified by MS sequencing (peptide residues 37 58), but another was identified by charge deconvolution of the multiply charged isotopic envelope (peptide residues 24 71) Results indicated that the sites of autoproteolytic cleavage differed between Subtype F s and CRF01_A/Es, and are shown in the maps in Figures 3 23 and 3 24. The arrows denote HIV 1PR peptide fragments identified by mass spectrometry. Note, for CRF01_A/ E, there currently exist a few gaps in the sequence, and work is ongoing to identify these peptide fragments. Conclusions Affect of Inhibitors on CW EPR Line Shapes of HIV 1PR Subtype F and CRF01_A/E It is known that there exist dramatic differences be tween the flap motion of free and inhibitor bound protease; however, the spectra collected in this experiment reported very minor changes in the spectral line shape of protease in the apo form and with various inhibitors. Thus, it can be concluded that th e CW EPR does not report on the motion of the flaps of these protease

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147 constructs. For this reason, the system will be studied using a pulsed technique, called double electron electron resonance, to be discussed in depth in the following chapter. Monitorin g the Autoproteolysis of HIV 1 Protease by SDSL EPR and Mass Spectrometry The CW EPR methodology has proven to be very useful in monitoring the slow autoproteolysis or degradation of a protein without depletion of the sample. By evaluating the EPR spectra l line shape with respect to I HF plots of b(t), fit with a two component exponential growth function were generated, providing a term that is proportional to the amount of uncleaved protein in the sample. Many experimental techniques, such as NMR, EPR a nd ITC, require protein concentrations near 150 M, thus it is important to examine the rates of autoproteolysis at that concentration. I n all cases, the normalized intensity of to I HF is observed to increase over time, indicating protease degradation and unfolding. Additionally, the normalized intensity of I HF increased dramatically with increasing storage temperature, which indicates a temperature dependent autoproteolytic process. T he normalized spectral intensit ies I HF were converted to mole perce nt of proteolys ed, unfolded protease ( b) and plotted as a function of time The degradation constant t 1 (in days) was extracted for each sample as a measure of the rate of conversion of intact, properly folded protease to unfolded/cleaved protease. These resu lts also indicated that with a quick purification and addition of inhibitor or substrate, autoproteolysis can be reduced substantially, if not eliminated; thus, SDSL EPR experiments on active protease seem feasible. The presence of an inhibitor, which was added immediately upon completion of protein purification and prior to refolding, impeded the autoproteolytic activity of the samples, as evidenced by almost no change in the intensity of I HF over the course of the 30 days, demonstrating, via SDSL and CW EPR, that HIV 1PR can be stabilized against autoproteolysis via the timely addition of inhibitor, even at the high concentrations necessary for spectroscopic

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148 studies. Mass spectrometry revealed that in both constructs, the N terminus is sensitive to syste matic degradation, but there are also specific cleavage sites within the proteins. The most obvious sites of autoproteolysis for Subtype F are after L23, D30, G52, I63, and T73. The cleavages after L23 and D30 are conserved in CRF01_A/ E, but a new proteoly tic site appeared after Q61.

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149 Figure 3 19 Mass spectrum for HIV 1PR CRF01 A/E K55MSL, 1.5 pmol/ L in 50:50 ACN/H2O + 0.1% formic acid.

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150 Figure 3 20 Mass spectrum for HIV 1PR CRF01 A/E K55MSL 6.0 pmol/ L in 50:50 ACN/H2O + 1% formic acid

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151 Fi gure 3 21 Mass spectrum for HIV 1PR Subtype F s K55M SL 1.5 pmol/ L in 50:50 ACN/H2O + 0.1% formic aci d.

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152 Figure 3 22 Mass spectrum for HIV 1PR CRF01 A/E K55M SL 6.0 pmol/ L in 50:50 ACN/H2O + 1% formic acid

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153 Figure 3 23. Sites of autoproteoly tic cleavage in HIV 1PR s ubtype Fs K55M SL Figure 3 24. Sites of autoproteolytic cleavage in HIV 1PR CRF01 A/E K55M SL

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154 CHAPTER 4 PULSED ELECTRON PARAMAGNETI C RESONANCE STUDIES OF HIV 1 PROTEASE Introduction The previous chapter summarized results from continuous wave electron paramagnetic resonance ( CW EPR ) studies of spin labeled apo and inhibited HIV 1 protease (HIV 1PR) constructs of Subtype F and CRF01_A/E Although CW EPR has provided a means for monitoring au to proteolysis of active protease, we find that the nitroxide line shapes are dominated by the intrinsic motion of the spin label and thus do not provide a means to report on changes in flap motion and dynamics In this chapter, results from double electron electron resonance (DEER) exp eriments are reported and discussed DEER experiments provided a means to determine distance profiles between two spin labeled sites in the flaps which can be used to describe and quantify conformational sampling of protease constructs. Figure 4 1 Ribbon diagrams showing c rystal structure s of HIV 1PR in the (red) closed (PDB ID 2 PBX ) and (blue) semi open (PDB ID 1HHP ) flap conformations. T he active site hairpin flaps are shown In addition, the K55 C reporter sites are shown after modification with MTSL showing how the distance between the spin labels is expected to change as the flaps sample different conformations. Figure was originally made by Luis Galiano ( Ph.D 2008) and has been modified. S it e direc ted spin labeling (SDSL) with DEER electron paramagnetic resonance (EPR) spectroscopy was used to measure the distance between nitroxide labels attached at positions

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155 K55 C and K55 C 1 are ribbon diagrams from X ray crystal structu res of HIV (PDB ID 2BPX and IHHP, respectively) Each of these ribbon diagrams has been modified at positions A s the flaps undergo a conformational from about 33 to about 36 a net change of approximately 3 DEER distance measurements were successful in providing a description of the co nformational ensembles of the flap region of various subtypes of HIV 1PR (Galiano, Bonora et al. 2007; Galiano, Ding et al. 2009; Kear, Blackburn et al. 2009) Distance measurements by SDSL DEER EPR are based on th e magnitude of the magnetic dipolar coupling of the unpaired spins, which is proportional to 1/ r 3 where r is distance between the two spins (Pannier, Veit et al. 2000; Jeschke and Polyhach 2007) Distance profile s from spin labeled constructs of Subtype B si Subtype C si Subtype F si CRF01_A/E si and patient isolate V6 i and MDR769 i without ligand were analyzed in order to characterize the conformations and flexibility of the flap region of the pr otease and to iden tify what effect polymorphisms have on the conformational ensemble of HIV 1PR Additionally, in order to monitor differences in flap conformations upon inhibitor binding between constructs, HIV 1PR CRF01_A/E si constructs were also analyzed upon addition of inhibitors and a non hydrolysable substrate mimic CA p2 Chapter 1 provided a more detailed discussion on construct nomenclature and amino acid substitution code. In our naming scheme, a refers to sequences that have incorporated the Q7 K, L33I, and L63I substitutions that stabilize against autoproteolysis. The ( D25N ). Amino acid substitution code (e.g. D25N) is given by amino acid residue to be substituted out, followed by

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156 the residue number, followed by the amino acid to be substituted in (e.g. D25N the aspartic acid residue at position number 25 was mutated to an asparagine residue). Previous Work Initial work on DEER measurements of HIV 1PR was performed by former Fanucci group member Dr. Luis Galiano ( Ph.D 2008) At that time, we did not ha v e the capability here at the University of Florida ( UF ) to collect DEER data, and experiments were performed in the lab of Peter Fajer at the National High Magnetic Field Lab (NHMFL) wit h the help of post doctor al res earch fellow Marco Bonora This original data was collected at X band on a Brker EleXsys E580/E680 equipped with the ER 4118X MD5 Dielectric Ring Resonator Dr. demonstrated the success of the DEER methodol ogy when applied to the study of flap conformations in Subtype B si HIV 1PR These results showed clear differences in the most probable distance between the flaps and the flexibility of the flaps in the presence and absence of the protease inhibitor Riton avir (Galiano, Bo nora et al. 2007) Figure 4 2. DEER results of subtype B HIV 1P R with (grey) and without (black) the FDA approved inhibitor Ritonavir ; (A) d ipolar modulated echo data and (B) resulting distance distribution profile. The dipolar modulated echo data fo r the apo constr uct (Figure 4 2A black ) is noticea bl y differen t than that of the protease in the presence of protease inhibitor Ritonavir (Figure 4 2A,

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157 grey). The resulting distance distribution profiles (Figure 4 2B) report very different flap conformat ions and flexibility. Data shows that the average distance between the la beled sites on each flap shift by about 3 (from 36 to 33 ) upon addition of Ritonavir indicating that the flaps become locked in a close d position upon inhibitor binding Addi tionally, the breadth of the distance distribution profile decrease d dram ati cally upon addition of Ritonavir, indicating that the range of motion or flexibility of the flaps decreased as well. These results provided a glimpse into the structural mechanism of inhibitor resistance This work was noticed and expanded upon by the lab of Steve Kent at University of Chicago. Kent and co workers obtained both active and inactive (D25N) K55MTSL labeled HIV 1 protease constructs via total chemical synthe sis and re ported on interflap distances determined by DEER, in the presence of three peptidomimetic inhibitors. Each of the inhibitors, namely MVT 101, KVS 1, and JG 365, represent different stages of the enzyme catalyzed peptide bond hydrolysis reaction ; MVT 101 i s structurally similar to an earl y transition state, KVS 1 mimics the tetrahedral intermediate in the reaction and JG 365 mi mics a later transition state (Baca and Kent 1993; Torbeev, Mandal et al. 2008) The resultant distance distribution profiles demonstrated that the flaps adopted different catalytic properties throughout the course of the catalytic reaction. In the early stages of the reaction, the flaps are found predominantly in the closed conformation with little flexibility. As the reaction proceeds, data indicated that the flaps adopt a more open conformation with increased flex ibility perhaps aiding in product release (Torbeev, Raghuraman et al. 2008) Further work in the Fanucci lab performed by Luis Galiano with the assistance of Dr. Ralph Weber, Senior Applications Scientist for Br ker 's EPR division focused on understanding the role of secondary polymorphisms on acquired drug resistance by the protease. Specifically,

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158 the V6 and MDR769 drug resistance constructs were investigated V6 i s a clinical isolate from a pediatric patient treated with RTV and MDR 769 was isolated from a patient previou sly treated with IDV NFV, SQV and APV. Dr. Galiano was able to show that mutations that arise in response to PI treatment alter the flap conformations of the apoenzyme, thus affecting the c onformational ensemble of the protease. In order to provide structural insight into the experimental data, MD simulations were performed in the lab of Carlos Simmerl ing, and the trends are in excellent agreement. Subtype B adapts an average conformation similar to the semi open conformation described by X ray crystallographic studies. Conversely, MDR769 adopts a more open average conformation and V6 a more closed average conformation with respect to Subtype B. These results were important because they d emonstrated that drug induced polymorphisms, often secondary with respect to proximity to the active site, can affect flap conformations and flexibility, likely contributing to drug resistance. These findings that were featured in an issue of Chemical and Engineering News (Drahl 2009) will likely contribute toward the design of inhibit ors that can tolerate various point mutations while maintaining binding affinity. Subsequent wor k on Subtype B protease was performed by Mandy E. Blackburn, who compared the dis tance distribution profiles of HIV 1PR in the apo form with those in the presen ce of nine separate FDA approved protease inhibitors and a substrate mimic ( Figure 4 3 ) (Blackburn, Veloro et al. 2009) With this work came the discovery that with sufficiently high signal to noise ratio (SNR) in the dipolar modulated echo curve, information about the relative population distributions of the major flap conformations can be extracted in order to describe the energy landscape and conformational sampling of HIV 1P R (a detailed discussion of the described analysis method can be found in Chapter 2). Inhibited HIV 1PR distance profiles were

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159 split into two groups, those that showed a strong affect on flap closing (Figure 4 3A) and those that revealed a weak affect (Figure 4 3B) and d etailed analyses were performed to provide distinct relative population percentages for the conformational e nsemble of the flap s upon interaction with inhibitor or substrate (Figure 4 3 C) Inhibitors defined as having strong interactions have at least 70% of the conformational ensemble in the closed flap conformation IDV, NFV, and ATV moderate/ Figure 4 3 C shows that the flaps, in the presence of those inhibitors, were predominantly found in the semi open conformation. M oderate affects were seen for ATV, where approximately 40 % of the conformational ensemble is in the closed conformation. Weak affects were seen for IDV and NFV with less than 20% of the conformational ensemble in the closed conformation (Blackburn, Veloro et al. 2009) Figure 4 3 Distance distributi on profiles of subtype B HIV 1PR with inhi bitors that have shown a weak effect on flap closing. The apo distance profile is given in both groups for reference. Error is estimated at 2.5%.

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160 Compariso ns were made between the percentage of the closed conformer seen with DEER and enzymatic inhibition constants, thermodynamic dissociation constants, and the number of non water mediated hydrogen bonds identified in crystallographic complexes. No strong co rrelation seems to exist between the relative percent closed co n formation and the K i or K D values; however, t he DEER data does seem to be in agreement with th e number of non water mediated h ydrogen bonds between the inhibitor and the protease suggesting a correlation regarding inhibitor effectiveness. This work was also important because it showed that with a high signal to noise ratio (SNR) in the dipolar modulated echo curve, information can be gained regarding the relative conformational ensembles of t he flaps of HIV 1PR. Table 4 1 Comparison of relative percentage of closed flap conformation of HIV 1PR subtype B to p ublished v alues of K i K D and the number of non water mediated hydrogen bonds between inhibitor and protease con struct (Blackburn, Veloro et al. 200 9) Inhibitor Relative % K i K D # non water (abbreviation) c losed c onformation (nM) (pM) mediated Hydrogen bonds (5%) (Blackburn, Veloro et al. 2009) Saquinavir (SQV) 93 1.3 a 280 c 7 d Tipr anavir (TPV) 91 0.019 b 19 b 6 e Ritonavir (RTV) 90 0.7 a 100 c 7 f Darunavir (DRV) 87 0.010 b 10 b 6 d Lopinavir (LPV) 84 0.05 a 36 c 3 g Amprenavir (APV) 76 0.17 a 220 c 5 d Atazanavir (ATV) 41 0.48 a NA 3 b Indinavir (IDV) 14 3.9 a 590 c 3 d Nelfinavir (NFV) 14 1. 2 a 670 c 2 d *a (Clemente, Moose et al. 2004) b. (Muzammil, Armstrong et al. 2007) c. (Yanchunas, Langley et al. 2005) d. (Prabu Jeyabalan, King et al. 2006) e. (Nalam, Peeters et al. 2007) f. (Prabu Jeyabalan, Nalivaika et al. 2003) g. (Reddy, Ali et al. 2007) The SDSL DEER EPR resul ts presented in this chapter were collected in orde r to examine conformational sampling in the apoenzyme and to determine how natural ly evolved and drug induc ed polymorphisms alter the effect of inhibitors on the conformational ensemble and flap flexibilit y of non B subtypes and drug resistant patient isolates from protease inhibitor exposed patients. The dipolar modulated echo data and resulting distance distribution profiles differed

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161 greatly among the apo protease constructs From detailed analysis of t he echo data, we reported differences in populations of four distinct flap conformations, namely wide open, semi open, closed, and tucked/curled. These results demonstrated that natural and drug induced polymorphisms in the amino acid sequence of various subtypes and patient isolates whether because of naturally occurring amino acid substitutions or drug pressure selected mutations alter the average flap conformations and flexibility of the flaps These results, which may indicate how select mutations ma y play a role in viral fitness and drug resistance, were featured in an issue of AIDS Weekly (30 Nov. 2009) Materials and Methods Materials The chemicals, reagents, and supplies were obtained fro m Fisher Scientific (Pittsburg, Pennsylvania) and used as received, with a few noted exceptions. pET23 plasmid DNA was purchased from Novagen (Gibbstown, New Jersey) and sequence specific information is given in the appendix HiTrap Q HP Anion Exchange column, HiPrep 16/60 S ephacryl S 200 high resolution s ize e xclusion column was purchased from GE Biosciences (formerly Amersham, Pittsburg, Pennsylvania). HIV 1P R DNA was synthesized and subsequently purchased from DNA2.0 (Menlo Park, California). 4 m alei mido 2,2,6,6 t etramethyl 1 piperidinyloxy (4 m aleimido TEMPO, MSL) was purchased from Sigma Aldrich (St. Louis, MO ). (1 o xyl 2,2,5,5 tetramethyl 3 pyrroline 3 methyl) methanethiosulfonate spin label ( MTSL ) was purchased from Toronto Research Chemicals, I nc (North York, Ontario, Canada). The QuikChange site directed mutagenesis kit was purchased from Stratagene (La Jolla, California). 0.60 I.D. x 0.84 O.D. capillary tubes were purchased from Fiber Optic Center (New Bedford, Massachusetts). BL21*(DE3) pL ysS E. coli cells were purchased from Invitrogen (Carlsbad, California). NaOAc (C 2 D 3 O 2 ) D 2 O, d 8 glycerol were purchased from Cambridge Isotope Labs

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162 ( Andover, MA ). Ritonavir, I ndinavir, T ipranavir, Darunavir, A mprenavir, A tazanavir, N elfinivir, Saquinav ir, and L opinavir were generously received from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH ( Bethesda, Maryland) (NIH) The non hydrolysable substrate mimic, CA p2 (H Arg Val Leu r Phe Glu Ala Nle NH2 (R V L r F E A Nle NH2, r = reduced) was synthesized and purchased from the University of Florida Protein Chemistry Core Facility (Gainesville, Florida). Methods Details of protein c onstructs Six different HIV 1PR construc ts will be discussed in this chapter ; namely A/E s i (CRF01_A/E; D25N, K55C, C67A, C95A, Q7K, L33I, L63I), F si (Subtype F; D25N, K55C, C67A, C95A, Q7K, L33I, L63I ), B s i (Subtype B; D25N, K55C, C67A, C95A, Q7K, L33I, L63I ), C s i (Subtype C ; D25N, K55C, C67A, C95A, Q7K, L33I, L63I ), V6 i ( Patient isolate V6 ; D25N, K55 C, C67A, C95A), and MDR769 i ( multi drug resistant patient isolate MDR769 ; D25N, K55 C, C67A, C95A). V6 was isolated fro m a pediatric patient previously treated with the protease inhibitor Ritonavir and MDR769 is a multi drug resistant protease isolated from a patient previously treated with Saquinavir, Indinavir, Amprenavir and Nelfinivir. All constructs were labeled wi th (1 Oxyl 2,2,5,5 Tetramethy l Pyrroline 3 Methyl) methanethiosulfonate spin label ( MTSL ) at the K55 C and K55 C sites. Structures of the spin labels and spin labeled side chains are shown in Figure 2 10 HIV 1PR variant constructs contain the following polymorphisms relative to the LAI consensus sequence of Subtype B: Subtype C T12S, I15V, L19I, M36I, S37A, H69K, N88D, L89M, I93L Subtype F I15V, E35D, S37N, R41K, R57K CRF01_A/E I13V, E35D, M36I, S37N, R41K, H69K, and L89M V6 K20R, V32I, M36I A71V, V82A, L90M MDR769 L10I, M36V, M46L, I54V, I62V A71V, V82A, I84V, L90M

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163 Tables 4 2 (residues 1 50) and 4 3 (residues 51 99) giv e details of protein sequences for each construct. H ighlighted in grey a re the positions of the stabilizing mutations of the D25N active site mutation s of the K55C reporter site mutation s and of the CYS to ALA mutations required to facilitate SDSL Italicized and in yellow are the residues on each variant that differ from those in the LAI consensus sequence of Subtyp e B. Figure 4 4 shows ribbon diagrams of HIV 1PR structure highlighting the sites of the protein constructs where polymorphisms occur. Table 4 2 HIV 1 P R v ariant s equence a lignment r esidues 1 50 1 50 Subtype B: PQITLW K RPLVTIKIG GQLKEALL N TGADDTV I EEMSLPGRWKPKMIGGI Subtype C: PQITLW K RPLV S IK V GGQ I KEALL N TGADDTV I EE IA LPGRWKPKMIGGI Subtype F: PQITLW K RPLVTIK V GGQLK EALL N TGADD TV I E D M N LPG K WKPKMIGGI CRF01_A/E: PQITLW K RPL VT V KIGGQLK EALL N TGADDTV I E DIN LPG K WKPKMIGGI V6: PQITLWQRPLVTIKIGGQL R EALL N TGADDT I F EE I SLPGRWKPKMIGGI MDR769: PQITLWQRP I VTIKIGGQLKEALL N TGADDTVLEE VN LPGRWKPK L IGGI Table 4 3 HIV 1P R v ariant s equence a lignment r esidues 51 99 51 99 Subtype B: GGFI C VRQYD QI I IEI A GHKAIGTVLVGPT PVNIIGRNLLTQIG A TLNF Subtype C: G GFI C VRQYDQI I IEI A G K KAIGTVLVGPTPVNIIGRN M L TQ L G A TLNF Subtype F: GGFI C V K QYDQI I IEI A GHKAIGTVLVGPTPVNIIGRNLLTQIG A TLNF CRF01_A/E: GGFI C VRQYD QI I IEI A G K KAIGTVLVGPTPVNIIGRN M LTQIG A TLNF V6: GGFI C VRQYD QI P IEI A GHK V IGTVLVGPT P A NIIGRNL M TQIG A TLNF MDR769: GGF V C VRQYDQ VP IEI A GHK V I GTVLVGPTP A N V IGRNL M TQIG A TLNF Figure 4 4 R ibbon diagrams of HIV 1PR (PDB ID: 2PC0) with amino acid differences relative to the LAI consensus seque nce highlighted by colored spheres All diagrams were rendered with VMD (Humphrey 1996)

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164 Expression of HIV 1 p rotease The over e xpression of e ach protein construct was c arried out in the pET 23a vector from Novagen Expression of Subtype F and CR F01_A/E constructs was described in Chapter 3 and expression of Subtype B and C proceeded via a similar method. After induction with IPTG, the over e xpression of MDR769 and V6 constructs was carried out at 20 C for longer periods of time, approximately 10 hours each due to lower expression levels Purification of HIV 1 p rotease Purification of HIV 1 PR was carri ed out as described in Chapter 3, with the exception of a change in the pH of the anion exchange buffer. The pH of this buffer is dependent upon the isoelectric point (pI) of the protein which was calculated using the isoelectric point calculator program on EXPASY ( http://www.expasy.ch/tools ) Th e F, CRF01 _A/E V6, and MDR769 constructs are 9.39, 9.59, 9.28, 9.53, 9.12, and 9.06, respectively; thus the respective pHs of the anion exchange buffer were 8.85, 9.05, 8.75, 9.05, 8.65, and 8.50. Using this purification scheme, we are able to produce protein that was estimated to be >95 % pure by SDS PAGE. Spin label ing Spin labeling was carried out as described in Chapter 3 All DEER experiments were carried out using MTSL spin label When spin labeling was needed, the purified and refolded protein sample was buff er exchanged into 10 mM Tris HCl, pH 6.9 (further details of spin labeling discussed in next section) after the final purification step. Approximately 1 mg of spin label was dissolved in 1 00 L ethanol, and added to approximately 40 mL of HIV 1 PR in 10 mM Tris HCl, pH 6.9. The spin labeling reaction was carried out in the dark (via wrapping reaction tube in aluminum foil) at room temperature for approximately 4 6 hours followed by 6 8 hours at 4 C for inactive (D25N) constructs, and at 4 C for approxima tely 8 12 hours for active

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165 (D25) constructs. At this time, t he sample solution was centrifuged at 12000 rpm for 20 minutes at 4 C to remove solid impurities and aggregated proteins. The sample was then buffer exchanged into 2 mM NaOAc, pH 5, and concent rated to OD 280 =1.25. Buffer requirements To extend the spin memory relaxation time, T m, which improves the signal to noise ratios (SNR) of the experimental echo curves, samples were buffer exchanged into d euterated solvent composed of 2 mM Na OAc (C 2 D 3 O 2 ) in D 2 O, pH 5.0, with 30% d 8 glyc erol (used as a glassing agent). The buffer exchange process is carried out using a 5 mL HiTrap Desalting column from GE Healthcare (packed with Sephadex G25) which is first washed successively with 3 4 column volume s (15 20 mL) of nanopure water (nH 2 O), 1M NaCl, nH 2 O, 0.5 M NaOH, nH 2 O, and NaOAc (pH 5). The column is then equilibrated with 10 mL deuterated NaOAc (pH 5). 1 mL sample (OD280 = 2.5 to 2.7) is then injected, followed by 0.5 mL of deuterated NaOAc; all flow through to this point is discarded. Another 1.5 mL of deuterated NaOAc is injected while collecting the buffer exchanged sample in another tube. Finally, 0.5 mL of nH 2 O is injected while collecting the last 0.5 mL of buffer exchanged sample The 2 mL of buffer exchanged sample is then concentrated to the appropritate volume. Circular d ichroism s pectroscopy To demonstrate that HIV 1 PR constructs retained proper secondary structure after spin labeling circular dichroism (CD) experiments were perf ormed as described in Chapter 3. Typical parameters used for circular Dichroism experiments are summarized in Table 3 10. The results of these exp eriments are given in Figure 4 5 Results match published circular dichroism results for HIV 1PR, indicatin g proper folding of the protease.

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166 Figure 4 5 Circular d ichroism spectra for spin labeled HIV 1 PR, subtype B LAI consensus sequence (cyan), subtype C (red), CRF01_A/E (green), subtype F (blue), V6 (black), and MDR769 (purple). DEER e xperiments P ulsed EPR data were collected on a Brker EleXsys E580 EPR spectrometer (Billerica, MA) equipped with the ER 4118X MD 5 dielectric ring resonator at a temperature of 65 K (cooled via liquid helium) and 4 pulse DEER sequence described in Chapter 2. DEER data co llection was typically preceded by a series of preliminary experiments also described in Chapter 2, designed to accurately determine the center field, T m the d 0 (time in ns at which the echo begins) and pulse gate (the breadth of the echo in ns), and app ropriate positions for the resonance approximately 26 Gauss below the central resonance DEER d ata a nalysis Data analysis was carried out as descri bed previously by Blackburn et al. ( Blackburn, Veloro et al. 2009) The raw experim ental data was processed via Tikhonov Regularization using DeerAnalysis2008 software, available at http://www.epr.ethz.ch/software/index D istance

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167 prof iles were reconstructed with a series of Gaussian functions via an in house Matl ab based program called DeerSim. Population v alidation Population validation was performed as discussed previously by Blackburn et al. (Blackburn, Veloro et al. 2009) The validity of each popu lation with a relative percentage of 10 % or below was tested via population suppression using DeerSim and DeerAnalysis2008. This concept is discussed in detail in Chapter 2. Results and Discussion Subtype P olymorphisms F ound A mong S ubtypes B, C, F, CRF01_A/E and P atie nt I solates V6 and MDR769 C onfer A ltered F lap C on f ormations and F lexibility in the Apo P rotease Introduction As discussed in detail in previous chapters, HIV 1 is categorized into different groups, subtypes (or clades) and circulating r ecombinant forms (C RFs). G roups refer to distinctive viral lineages, subtypes are specific taxonomic groups within a lineage, and CRFs are recombinant forms of the virus comprised of different viral strains (Kantor, Katzenstein et al. 2005) Each subtype and CRF is defined by a unique set of naturally occurring polymorphisms. Previous SDSL DEER results have provide d detailed information about the flap conformations sampled in Subtype B. Distinctive flap c onformations have been desc ribed and are referred to as curled, closed, semi open, and wide open Each of these conformations has been detected in the distance profile of Subtype B protease and has been modeled with molecular dynamics (MD) simulations (Galiano, Bonora et al. 2007; Ding, Layten et al. 2008; Kear, Blackburn et al. 2009; Torbeev, Raghuraman et al. 2009) Protease inhibitors used in treatment of HIV 1 are often designed with respect to subtype B (Wlodawer and Vondrasek 1998) thus it is of great importan ce to understand how subtype polymorphisms affect protein structure and flexibility and

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168 thus the efficacy of inhibitors (Rose, Craik e t al. 1998; Velazquez Campoy, Vega et al. 2002; Clemente, Coman et al. 2006; Coman, Robbins et al. 2007; Sanches, Krauchenco et al. 2007; Bandaranayake, Prabu Jeyabalan et al. 2008; Coman, Robbins et al. 2008) Zero time s election Figure 4 6 Dipolar modulated echo curves u sed for zero time selection of A) s ubtype B si B) s ubtype C si C) s ubtype F si D) CRF01_A/E si E) V6 i and F) MDR769 i The first step in DEER data analysis by TKR is to accurately determine the zero time. As discussed in Chapter 2, DEER data was collected with a small amount of negative time (Figure 2 17A). If the incorrect zero time is selected, the distance distribution will be shifted towards either smaller or greater distances (Figure 2 17B). DEER data in the form of a dipolar 300 to 300 and plotted in Origin8.0 (Figure 4 6), then fit to a function called GaussAmp (the amplitude version of the Gaussian peak function) of the form given in Equation 4 1, where y 0 is the y offs et, A is the amplitude, y is

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169 equal to y 0 + A, w = the full width at half max, and xc is the center of the function; thus x c is equal to the zero time. Table 4 4 reports the zero times chosen for DEER data analysis. (4 1) Table 4 4. Zero times chosen for apo data analysis. Subtype Zero time B si 110 ns C si 115 ns F si 305 ns CRF01_A/E si 108 ns V6 i 108 ns MDR769 i 307 ns Background subtracted dipolar modulated echo curves Background subtracted ec ho data with overlaid TKR fits are shown for data analyzed without and with a long pass filter, respectively, in Figure 4 7A and 4 7B. The long pass filter, an optical filter that selectively attenuates shorter wavelengths, was applied using DeerAnalysis2 008 TKR analysis software. Calculated values of SNR are given in Table 4 5. The following sections provide details for all data analysis, including: A) Long pass filtered and background subtracted dipolar echo curve (black) overlaid with the T KR regene rated echo curve from DeerAnalysis (red) and with the reconstructed echo curve from DeerSim (blue) corresponding to the sum of distance profiles comprising the Gaussian populations shown in D. B) L curve utilized to choose the appropriate regularizatio n parameter. C) TKR distance profile (red) overlaid with summed Gaussian population profile (blue). D) Individual populations in Gaussian reconstruction of the TKR distance profile. E) Pake dipolar pattern resulting from Fourier transform of the backgro und subtracted dipolar echo curve (black) and the TKR fit echo curve from DeerAnalysis (red).

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170 Additionally, a t able is provided which summarizes values determined from both TKR analysis Gaussian reconstruction P opulation validation procedures and a Fi gure summarizing the population validation process are given, and include: A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population marked B) Individual populations for the Gaussian reconstruction. C J, where applicable) Background subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Figure 4 7. Background subtracted t ime domain echo data (bl ack) with fits generated by TKR for both A) non optically filtered and B ) l ong pass filtered data Table 4 5. Values of signal:noise ratios (S NRs) for background subtracted echo data. Construct Raw SNR Long pass filtered SNR Subtype B 20 28 Subtype C 20 21 Subtype F 13 20 CRF01_A/E 13 20 V6 13 23 MDR769 11 19

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171 Data analysis and population validation process: Subtype B s i Figure 4 8 Data analysis for HIV 1PR s ubtype B si a po. A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D, B) L c urve ( 10), C) d istance profile from TKR analysis (red) overlaid with the summed Gaussian p opulation profile (blue dashed), D) i ndividual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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172 Figure 4 9. Populati on validation process for HIV 1P R s ubtype B si apo A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue das hed). B ) Individual populations for the Gaussian reconstruction. D J) Background subtracted dipola r echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Table 4 6 Results of Gaussian reconstruction and population validati o n procedures for B si Subtype B si Apo R ( ) FWHM ( ) TKR % Final % Unassigned 22.2 3.0 2 0 Tucked 28.5 4.0 5 5 Closed 33.3 3.9 23 24 Semi open 36.1 5.2 65 67 Wide open 40.4 2.8 5 4

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173 Data analysis and population validation process: Subtype C si F igure 4 10 Data analysis for HIV 1PR s ubtype Csi a po. ( A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. ( B) L curv e ). ( C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). ( D) Individual populations necessary for Gaussian reconstruction process, and ( E) Pake dipolar pattern

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174 Figure 4 11 Popu lation validation proc ess for HIV 1 P R s ubtype C si apo A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue das hed). B ) Individual populations for the Gaussian reconstruction. D J) Background subtracted di polar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Table 4 7 Results of Gaussian reconstruction and population validation procedures for C si Subt ype C si Apo R ( ) FWHM ( ) TKR % Final % Tucked 29.7 3.1 13 13 Closed 33.3 2.8 8 8 Semi open 36.7 4.0 52 52 Wide open 40.2 3.3 27 27

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175 Data analysis and population validation process: Subtype F si Figure 4 12 Data analysi s for HIV 1PR s ubtype Fsi a po. A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve ). C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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176 Figu re 4 13 Populati on validation process for HIV 1P R s ubtype F si apo A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue das hed). B ) Individual populations for the Gaussian reconstruction. D J) Background subtracted dipola r echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Table 4 8 Distance distribution profile for apo HIV 1PR Fsi Subtype F si Apo R ( ) FWHM ( ) TKR % Final % Tucked 28.8 4.8 12 12 Closed 32.1 4.9 16 17 Semi open 35.8 6.8 68 71 Wide open 46.5 5.4 2 0 Unassigned 53.0 5.9 2 0

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177 Data a na lysis and population v alidation : CRF01_A/E si Figure 4 14 Data analysis for HIV 1PR CRF01_A/Esi Apo. A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve ). C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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178 Figure 4 15 Population validation process for HIV 1 P R CRF01_A/E si apo A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue das hed). B ) Individual populations for the Gaussian reconstruction. D J) Background subtracted dipolar e cho curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Table 4 9 Distance distribution profile for HIV 1PR A/Esi CRF01_A/Esi Apo R ( ) FWHM ( ) TKR % Final % Curled 21.2 3.1 1 0 Tucked 29.7 5.2 13 14 Closed 33.3 5.4 25 25 Semi open 36.7 6.5 59 61 Wide open 40.2 4.7 2 0

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179 Data analysis and p opulation v alidation : V6 i Figure 4 16 Data analysis for HIV 1PR pa tient isolate V6i a po. A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve ). C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern B A C

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180 Figure 4 17 Population va lidation proc ess for HIV 1 P R V6i apo A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue das hed). B ) Individual populations for the Gaussian reconstruction. D J) Background subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Table 4 10 Distance distribution profile for V6i V6i Apo R ( ) FWHM ( ) TKR % Final % Tucked 29.3 3.85 8 8 Closed 33.2 6.0 10 10 Semi open 36.0 6.5 82 82

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181 Data a na lysis and population v alidation : MDR769 i Figure 4 18 Data analysis for HIV 1PR patient isolate MDR769i a po. A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve ). C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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182 Figure 4 19 Population va lidation proc ess for HIV 1P R MDR769i apo A) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue das hed). B ) Individual populations for the Gaussian reconstruction. D J) Background subtracted dipolar echo cu rve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue).

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183 Table 4 11 Distance distribution profile for MDR769i MDR796i Apo R ( ) FWHM ( ) TKR % Final % Una ssigned 20.4 3.4 7 0 Curled 26.3 3.4 7 7 Closed 33.4 3.5 7 9 Semi open 36.4 5.4 74 79 Wide open 44.9 3.3 5 5 Polymorphism induced shifts in the conformational ensemble The distance profiles for each of the apo proteases from s ubtypes B, C, F, CRF01_A/E, V6, and MDR 769, are shown in Figure 4 20A (Kear, Blackburn et al. 2009) For each of the constructs, a peak centered near 33 corresponding to the closed co nformation is required for regeneration of the TKR distance profile. The semi open conformation, with a distance of 36 was found to be the major sub population required to regenerate each of the distance profiles. This conformation was assigned from mo lecular dynamic ( MD ) simulations A sub population centered at 2530 is required for adequate fitting of the distance profiles. These distances correspond to conformations in which the flaps are tucked in towards one another or into the active site poc ket (Scott and Schiffer 2000; Heaslet, Rosenfeld et al. 2007) Distances closer to 25 are assigned to a curled conformation, while distances closer to 30 are assigned to a tucked flap conformation. Finally, a pop ulation with average distances of 40 45 is needed to regenerate each of the d istance profile s for the apo proteases. These distances correspond to a wide open conformat ion of the flaps which has seen in MD simulations of Subtype B (Ding, Layten et al. 2008) Figure 4 20 B shows the relative percentages of each of the sub populations utilized in the Gaussian reconstruction of each of the T KR distance profiles, and Figures 4 21 and 4 22 show this same data, but b roken into individual conformations and contructs, respectively. Figure 4 23 shows individual overlays of the apo Subtype B distance profile with

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184 Figure 4 20 DEER results for HIV 1P R s ubtypes B, C, F, CRF01_A/E, and drug resist ant patient isolates in the apo; A) d istance distribution profiles of each construct, and B) results of population analysis. Figure 4 21 R elative percentage of A) tucked/curled, B) closed, C) semi open, and D) wide open co nformations of protease constructs in the apo form

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185 Figure 4 2 2 Individual plots showing relative percentage of tucked/curled, closed, semi open and wide open confo rmations in the apo form of A) s ubtype B, B) s ubtype C, C ) s ubtype F, D) CRF01_A/E, E ) V6, and F) MDR769 variants. Table 4 12. Summary of distance parameters obtained from DEER distance profiles of HIV 1PR constructs. Construct Range (Span) Most Probable distance Average Distance ( B si 24 45 (21) 35.2 35.2 C si 25 45 (20) 36.9 36.5 F si 24 45 (21) 35.1 34.3 CRF01_A/E si 25 45 (20) 34.8 35.2 V6 i 25 45 (20) 35.8 35.2 MDR769 i 22 49 (27) 36.3 35.9 The different in the overall shape and breadth of the distance profiles shown in Fig ures 4 2 0 A and 4 23 indicate that the individual polymorphisms that define various subtypes, CRFs, and patient isol ates have a drastic impact on ave rage flap conformation and flexibility. Table 4 12 lists values of the overall span, the most probable distance and the average distance for each construct. The span of flap motion was calculated using the derivative of th e distance profile, and the double derivative was used to help locate the centers of the individual Gaussian peaks for

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186 the reconstruction procedure, as shown in Figure 4 24. The most probable distance is simply the distance corresponding to the most inten se point in the distance profile (Kear, Blackburn et al. 2009) Figure 4 23 D istance distribution pro file overlays of s ubtype B and A) s ubtype C, B) CRF01_A/E, C) s ubtype F, D) V6 and E) M DR769.

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187 Figure 4 24. A) Derivative spectra of distance profiles of each variant used to calculate the span of the distance profile, and B) s econd derivative spectra of distance profiles of each variant used to help identify the centers of the individua l Gaussian peaks for the reconstruction procedure. From analysis of the relative percentages of each sub population s, the a ffects of natural and drug pressure selected polymorphisms on the average flap conformation can be understood as shifti ng the conformational ensemble of the system and changing the fl ap flexibility. The distance profile for Subtype C indicates a relatively large percentage of wide open flap conformation. It is possible that this difference could be attributed to the pres ence of three unique polymorphisms located at positions 12, 19, and 93 Changes in flexibility are inferred from the breadth of each of the sub populations. T he breadths of the closed populations of Subtype F, CR01_A/E and V6 have broader than average br eadths seen for the other apo constructs indicating either an increase in flap flexibility or flap instability for the closed conformation (Kear, Blackburn et al. 2009) T he distance profil es for V6 and MDR769 differ slightly from those in earlier report (Galiano, Ding et al. 2009) ; however, the data reported here are consistent with the conclusion that MDR769 has a larger relative percentage of wide open conformation than

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188 Subtype B. The distance profile for V6 shows a greater relative percentage of V6 in the tucked/curled conformation, however the average value for flap conforma tion matches within error that of B I nhibitor I n duced F lap C losure i n CRF01_A/E C onstructs As previously described, t here are currently 9 FDA approved protease inhibitors that are administered to HIV 1 pat ients in the form of a cocktail. These PIs were designed with respect to subtype B (Wlodawer and Vondrasek 1998) ; thus, however the efficacy of the inhibitors on other subtypes, circulating recombinant forms (CRFs), and patient isolates differ dramatically. It is thus of great importance to understand how variant specific po lymorphisms alter protein structure and flexibility and thus efficacy of inhibitors (Rose, Craik et al. 1998; Velazquez Campoy, Vega et al. 2002; Clemente, Coman et al. 2006; Sanches, Krauchenco et al. 2007; Bandaran ayake, Prabu Jeyabalan et al. 2008; Coman, Robbins et al. 2008) Additionally, it would be very beneficial to identify which inhibitors or combinations of inhibitors effectively close the flaps of each individual variant, thereby possibly reducing the d rug load given to each HIV 1 patient. Apo data was reported and discussed in the previous section and will not be repeated here. This section specifically reports on DEER data for HIV 1PR CRF01_A/E with each of nine FDA approved protease inhibitors and t he non hydrolyzable substrate mimic CA p2. The following sections provide all details for data analysis and population validation, as de scribed in the previous section. CRF 01_A/E si dipolar modulation zero time determination The proper zero times were de termined as described previously. Figure 4 25 shows the truncated dipolar evolution curves fit to a GaussAmp function of the form shown in Equation 4 1. Table 4 13 reports on the zero times chosen for CRF01_A/Esi data analysis.

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189 Table 4 13. Zero times c hosen for CRF01_A/Esi data analysis. Inhibitor/Substrate Zero time RTV 105 ns IDV 113 ns LPV 311 ns TPV 309 ns SQV 308 ns DRV 311 ns NFV 307 ns ATV 311 ns APV 311 ns CA p2 106 ns Figure 4 25 Truncated d ipolar modulated echo curves used for zero time selection of CRF01_A/E with A) RTV, B) IDV, C) LPV, D) TPV, E), SQV, F) DRV, G) NFV, H) ATV, I) APV an d J) CA p2

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190 CRF01_A/E si with CA p2 Figure 4 26 Data analysis for HIV 1PR CRF01_A/E wit h CA p2 A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) ov erlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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191 Figure 4 27 Population validation process for HIV 1P R CRF01_A/E with CA p 2 B ackgro und subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned, B) wide open and unassigned, and C) curled, tucked, semi open wide open, and unassigned suppressed Table 4 14. Distance distribution profile for CRF01_A/E with CA p2 CRF01_A/Esi CA p2 R ( ) FWHM ( ) TKR % Final % Curled 26.2 1.5 7 7 Tucked 30.5 1.8 8 8 Closed 33.1 2.5 75 77 Semi open 36.7 1.8 5 5 Wide open 42.4 2.0 3 3 Wide open 48.7 2.0 2 0

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192 CRF01_A/E si with NFV Figure 4 28 Data analysis for HIV 1PR CRF01_A/E NFV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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193 Figure 4 29 Population validation process for HIV 1P R CRF01_A/E NFV B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the d istance profile with one or more populations suppressed (blue) ; with A) curled, B) curled and wide open, and C) curled, tucked, and wide open suppressed. Table 4 15. Distance distribution profile for CRF01_A/E NFV CRF01_A/Esi NFV R ( ) FWHM ( ) T KR % Final % Curled 26.6 2.9 4 0 Tucked 30.6 5.1 7 8 Closed 32.2 5.0 26 30 Semi open 36.1 4.9 60 62 Wide open 41.0 4.7 3 0

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194 CRF01_A/E si with TPV Figure 4 30 Data analysis for HIV 1PR CRF01_A/E TPV A) Back gro und subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the sum med Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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195 Figure 4 31 Population validation process for HIV 1P R CRF01_A/E TPV B ackground subtracted dipolar ec ho curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned peaks, B) wide open and unassigned peaks, and C) tucked, wide open, and unassigned pea ks suppressed. Table 4 16. Distance distribution profile for CRF01_A/E with TPV CRF01_A/Esi TPV R ( ) FWHM ( ) TKR % Final % Unassigned 17.5 1.5 1 0 Unassigned 22.8 1.5 1 0 Curled 26.1 1.7 10 10 Tucked 30.6 2.0 2 3 Close d 32.9 2.3 82 85 Wide open 40.2 1.0 2 2 Unassigned 45.5 2.0 2 0

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196 CRF01_A/E si with LPV Figure 4 32 Data analysis for HIV 1PR CRF01_A/E LPV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessar y for Gaussian reconstruction process, and E) Pake dipolar pattern

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197 Figure 4 33 Population validation process for HIV 1P R CRF01_A/E LPV B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) wide open, and B) semi open and wide open suppressed. Table 4 17. Distance distribution profile for CRF01_A/E LPV CRF01_A/Esi LPV R ( ) FWHM ( ) TKR % Final % Curled 26.0 2.1 11 11 Closed 32.8 3.1 80 81 Semi open 37.4 2.7 8 8 Wide open 43.4 2.0 1 0

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198 CRF01_A/E si with SQV Figure 4 34 Data analysis for HIV 1PR CRF01_A/E SQV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Indivi dual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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199 Figure 4 35 Population validation process for HIV 1P R CRF01_A/E SQV A, B ) B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) a nd the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) wide open, and B) semi open and wide open suppressed. Table 4 18. Distance distribution profile for CRF01_A/E SQV CRF01_A/Esi SQV R ( ) FWH M ( ) TKR % Final % Curled 26.1 1.8 11 11 Closed 32.7 2.5 78 80 Semi open 36.4 2.0 9 9 Wide open 43.2 1.6 2 0

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200 CRF01_A/E si with ATV Figure 4 36 Data analysis for HIV 1PR CRF01_A/E ATV A) Back ground subtracted dipolar ech o curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population pr ofile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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201 Figure 4 37 Population validation process for HIV 1P R CRF01_A/E ATV A ) B ackground subtracted dipolar echo curve (black) overl aid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Table 4 19. Distance distribution profile for CRF01_A/E ATV CRF01_A/Esi ATV R ( ) FWHM ( ) TKR % Final % Closed 32 .9 5.3 58 58 Semi open 36.5 5.5 34 34 Wide open 40.2 4.5 8 8

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202 CRF01_A/E si with DRV Figure 4 38 Data analysis for HIV 1PR CRF01_A/E DRV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curv e (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary fo r Gaussian reconstruction process, and E) Pake dipolar pattern

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203 Figure 4 39 Population validation process for HIV 1P T CRF01_A/E DRV B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the d istance profile with one or more populations suppressed (blue) ; with A) wide open, B) semi open and wide open, C) tucked and wide open suppressed. Table 4 20. Distance distribution profile for CRF01_A/E DRV CRF01_A/Esi DR V R ( ) FWHM ( ) TKR % Fi nal % Curled 25.9 1.35 10 10 Tucked 28.9 1.2 6 6 Closed 33.0 2.15 78 79 Semi open 36.3 1.2 5 5 Wide open 42.3 1.3 1 0

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204 CRF01_A/E si with APV Figure 4 40 Data analysis for HIV 1PR CRF01_A/E APV A) Back ground subtract ed dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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205 Figure 4 41 Population validation process for HIV 1P R CRF01_A/E APV B ackground subtracted dipolar echo curve (b lack) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned and wide open, B) curled, tucked, and unassigned peaks, and C) tucked, wide open, and unassigned, and D) tucked, wide open, and unassigned peaks suppressed. Table 4 21. Distance distribution profile for CRF01_A/E APV CRF01_A/Esi APV R ( ) FWHM ( ) TKR % Final % Unassigned 22.4 1.0 1 0 Curled 26.15 1.9 5 5 Tucked 30.55 1.4 2 0 Closed 32.85 3.0 65 68 Semi open 36.15 3.4 21 22 Wide open 40.7 2.3 5 5 Unassigned 45.35 1.5 1 0

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206 CRF01_A/E si with RTV Figure 4 42 Data analysis for HIV 1PR CRF01_A/Esi with RTV A) Back ground subtracted dipolar echo c urve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profi le (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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207 Figure 4 43 Population validation process for HIV 1P R CRF01_A/E si with RTV B ackground subtracted dipolar echo curve (black) over laid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue). Table 4 22. Distance distribution profile for CRF01_A/Esi with RTV CRF01_A/Esi RTV R ( ) FWHM ( ) TKR % Final % Curle d 26.3 2.4 14 14 Closed 32.9 2.8 57 57 Semi open 35.9 3.4 22 22 Wide open 40.8 2.5 7 7

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208 CRF01_A/E si with IDV Figure 4 44 Data analysis for HIV 1P R CRF01_A/E si with IDV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlaid with the summed Gaussian population profile (blue dashe d). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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209 Figure 4 45 Population validation process for HIV 1P R CRF01_A/E. B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (re d) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned and B) unassigned and curled suppressed. Table 4 23. Distance distribution profile for CRF01_A/Esi with IDV CRF01_A/Esi IDV R ( ) FWHM ( ) TKR % Final % Unassigned 16.7 5.0 6 0 Curled 27.0 5.2 11 11 Closed 33.1 6.4 2.8 30 Wide open 36.0 7.3 55 59 A comparison of distance profiles from CRF01_A/E si with various inhibitors The distance profiles err or analyses, and conformational ensemble report for CRF01_A/E with each of nine FDA approved inhibitors and the substrate mimic CA p2 are sho wn above. Figure 4 46 show s the dis tance distribution profiles for each inhibitor, and Figure 4 47 (A) shows those that demonstrated a affect on flap closure Figure 4 48 shows a graph of each of the sub populations utilized in the Gaussian reconstruction of each of the TKR distance profiles summarizing the d ifferences in the conformational ensembles of the CRF01_A/E prot ease, with respect to inhibitor. Indinavir, Nelfinavir, and Atazanavir did not induce a drastic change in the relative percent closed flap conformation with respect to apo, thus were placed i

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210 Figure 4 46. Overlay of distance distribution profiles for CRF01_A/Esi in the apo form and with each of 9 FDA approved protease inhibitors and CA p2 substrate. Figure 4 47 Distance distribution profiles of CRF01_A/Esi with inhibitors that exhibited an (A) Figure 4 48 P opulatio n analysis for CRF01_A/E Error is estimated at 2.5%.

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211 Inhibitor I nduced F lap C losure in Subtype F si This secti on reports on DEER data for HIV 1PR Subtype F with each of nine FDA appro ved protease inhibitors and CA p2 substrate mimic The following sections provide details for data analysis and population validation, including l ong pass filtered and background sub tracted dipolar echo curve (black) overlaid with the T KR regenerated echo curve from DeerAnalysis (red) and with the reconstructed echo curve from DeerSim (blue) corresponding to the sum of distance profiles comprising the Gaussian populations needed to re generate the profile, the L curve TKR distance profile (red) overlaid with summed Gau ssian population profile (blue), i ndividual populations in Gaussian reconstruct ion of the TKR distance profile, P ake dipolar pattern and a t able which summarizes values determined from both TKR analysis Gaussian reconstruction and all appropriate p opulation validation figures. Subtype F si dipolar modulation zero time determination The proper zero times were determined as described previously. Shown in Figure 4 49 are t 300 to 300, each fit to a GaussAmp function (the amplitude version of the Gaussian peak function) of the form shown in Equation 4 1. Table 4 24 reports on the zero times chosen for Subtype F si dat a analysis determined by the aforementioned fitting. Table 4 24. Zero times chosen for F si data analysis. Inhibitor/Substrate Zero time RTV 307 ns IDV 308 ns LPV 311 ns TPV 308 ns SQV 307 ns DRV 107 ns NFV 311 ns ATV 310 ns APV 306 ns CA p2 108 ns

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212 Figure 4 49. Truncated dipolar modulated echo curves used for zero time selection of CRF01_A /E with A) RTV, B) IDV, C) LPV, D) TPV, E) SQV, F) DRV, G) NFV, H) ATV, I) APV, an d J) APV.

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213 Subtype F si with RTV Figure 4 50. Data analysis for HIV 1PR Subtype Fsi with RTV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian popul ations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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214 Figure 4 51. Population v alidation process for HIV 1 P R Fsi with RTV B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) un assigned and B) unassigned and curled suppressed. Table 4 25. Distance distribution profile for Subtype Fsi with RTV Subtype Fsi RT V R ( ) FWHM ( ) TKR % Final % Curled 26. 5 2.0 17 17 Tucked 29.2 1.3 3 0 Closed 33.1 2.5 71 7 1 Semi open 36.1 2.0 3 0 Wide open 41.4 2.1 6 9

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21 5 Subtype F si with IDV Figure 4 52. Data analysis for HIV 1PR s ubtype Fsi with IDV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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216 Figure 4 53. Population validation process for HIV 1 P R s ubtype Fsi with IDV Background subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the di stance profile with one or more populations suppressed (blue). Table 4 26. Distance distribution profile for s ubtype Fsi with IDV Subtype Fsi ID V R ( ) FWHM ( ) TKR % Final % Unassigned 23.5 4.1 3 0 Curled 27.0 4.8 8 9 Closed 33. 0 6.1 41 44 Semi open 36.1 6.8 43 47 Unassigned 51.6 5.1 5 0

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217 Subtype F si with LPV Figure 4 54. Data analysis for HIV 1PR s ubtype Fsi with LPV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated ec ho curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations neces sary for Gaussian reconstruction process, and E) Pake dipolar pattern

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218 Figure 4 55. Population validation process for HIV 1P R Fsi with LPV B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve fo r the distance profile with one or more populations suppressed (blue) ; with unassigned population suppressed. Table 4 27. Distance distribution profile for s ubtype Fsi with LPV. Subtype Fsi LPV R ( ) FWHM ( ) TKR % Final % Unassigned 22.9 1.3 6 0 Curled 25.8 1.9 7 8 Tucked 28.9 1.5 10 1 0 Closed 33.1 2.5 67 71 Wide open 41.7 2.1 10 11

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219 Subtype F si with TPV Figure 4 56. Data analysis for HIV 1PR s ubtype Fsi with TPV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (b lue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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220 Figure 4 57. Population validation process for HIV 1P R Fsi with TPV B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned and B) unassigned and curled suppressed. Table 4 28. Distance distribution profile for s ubtype Fsi with TP V Subtype F si TP V R ( ) FWHM ( ) TKR % Final % Unassigned 18.6 1.0 2 0 Curled 25.9 1.0 2 0 Tucked 29.0 1.0 1 0 Closed 32.9 2.1 8 3 88 Wide open 41.3 1.9 12 12

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221 Subtype F si with SQV Figure 4 58. Data analysis for HIV 1PR s ubty pe Fsi with SQV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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222 Figure 4 59. Population validation process for HIV 1P R Fsi with SQV B ack ground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned and B) unassigned and curled suppressed. Table 4 29. Dista nce distribution profile for s ubtype Fsi with SQV Subtype F si SQV R ( ) FWHM ( ) TKR % Final % Curled 26.5 1.8 13 15 Closed 33.2 3.0 74 75 Wide open 42.0 3.0 10 10 Unassigned 50.0 2.6 3 0

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223 Subtype F si with DRV Figu re 4 60. Data analysis for HIV 1PR s ubtype Fsi with DRV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curv e. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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224 Figure 4 61. Population validatio n process for HIV 1PR s ubtype F with DRV. Background subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned, B) semi ope n and unassigned suppressed. Table 4 30. Distance distribution profile for s ubtype Fsi with DRV Subtype F si DRV R ( ) FWHM ( ) TKR % Final % Unassigned 21.5 1.6 6 0 Curled 26.8 1.7 15 16 Tucked 30.5 4.7 17 19 Closed 33.4 2 .2 60 65 Semi open 38.1 1.3 3 0

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225 Subtype F si with NFV Figure 4 62. Data analysis for HIV 1PR s ubtype Fsi with NFV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed ech o curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction pro cess, and E) Pake dipolar pattern

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226 Figure 4 63. Population validation process for HIV 1P R Fsi with NFV B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or m ore populations suppressed (blue) ; with A) unassigned and B) unassigned and curled suppressed. Table 4 31. Distance distribution profile for s ubtype Fsi with NFV. Subtype Fsi NFV R ( ) FWHM ( ) TKR % Final % Curled 25.0 3.3 4 0 Tucked 29 .3 4.3 10 10 Closed 33.1 5 .2 27 29 Semi open 36.2 5.4 50 54 Wide open 40.0 3.1 7 7 Unassigned 59.0 5.0 2 0

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227 Subtype F si with ATV Figure 4 64. Data analysis for HIV 1PR s ubtype Fsi with ATV A) Back ground subtracted dip olar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian popul ation profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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228 Figure 4 65. Population validation process for HIV 1P R Fsi with ATV B ackground subtracted dipolar echo curve (black) ov erlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned and B) unassigned and curled suppressed. Table 4 32. Distance distribution profile for s ubtype Fsi with A TV Subtype Fsi ATV R ( ) FWHM ( ) TKR % Final % Unassigned 21.4 2.6 2 0 Tucked 28.9 5.2 13 13 Closed 33.0 5.6 56 57 Semi open 36.0 4.9 19 1 9 Wide open 40.5 4.5 10 11

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229 Subtype Fsi with APV Figure 4 66. Data an alysis for HIV 1PR Fsi with APV A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gaussian populations shown in D. B) L curve. C) Distance profile fr om TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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230 Figure 4 67. Population validation process for HIV 1P R Fsi with APV. B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppressed (blue) ; with A) unassigned and B) unassigned and curled suppressed. Tabl e 4 33. Distance distribution profile for s ubtype Fsi APV. Sutype Fsi APV R ( ) FWHM ( ) TKR % Final % Unassigned 17.5 2.0 3 0 Curled 26.4 3.0 12 12 Tucked 28.9 2.8 8 8 Closed 33.5 3.5 67 7 0 Wide open 40.3 2 .6 10 1 0

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231 Subtype F si with CA p2 Figure 4 68. Data analysis for HIV 1PR s ubtype Fsi with CA p2 A) Back ground subtracted dipolar echo curve (black) overlaid with the TKR regenerated echo curve (red) and re constructed echo curve (blue) comprising the Gauss ian populations shown in D. B) L curve. C) Distance profile from TKR analysis (red) overlain with the summed Gaussian population profile (blue dashed). D) Individual populations necessary for Gaussian reconstruction process, and E) Pake dipolar pattern

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232 Figure 4 69. Population validation process for HIV 1P R s ubtype Fsi with CA p2 B ackground subtracted dipolar echo curve (black) overlaid with the TKR fit (red) and the modified echo curve for the distance profile with one or more populations suppresse d (blue) ; with A) unassigned 16 B) unassigned 19.6 C) both unassigned 16 and 19.6 D) both unassigned and wide open, E) both unassigned, semi open and wide open, and F) both unassigned, tucked, semi open and wide open su ppressed. Table 4 34. Dista nce distribution profile for s ubtype Fsi with CA p2. Subtype Fsi CA p2 R ( ) FWHM ( ) TKR % Final % Unassigned 16.0 1.0 1 0 Unassigned 19.6 1.2 4 0 Curled 26.1 1.3 13 14 Tucked 30.9 1.4 3 3 Closed 33.1 2.2 72 76 Sem i open 36.0 1.3 4 4 Wide open 41.5 1.3 3 3

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233 A comparison of distance profiles from Subtype F with various inhibitors Figure 4 7 0 P o pulation analysis for s ubtype Fsi. Error is estimated at 2.5%. Conclusions The DEER results reported i n this work show that natural and drug pressure selected polymorphisms within subtypes CRFs, and patient isolates of HIV 1 protease alter the average flap conformations and flexibility of the apo protease Each of the apo distance profiles is strikingly different, and the changes can be described as shifts in the conformational ensemble of the protease, comprising of four distinctive sub populations of HIV 1PR conformations Additionally, certain constructs showed enhanced flexib ility or structural insta bility. These important differences may play a role in viral fitness and drug resistance (Kear, Blackburn et al. 2009) Additionally, t he distance distribution profiles for CRF01_A/E proteas e showed a similar trend with respect to inhibitor induced flap closure as previously reported work by Blackburn et al. for subtype B protease. Indinavir, N elfinavir, and A tazanavir had a weak affect on flap

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234 closure, while R itonavir, L opinavir, T ipranavir S aquinavir, A mprenavir, D arunavir, and CA p2 each had a strong affect on flap closure (Blackburn, Veloro et al. 2009)

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235 CHAPTER 5 SOLUBLE EXPRESSION A ND PURIFICATION OF M ULTIPLY DISULFIDE BO NDED PROTEINS FROM ESCHER ICHIA COLI Introduction Prorenin is the inactive zymogen of renin, which is an aspartic protease that plays a vital role in blood pressure regulation by catalyzing the first and rate limiting step in the activation pathway of its substrate angiotensinogen. D iscovered and characterized in 1898 by Robert Tigerste dt of t he Karolinska Institute of Stoc k holm (Tigerstedt and Bergman 1898; Phillips and Schmidt Ott 1999) renin is secreted via two separate pathways, a constitutive secretion of prorenin and a regulated pathway for secretion of the mature enzyme (Pratt, Flynn et al. 1988) Renin is a highly specific protease that hydrolyzes angiotensinogen into angiotensin I with a K M = 1 M in the absence of ATP6AP2 and 0.15 M in the presence of membrane bound ATP6AP2. Angiotensin I is then further hydrolyzed by angiotensin converting enzyme (ACE) into vasoactive angiotensin II (Nguyen and Sraer 2002; Fujino, Nakagawa et al. 2004) This pathway is part of the Renin Angiotensin System (RAS), a key modulator of bloo d plasma, lymph, and interstitial fluid volume, arterial vasoconstriction, blood pressure, and cardiac and vascular functi on As such, many hypertension drugs function by regulating blood pressure at various points in the RAS Prorenin circulates through the plasma until it reaches the secretory granules, where the pro segment is cleaved and active renin is release d Curiously, prorenin is found in much higher concentrations in the bloodstream than renin, representing a likely enzymatic control mechanism (Danser 2003; Morris 2003; Marathias, Agroyannis et al. 2004; Schweda and Kurtz 2004; Berecek, Reaves et al. 2005) The primary structure of prorenin consists of 452 amino acid residues, of which 46 correspond to t he pro sequence. The enzyme functions as a monomer with aspartate active site residues located at positions D 104 and D 292 (using preprorenin numbering system) Reports

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236 indicate that human renin is glycosylated (N linked) at the N 71 and N141 positions, an d that those post translational modifications are necessary for proper secretion from mammalian cells (Rothwell, Kosowski et al. 1993) Additionally, three disulfide bonds are formed from six CYS residues at positions 117 124, 283 287, and 325 362 (Imai, Miyazaki et al. 1983; Hardman 1984) A ribbon diagram rendered in VMD, showing the crystal structure of re nin is provided in Figure 5 1. Catalytic aspartate residues are shown in the active site as purple space filling model residues. Disulfide bonds are shown v ia blue space filling model residues. Figure 5 1. Crystal structure of human renin (PDB ID 2REN). I n vitro, conversion of prorenin into active renin appears to be a two step process involving the generation of an intermediary form of activate d prorenin, where the active site is exposed but the pro segment has not yet been proteolytica lly cleaved from the enzyme The first step is thought to be an acid induced activation that occurs as the result of a conformational change in the pro segment In the inactive form (based upon structural homology to pepsinogen) the pro segment is expected to be folded over the active site and in tertiary contact with o ther regions of the protein (Derkx, Schalekamp et al. 1987; Dunn 2002) At low pH, it is believed that a series of residues become protonated, thus breaking three salt bridges that are implicated in holding the

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237 pro segment over the active site and resulting in an un structured conformation of the pro segment that is no longer in contact with the active site. This conformer is referred to as the acid activated form of prorenin. This first step has been shown via enzymatic assays to be reversible via a pH switch back t o neutral conditions. The second step in the activation of prorenin to renin is the proteolytic removal of the pro segment resulting in active renin (Derkx, Schalekamp et al. 1987) Physiologic activation mechanisms have not yet been identified nor has the identity of the activating enzyme been determined. Currently, very little structural data on prorenin is available in the literature. This is likely related to the fact that current methods for recombinant expression of aspartic proteases h ave been plagued by difficulty. Current methods of isolating prorenin include recombinant expression and secretion from Chin ese Hamster Ovary (CHO) (Mercure, Thibault et al. 1995) and H uman Embryonic Kidney (HEK) cells, and t he Baculovirus (BEV) system In comparison to these methods, bacterial expression offers a relatively inexpensive, quick and high yield system; however, most aspartic proteases including prorenin, have multiple disulfide bonds, h ence these proteins, when cloned using the E. coli system, are usually expressed as inclusion bodies. Inclusion body proteins require that they be denatured and then refolded in order to obtain properly folded, functional protein. Refolding, however, does not ensure that the protein will be both properly refo lded and active ( Nishimori Kawaguchi et al. 1982; Imai, Cho et al. 1986; Kaytes, Theriault et al. 1986; Masuda, Nakano et al. 1986; Lin Wong et al. 1989; Yamau chi, Nagahama et al. 1990; Chen, Koelsch et al. 1991) The expression method described within this paper overcomes these difficulties. The bacterial system does not produce glycosylated prorenin; however, structural and enzymatic studies have shown th at the non glycosylated form is an adequate structural and

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238 functional model. The crystal structures for both the glycosylated and non glycosylated renin are quite similar, indicating that glycosylation has little effect on the st ructure A previous site directed mutagenesis study, where both of the N glycosylation sites were replaced by alanine, has shown that lack of glycosylation has no effect on the spec ific activity of the enzyme (Hori, Yoshino et al. 1988; Rahuel, Priestle et al. 1991) A bacterial expression methodology aimed at producing soluble, properly folded and functional prorenin, activat a ble by pH or enzyma tic removal of the pro segment, was investigated using thioredoxin f usion construct methodology. As described in detail in Chapter 2, t he cytoplasmic reducing potential of E. coli often causes accumulation of proteins with disulfide bonds into insoluble i nclusion bodies. Once in inclusion bodies, proteins require denaturation and refolding steps that often result in improperly folded protein Most remarkably, f usion constructs with E. coli thioredoxin (trxA) can eliminate the formation of inclusion bodie s by aid ing in proper folding of water soluble proteins containing disulfide bonds (Vallie, DiBlasio et al. 1993) Thioredoxin is small (11.6 kD), thermally stable, and is involved in a variety of cellular functions, including the reduction of protei n disulfides and sulfate metabolism. A ribbon diagram showing the x ray crystal structure of thioredoxin is provided in Figure 5 2, showing the two native cysteine residues C32 and C35, involved in a disulfide bond Under physiological conditions, thio redoxin is found under an equilibrium of both the oxidized (disulfide form) and reduced (dithiol form) enzyme. The mechanism by which thioredoxin fusion constructs function is thiol disulfide exchange, a simple chemical reaction in which a free thiolate g roup attacks a sulfur atom within a disulfide bond and the principle reaction by which disulfide bonds are formed and rearranged in a protein. The or iginal disulfide

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239 bond is broken and its other sulfur atom is released as a free thiolate and a new disulf ide bond forms between the attacking thiolate and the original sulfur atom. Figure 5 2 Ribbon diagram showing x ray structure of oxidized thioredox in, PDB ID 2TRX. Materials and Methods Materials The chemicals, reagents, and supplies were obtained from Fisher Scientific (Pittsburg, Pennsylvania) and used as received, with a few noted exceptions pET32 a DNA Ni NTA His b ind r esin, and Xarrest agarose were purchased from Novagen (Gibbstown, New Jersey). HiTrap Chelating HP column HiTrap Q HP a nion e xchange column, and HiPrep 16/60 Sephacryl S 3 00 h igh resolution s ize e xclusion column s were purchased from GE Biosciences (formerly Amersham Pittsburg, Pennsylvania) Prorenin DNA was synthesized and subsequently purchased fro m DNA2.0 ( Menlo Park, California) The QuikChange site directed mutagenesis kit and PfuUltra DNA polymerase were purchased from Stratagene (La Jolla, California). BL21 (DE3) and Origami(DE3) E. coli cells were purchased from Invitrogen (Carlsbad, Californ ia). Spin MiniPrep Kit, PCR p urification k it, g el e xtrac tion k it, and b uffers P1 and P2 were purchased from Qiagen ( Valencia, California ) The dNTP mix was purchased from Bioline (Taunton, Massachusetts ) The oligonucleotide primers were purchased from IDT (Coralville,

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240 Iowa ) The 1 kb DNA ladder, pre stained protein marker, restriction enzymes and buffers, T4 DNA ligase and buffer, and B SA were purchased from New England Biolabs ( Ipswich, Massachusetts ) Criterion pre cast protein gels, broad range mol ecular weight marker, and Laemmeli sample buffer were purchased from BioRad ( Hercules, California). The GelCode Blue stain and Cooma ssie Plus Protein Assay Reagent were purchased from Pierce ( Rockford, Illinois ). Renin substrate 1 (R 2931) and h uman reco mbinant renin (R 2779) were purchased from Sigma ( St. Louis, Missouri ). Bo vine serum albumin standards were purchased from Thermo Scientific ( Rockford, Illinois) Methods Cloning of p rorenin Table 5 1 P rorenin DNA sequence flanked (N terminal) with NcoI restriction site and Factor Xa cutsite and (C terminal ) stop codons and BamHI restriction site. ccatgg atcgatggtcgc ctgccaaccgatactactacttttaaacgcatcttcctgaaa cgta tgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggtcctgaatg gagccaaccgatg aaacgcctgaccctgggcaacactacctcttctgtgatcctgactaactac atggacacgcaatattacggcgaaattggcattggtaccccgccgcagaccttcaaggttgttt ttgacaccggctctagcaacgtatgggtgccttcttccaagtgttctcgtctgtacactgcatg cgtttaccacaaactgtttgatgcgtctgactcctctagctacaaacacaatggtaccgaactg accctgcgttatt ctaccggtaccgtttctggtttcctgagccaagatatcattactgttggcg gtatcaccgtaacgcagatgttcggcgaagttaccgaaatgccagcgctgccgttcctggctga attcgacggtgttgtaggtatgggttttattgaacaagcgatcggtcgtgtaactccgatcttc gacaacattattagccagggtgttctgaaagaagatgtgttctctttttactataaccgtgatt ctgaaaactccca atctctgggcggccagatcgtgctgggtggctctgatccgcagcactacga gggcaactttcactacatcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtt tctgttggctcttctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgcta gctacatctccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacg tctgttcgattat gtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttccaaaa agctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacctgggcgct gggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataaccgcatcggtttc gctctggcgcgt ggttcc taataa ggatcc The E. coli codon and expression optimized gene for prorenin ( sequence given in Table 5 1 ) was purchased from D NA 2.0 and received in pJ2:G02057 vector (Figure 5 3). The gene was flanked with an NcoI restriction site (yellow)

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241 The pJ2:G02057 vector features Kanamycin resistance gene and the pUC ori origin of replication, given in blue in Fig ure 5 3, and the prorenin gene flanked by NcoI and BamHI DNA restriction sites, given in red in Figure 5 3. Figure 5 3 pJ2:G02057 storage vector in which prorenin DNA was obtained. Kan amycin resistance gene and pUC ori are shown in blue and prorenin g ene is shown in red. Prorenin gene was flanked by NcoI and BamHI cutsites. Figure courtesy of DNA2.0 The prorenin gene was removed from pJ2:G02057 via restriction digestion with NcoI and BamHI cutsites by standard protocol. In addition, the pET32a vecto r (map shown in Figure 5 4 ) was prepared for sub cloning of prorenin by double cleavage with the same enzymes. The pET32 vector series is designed for cloning and high level expression of proteins fused with the 109 amino acid Trx t ag thioredoxin protein The products of both digestio ns were run on a 1% agarose gel and the linearized vector and prorenin gene were subsequently excised from the gel and purified via the Qiagen Gel Extraction and Purification Kit. The purified products were

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242 ligated together using T4 DNA Ligase according to standard ligation procedure. This vector sub cloned with the prorenin gene construct will now be referred to as pET32a_XaPR. This plasmid was transformed into XL1Blue strain E. coli cells and subsequently purified using t he Qiagen mini prep plasmid prep kit and checked by sequencing Samples were run on a 1% agarose gel to check size and purity of cloning product (shown in Figure 5 5 ). Figure 5 6 provides a cartoon representation of the fusion construct, showing the posi tions of the two fusion partners, prorenin and thioredoxin, separated by Factor Xa and enterokinase cleavage sites, a 6 His tag, and an S tag. Figure 5 4. pET 32a(+) vector map. TrxA gene, illustrated by a black arrow, is downstream of the multiple clon ing site that contains both the NcoI and BamHI restriction sites. Figure courtesy of Novagen.

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243 Figure 5 5 1% agarose gel used to confirm purity of pET32a_XaPR plasmid prior to sequencing Lane 1: 2 L of 1kb DNA ladder. Lane s 2 7 : 2 L of purifi ed pET32a_XaPR. Figure 5 6. Domain diagram of the prorenin thioredoxin fusion construct. Expression of p rorenin trx fusion construct Origami B(DE3) and B L21(DE3) strain E. coli cells were transformed independently with pET32a _XaPR DNA by standard he at shock methodology and subsequently plated on L uria B ertani (LB) agar plate s containing 100 mg/mL ampicillin, 20 mg/mL tetracycline, and 10 mg/mL kanamycin (for Origami), and 100 mg/mL ampicillin (for BL21), for selection. 1 mL liquid culture was grown in sterile LB media at 37 C and 200 RPM to an approximate OD 600 = 0.6 then added to a 3 L Fernbaugh flask containing 1 L sterile LB media C ultures were grown a t 37 C until the OD 600 was approximately 0.8. Cultures were induced for over expression b y adding 1 mL of 0.8 M isopropyl D thiogalactoside (IPTG) at 20 C and incubated for 24 hours at 2 00 RPM. 1 2 3 4 5 6 7 Size (Kb) 10 3 1 0.5

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244 Harvesting of c ells and c ollection of s oluble p rotein After over expression, t he 1 L culture was c entrifuged for 15 m inutes at 5000 RPM at 4 C in order to isolate the cell pellet. Harvested cells were resuspended in 30 mL buffer consisting of 0.02 M sodium monophosphate, 0.4 M sodium chlori de, and 40 mM imidazole, pH 7.4 and 30 L low concentration protease inhibitor cocktail (set VII from Calbio chem) was added. Cells were subsequently lysed using sonication in 5 second on/off pulses for approximately 7 minu tes and passage through a French Pressure Cell (3x at 1000 psi ). Pulsed sonic ation was performed in an attempt to lyse the cells without ca using significant shearing of the proteins. Lysed cells were then centrifuged at 3000 RPM for 10 minutes and then again at 18,500 x g for 15 minutes at 4 C to remove unwanted cellular components and insoluble proteins. The supernatant, which now contain ed the fusion construct and other soluble cell content was collected for further purification of prorenin thioredoxin fusion construct Purification of f usion c onstruct gel electrophoresis, and protein concentration estimates All chromatographic steps d escribed in subsequent sections were performed using an AKTA Prime (GE Healthcare) monitoring eluent absorbance at 280 nm and conductivity in milli Siemens (mS). Numerous purification schemes were examined. Reported herein are the methods involved in the most successful of those schemes. All protein gel electrophoresis on prorenin and prorenin thioredoxin fusion construct was performed using 18% Tris glycine pre cast Criterion gels, run at 180 volts Protein concentrations were determined using Coomassi e Plus Protein Assay Reagent (Pierce) with bovine serum albumin (BSA) standards (Thermo Scientific). HiTrap TM Chelating HP a ffinity c hromatography The soluble protein mixture was loaded onto a 5 mL Ni charged HiTrap TM Chelating HP column (Amersham B ioscie nces) and eluted over a 75 mL, 0.03 1 M imidazole gradient at 5

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245 mL/min flow rate 1 mL fractions were collected during elution Fractionated e luted protein was analyzed via SDS PAGE ( 4 20% Tris glycine pre cast Criterion gel), run at 180 volts for appr oximately one hour, and visualized with GelCode Blue stain (Pierce). HiTrap TM Q H P anion exchange c hromatography F ractions containing the target protein were collected and pooled and added to 200 mL of 0.02 M Bis tris, pH 8 for anion exchange chromatog raphy A 5 mL HiTrap TM Q HP anion e xchange column was equilibrated with 3 5 column volumes of 0.02 M Bis tris, pH 8 following by 3 5 volumes 1 M NaCl 0.02 M Bis tris, pH 8 and then 3 5 volumes 0.02 M Bis tris, pH 8 The protein mixture w as loaded on to the column and eluted over a 75 mL 0 to 1 M NaCl gradient at a flow rate of 5 mL/min and 1 mL fractions collected. Fractions were run on SDS PAGE using a 4 20% Tris glycine pre cast Criterion gel This chromatographic step typically resulted in pr orenin thioredoxin fusion construct of greater than 90% purity, as es timated by SDS PAGE. Buffer exchange by d esalt ing c olumn In preparation for removal of the thioredoxin fusion partner by Factor Xa cleavage, the protein solution was buffer exchanged into 100 mM NaCl, 50 mM Tris HCl 5 mM calcium chloride, pH 8 All buffer exchange steps were carried ou t via a desalting column and standard buffer exchange procedure. The c olumn was equilibrated with this buffer and the protein solution was loaded onto the column at a rate of 10 mL/min and fractions collected. Other methods of buffer exchanged we re compared for efficacy, including dialysis, but none were found to be as effective as the methodology first described above. Concentrating of p rotein samples In preparation for removal of the thioredoxin fusion partner by Factor Xa cleavage, t he prote in solution was concentrate d to the optimal concentration of approximately 0.5 mg/mL.

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246 All concentration steps were carried out using the Millipore Amicon Ultra 4 Centrifugal Filter Devices C oncentration s were determined using a standard Bradford assa y C entrifugation steps were performed at 4000 RPM. Several different protein concentrations were analyzed before settling on an optimum, including 0.25 3 mg/mL. Cleavage of Fusion Construct Approximately 1 mL of pro renin at an approxi mate concentrati on of 0.5 mg/mL was incubated at 4 C for 3 days with 6 30 L (dependent upon amount of fusion construct present 1 U nit ) of NEB Factor Xa protease ( 1 mg/mL), 60 L of glycerol, and 35 L of Bovine Serum Albumin ( BSA ) (2 mg/mL) Other conditions were tested, and these were found most optimal. After completion of the reaction the reaction produ cts prorenin ( 45 kD ) and thioredoxin ( 12 kD ) were visualized using SDS PAGE ( 18 % Tris glycine pre cast Criterion gel), and the amino acid sequence of prorenin is provided in Table 5 2. Table 5 2 Prorenin amino acid sequence LPTDTTTFKRIFLKCRM PSIRESLKERGVDMARLGPEWSQPMKRLTLGNTTSSVILTNYMDTQY YGEIGIGTPPQTFKVVFDTGSSNVWVPSSKCSRLYTACVYHKLFDASDSSSYKHNGTELTLRYS TGTVSGFLSQDIITVGGITVTQMFGEVTEMPALPFLAEFDGVVGMGFIEQAIGRVTPIFDNIIS QGVLKEDVFSFYYNRDSENSQSLGGQIVLGGSDPQHYEGNFHYINLIKTVWQIQMKGVSVGSST LLCEDGCLALVDTGASY SGSTSSIEKLMEALGAKKRLFDYVVKCNEGPTLPDISFHLGGKEYTL TSADYVFQESYSSKKLCTLAIHAMDIPPPTGPTWALGATFIRKFYTEFDRRNNRIGFA LAR Quenching Factor Xa reaction with Novagen Xarrest agarose E xcess Factor Xa enzyme was sequestered using Novagen Xarrest agarose at a ratio of 10 0 L of slurry per 4 units Factor Xa protease. The agarose was previously equilibrated in 100 mM NaCl, 50 mM Tris HCl 5 mM calcium chloride, pH 8 T he products of the cleavage reaction were added di rectly to the agarose and incubated at room temperature for approximately 10 minutes, at which time t he reaction mixture was centrifuged at 1000 x g for 5 minutes and the

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247 super natant, containing thioredoxin and prorenin, was collected, while the Factor Xa remained bound to the agarose Purification of p roren in from Factor Xa protease and thioredoxin In a final purification step, p rorenin was purified away from thioredoxin using His bind (Ni NTA) resin The supernatant, which contained the cleaved protein, was adjusted to pH 7.5 with 1M Tris H Cl, pH 8.0, and added directly to equilibrated Ni NTA His bind resin (2mL resin slurry is required per 10mg His tagged protein). The reaction mixture was allowed to equilibrate for 10 m inutes at room temperature, then centrifuged at 1000 x g for 1m inute to pellet the res in. The supernatant then contains the pure recombinant prorenin, while the thioredoxin fusion tag remains bound to the resin. Circular d ichroism s pectroscopy Circular dichroism experiments with prorenin were run on an AVIV 202 Circular Dichroism Spect rometer (Steve Hagen laboratory, University of Florida) at 25 C. The monochromator w as set to a wavelength of 260 nm 190 nm with a bandwidth of 1 nm and a slitwidth of 0.569 nm. The protein was buffered in 50 mM phosphate and 2.5% glycerol, pH 7.0. A ctivation of p rorenin Prorenin was reversibly activated by a pH switch to acidic conditions. At pH 4.5, the pro segment was liberated from the active site, providing renin activity. Upon a pH switch back to 8.0, renin activity is lost as the pro segment forms salt bridges with enzyme portion of the protein. Prorenin a ctivity a ssay FRET based renin activity assays were run at pH 4.5, where the zymogen is expected to be in the acid activated conformation (pro segment liberated from active site), and also at pH 8.0, where prorenin is expected to be in the inactive conformation (pro segment blocking the active

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248 site due to presence of salt bridges) Buffer for all activity assays was 0.1 M NaCl, 0.05 M Tris base, pH 4.5 or pH 8.0. Lyophilized renin substrat e 1 was dissolved in high quality anhydrous dimethylsulfoxide (DMSO) to make a stock solution of 500 M substrate. Prior to activity assay, a fresh was prepared in 100 mM NaCl and 50 mM tris(hydroxymethyl) aminomethane ( Tris base), pH 8.0 The substrate solution was dispensed into a clean UV pass fluorescence cuvette and e ach assay was started by adding approximately 3% of the final volume of renin containing solution diluted in the assay buffer. E xcitation and emissio n monochro mators were set to 340 nm and 490 nm, respectively. Results and Discussion Sub c loning of P rorenin Gene into pET32a Expression V ector The E. coli codon and expression optimized gene for prorenin w as purchased from D NA 2.0 and received in the pJ 2:G02057 vector. For the purposes of sub cloning the gene into the ligation was carried out using standard procedures. Ligation was confirmed using DNA gel electrophoresis and subsequent sequ encing, thus sub cloning was successful. Over expression and Purification of Prorenin thioredoxin Fusion C onstruct pET32a_XaPR was transformed separately into both BL21(DE3) and OrigamiB(DE3) strain E. coli cells for a pilot scale expression experiment. Given in Figures 5 7 and 5 8 are pictures of the SDS PAGE gels showing the results of these experiments. The levels of expression of the prorenin thioredoxin fusion construct from BL21(DE3) and OrigamiB(DE3) are comparable. Scaling up of the expression procedure was carried out, from both strains, in 1 L LB and 24 hours of induction time at 20 C.

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249 Figure 5 7 Pilot expression o f prorenin thioredoxin fusion construct in BL21(DE3) strain E. coli cells. Lane 1: broad range protein ladder, Lanes 2 9: NOT INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 6, 8, 12, and 24 hours, respectively, Lanes 10 17: INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 6, 8, 12, and 24 hours, respectively. The apparent shift in size of the over expression product is an artifact created by the curvature of the gel during the photography step, as opposed to an increase in size of the protein itself. Figure 5 8 Pilot expression of prorenin thioredoxin fusion construct in OrigamiB(DE3) strain E. coli cells. Lanes 1 7: NOT INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 8, 12, and 24 hours, respectively, Lane 8: broad rang e protein ladder, Lanes 9 15: INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 8, 12, and 24 hours, respectively. The apparent shift in size of the over expression product is an artifact created by the curvature of the gel during the photography step, as opposed to an increase in size of the protein itself.

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250 The purification of the prorenin thioredoxin fusion construct was carried out via sequential chromatography using a nickel charged HiTrap TM Chelating HP a ffinity column followed by a HiTra p TM Q H P anion exchange column. Figures 5 9 and 5 10 show typical chromatograms corresponding to each of these separation steps. Because the fusion construct harbors a 6 His tag, it has high affinity for nickel charged column; as such, the protein is ret ained on the column and eluted over a slow gradient of the competitive inhibitor imidazole. Fractions containing the fusion construct were pooled and loaded onto an anion exchange column equilibrated according to the isoelectric point of the protein. Aft er these steps, prorenin thioredoxin was estimated to be >95% pure, as shown in Figure 5 11. Figure 5 9 Typical chromatogram from HiTrap TM Chelating HP nickel a ffinity column. Green trace shows progression of imidazole gradient. Red trace shows conductivity line in units of mS Blue line shows UV at 280 nm. Fractions 10 17 were collected for further purification.

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251 Figure 5 10 Typical chromatogram from HiTrap TM Q H P a nion e xchange column. Green trace shows progression of NaCl gradient. Red trace shows conductivity line in units of mS Blue line shows UV at 280 nm. Figure 5 11. 18% Tris glycine SDS PAGE gel demonstrating purity of prorenin thioredoxin fusion construct following sequential chromatographic steps.

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252 Enzymatic Removal of T hioredoxin Enzymatic removal of thioredoxin presented many difficulties. The prorenin thioredoxin fusion construct contains cleavage sites for both enterokinase and Factor Xa. The enterokin a se site is found in the native pET32a vector sequence, and t he Factor Xa site was incorporated by including the corresponding sequence in the gene that was sub cloned into the pET32a vector. All initial work was performed using enterokinase. It was demonstrated, however, that enterokin a se resulted in non specific cleavage of the prorenin thioredoxin fusion protein. As such, all subsequent work was performed using Factor Xa. Incubation with Factor Xa was successful in liberating prorenin from the thioredoxin fusion partner as shown in Figure 5 12 ; however, an es timated 80 90% of prorenin remaining in the sample became insoluble upon cleavage from thioredoxin, drastically lowering the overall protein yield. Evidence was collected, however, suggesting that the remaining prorenin was properly folded and activatab le upon a pH switch to acidic conditions. Following the Factor Xa reaction, thioredoxin (with a 6 His tag) was successfully removed from the reaction tube using loose nickel resin. Figure 5 12. 18% Tris glycine SDS PAGE gel demonstrating purity of prorenin thioredoxin fusion construct (lane 2) and prorenin (lane 3) after separation from thioredoxin. Broad range protein marker is shown in lane 1 for size reference.

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253 Evidence of P roper F olding A circular dichroism spectrum was obtained in order to d etermine the relative secondary structure of recombinant wild type pro renin; s pectrum is shown in Figure 5 13 and r esults of secondary structure analysis are reported in Table 5 3. This data reports a secondary structure consisting of 17.4% helix 30.4% sheet, 22% turn, and 29.5% unstructured (51.5% random coil). To date, no crystal structure of prorenin exists for comparison; however, a prediction from EXPASY Secondary Structure Prediction program ( http://www .expasy.ch/tools ) reports a similar theoretical secondary structure Figure 5 13. Circular dichroism spectrum of prorenin after liberation from thioredoxin fusion construct and separation from thioredoxin. Table 5 3 Secondary structural data for prorenin (obtained from circular dichroism analysis), renin, pepsinogen, and pepsin (each from analysis of crystal structure data), and theoretical prorenin structure analysis (as determined by EXPASY secondary structure prediction t ool). Prorenin Prorenin Renin Pepsinogen Pepsin (Experimental) (Theoretical) helix 17% 14% 10% 26% 15% sheet 30% 32% 40% 20% 46% Coil/turn 52% 54% 50% 54% 39% Comparisons were al so been made with the crystal structures of renin, pepsin (a similar aspartic protease), and pepsinogen (the inactive zymogen of pepsin). The similarity of these values p rovides strong evidence that the thioredoxin fusion system is successful in pr oducing soluble, properly folded prorenin from a bacterial system

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254 Ren in A ctivity M easurements of pH a ctivated P rorenin Renin activity in pH activated prorenin was measured by monitoring the cleavage kinetics of the fluorescently labeled peptide substrate analog Renin Substrate I (R2931) purchased from Sigma The assay utilizes fluorescence resonance energy transfer (FRET) to produce a spectroscopic response to the enzymatic cleavage of the substrate by pH activated prorenin. Renin Substrate I is the sma ll peptide Arg Glu (EDANS) Ile His Pro Phe His Leu V al Ile His Thr Lys (DABCYL) Arg. The peptide is the normal substrate for renin with the fluorophore 5 (aminoethyl)aminonaphthalene sulfonate (EDANS) linked at one end and the non fluorescent chromophore dimethylaminoa zo benzene 4 carboxylate (DABCYL ) linked at the other end The absorbance of the DABCYL chromophore overlaps with the excited state fluorescence of the EDANS fluorophore thereby quenching the EDANS via a FRET response, as shown in Figure 5 1 4 Proteolytic cleavage of the substrate results in spatial separation of th e fluorophore and acceptor, restorin g the fluorescence of the EDANS. As such, a gain in fluorescence over time can be exploited as a means to assess activated prorenin/renin acti vity. Figure 5 14. DABCYL absorption overlaps with the EDANS fluorescence thereby quenching the fluorescence through fluorescence resonance energy transfer Figure courtesy of Sigma.

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255 Assays were run at pH 4.5 where prorenin is expected to be in a ctive conformation (pro segment liberated from active site) as well as pH 8.0, where the pro segment is thought to be physically blocking the active site, thus rendering the protease inactive and the results are shown in Figure 5 15. At pH 8.0, no incre ase in fluorescence signal is seen. Figure 5 15 Renin Su bstrate I activity screening; A) activity screen at pH 8.0, where prorenin is thought to be in its inactive conformation. No inc rease in fluorescence is seen. B) Activity screen at pH 4.5, wher e the pro segment is liberated from the active site of the enzyme. An increase in fluorescence demonstrates that prorenin can be activated at acidic pH. Upon lowering the pH to 4.5, t he increase seen in fluorescence signal over time demonstrates that th is system produces prorenin that can be activated at acidic pH. In conjuction with CD data, the results from the activity assay suggest that the thioredoxin fusion construct expression methodology is successful in producing active, properly folded prorenin albeit in small yield at the present time Cysteine M utagenesis of Prorenin DNA for Possible EPR S tudies The s ite directed spin labeling EPR methodology may be useful to characterize the conformational changes in the pro segment of prorenin that occur as a function of either pH or site specific mutations. Analysis of the EPR line shapes from spin labels incorporated into the pro segment could be used to validate the hypothesis that prorenin can undergo a reversible, non

PAGE 256

256 proteolytic acid induced activat ion, which, based upon enzymatic assays, suggests that the pro segment undergoes a conformational change thus exposing the active site of the enzyme. No structural evidence (X ray or spectroscopic) of this conformational change has ever been presented. T o this end, numerous CYS mutants were generated by site directed mutagen e sis using t he Eppendorf Mastercycler P ersonal thermocycler All primers were designed using PrimerX ( http://www.b ioinformatics.org/primerx/cgi bin/DNA_1.cgi ) and synthesized and purchased from Integrated DNA Technology (IDT DNA, http://www.idtdna.com ) C areful attention was paid to primer length (30 45 bases), GC content (40 50%), and melting temperature (T m ) (60 70 C). Table 5 4 shows the specific sequences, lengths, GC content, and T m of each of the primers used in these experiments and table 5 5 shows the thermal cycling parameters used for mutagenesis reactions Th e following mutants were successfully generated by the procedure described above: T7C, F8C, L13C, V28C, and G35C. All successful reactions were analyzed via DNA gel electrophoresis using 1% w/v agarose gel and DNA sequencing at University of Florida DNA S equencing Core. Table 5 4 PCR p rimers utilized to introduce mutations to prorenin Mutation Primer (5` 3`) T m ( C) m.w. %GC G35C Forward GTAGATATGGCACGTRCTGTGTCCTGAATGGAGCCAAC 65.7 11,429.4 51.3 Reverse GTTGGCTCCATTCAGGACACAGACGTGCCATATCTAC 65.7 11,309.4 51.3 L13C Forward CTTTTAAACGCATCTTCTGTAAACGTATGCCTTCCATCCG 63.9 13,038.5 41.8 Reverse CGGATGGAAGG CATACGTTTACAGAAGATGCGTTTAAAAG 63.9 13,038.5 41. 8 V28C Forward CTCTGAAAGAGCGTGGTTGTGATATGGCACCTCTGGG 67.1 11,516.5 54.0 Reverse CCCAGACGTGCCATATCACAACCACGCTCTTTCAGAG 67.1 11,223.3 5 4.0 T7C Forward CTGCCAACCCATACTACTTG TTTTAAACGCATCTTCCTG 63.6 1 1 827.7 40.3 Reverse CAGGAAGATGCGTTTAAAACAAGTAGTATCGGTTGGCAG 63.6 12,143. 9 40.3 F8C Forward CCGATACTACTACTTGTAAACGCATCTTCCTG 59.5 9,694.3 4 3.8 Reverse CAGGAACATGCGTTTACAAGTAGTAGTATCGC 59.5 9,952.5 43.8

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257 Table 5 5. Therm al c ycling parameters for site directed mutagen e sis reactions on prorenin Segment Cycles Temperature Time 1 1 95 o C 5 minutes 2 18 95 o C 1 minute 50 o C 1 minute 68 o C 8 minutes Expression and Purification of V28C Mutant Prorenin V28C prorenin mutant was expressed and purified using the procedure described previously. Figure 5 16 shows an SDS PAGE gel indicating the high purity of the prorenin thioredoxin fusion construct following anion ex change chromatography. However, in similar fashion to wild type, upon enzymatic removal of thio re doxin, a majority of the V28C prorenin mutant protein became insoluble, thus substantially lowering the final protein yield. Figure 5 16 SDS PAGE gel show ing purity of V28C mutant following anion exchange chromatography. Lane 1: broad range protein molecular weight marker, Lane s 2 12: fractions 7 18 respectively, Lane s 13 18 : fractions 28 through 33 respectively, and corresponding to prorenin thioredoxin fusion construct. Conclusions An innovative system to express soluble, properly folded prorenin from E. coli was evaluated. Prorenin thioredoxin fusion construct was successfully expressed and purified to 90 95% using sequential affinity and anion ex change chromatographic steps. Thioredoxin was

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258 successfully liberated from the fusion construct using Factor Xa protease; however, at this time a substantial portion of the protein sample became insoluble, thus lowering the final protein yield to very smal l levels. The remaining prorenin was examined for proper folding by circular dichroism spectroscopy and for renin activity via a FRET based assay. In conclusion, though total yield per liter E. coli growth is relatively low, t his methodology allows for the production of soluble, properly folded, activatable prorenin without the need for complicated denaturation and refolding steps which often yield misfolded or inactive protein. Circular dichroism studies confirmed proper folding of the protein, and act ivity assays showed that prorenin was activatable at low pH.

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259 CHAPTER 6 CONCLUSIONS AND FUTU RE DIRECTIONS Conclusions Prorenin and HIV 1 Protease (HIV 1PR) are both aspartic proteases with broad implications in the medical field. HIV 1PR is a viral protein necessary for HIV 1 maturation and prorenin is a zymogen whose activation is vital to the regulation of blood pressure via its role in the Renin Angiotensin System (RAS). As of 2008, the World Health Organization (WHO) has reported that the number of people living with HIV or AIDS was approximately 33.4 million, and the American Heart Association reported that the percentage of adults over the age of 20 living with hypertension was approximately 32% with approximately 24 ,000 deaths in the United St ates alone (Clemente, Moose et al. 2004) Chapter 1 provid es detailed background information relevant to both HIV 1PR and renin/prorenin. Techniques used to examine these proteases include circular dichroism spectroscopy (CD), mass spectrometry, continuous wave (CW ) EPR and pulsed double electron electron resonance (DEER) spectroscopy Detailed descriptions on each of these methodologies are given in Chapter 2. Chapter 3 describes results from CW EPR experiments of HIV 1PR. CW EPR investigations provided a means to monitor the autoproteolytic degradation of the active p rotease constructs, Subtype F and CRF01_A/E. Currently unpublished work performed by graduate student Angelo Mike Veloro demonstrated that substantial autoproteolysis of the active proteases V6 and MDR769, without the three stabilizing mutations Q7K, L33 I, and L63I, takes place during the course of the purification; thus, to date, most of our structural work has been focused on protease constructs that have incorporated the Q7K, L33I, and L63I substitutions. In ord er to continue investigations of active protease, we needed to understand how conditions during sample preparation and storage affect the autoproteolytic process As such, the rate of

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260 autoproteolysis of active spin label ed HIV 1PR constructs was monitored via EPR spectroscopy. Over time, a sha rp spectral component appear ed in the high field resonance line of the EPR spectrum This signal is attributed to a change in correlation time and provides a direct means of monitoring protein degradation. Q uantitative analysis of the normalized i ntensit y of the high field line provided a value proportional to the concentration of the proteolysed pepti de fragment containing the spin label This methodology has the advantage over HPLC or SDS PAGE that is non destructive and requires very little protein sa mple. Additionally, we were able to demonstrate that with a quick purification and timely addition of inhibitor, autoproteolysis can be greatly reduced, implying that DEER experiments can likely be repeating using active protease. Pulsed EPR methodologi es and site directed spin labeling (SDSL) were employed in order to characterize the conformational ensembles of HIV 1PR from various subtypes and patient isolates, including Subtypes B, C, F, CRF01_A/E, V6 and MDR769, as d escribed in Chapter 4. We find t hat the CW line shapes are dominated by the intrinsic motion of the spin label, and as such do not report on changes in flap motions and dynamics. However, t he pulsed EPR technique DEER also known as p ulsed e lectron double r esonance (PELDOR), yielded dip olar modulated echo curves that provided detailed information regarding changes in flap conformations and flexibility R esults were analyzed to provide distance distribution profiles that provided information regarding the distances between spin labels at positions K55C and ensemble of the protease flaps as well as the flexibility and dynamics of the flaps in the apo form and in the presence of FDA approved protease inhibitors and a non hydrolyzable substrate mimic CA p2.

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261 In addition to the work done on HIV 1P R, a significant portion of my graduate research experience was dedicated to developing a soluble expression system for prorenin using the thioredoxin fusion m ethodology, with partial success, as described in Chapter 5. The zymogen was successfully over expressed from both BL21(DE3) and OrigamiB(DE3) strain E. coli cells and the fusion construct was successfully purified using a series of chromatographic steps. T he cleavage of thioredoxin from p rorenin seemingly render ed prorenin unstable causing the protein to consistently crash from solution, substantially l owering the total protein yield Soluble p rorenin remaining after separation from thioredoxin fusion partner was e xamined using circular dichroism spectroscopy and secondary structures were calculated. No crystal structure of prorenin exists to date, but the secondary structural composition was comparable to that of pepsinogen, a similar aspartic protea se zymogen, leading to the conclusion that the small amount of prorenin remaining after cleavage from the fusion partner is likely properly folded. Additionally, a FRET based assay was used to show that the protein was indeed activated by pH. Overall, th e method was successful in providing a small amount of properly folded, pH activatable prorenin; however, the purification and cleavage processes need to be further optimized for future studies aimed at SDSL EPR, NMR, and crystallography Future D irections A Site Directed Spin labeling Approach to Studying HIV 1 Protease To date, research in the group has focused on proteases from three subtypes, a circulating recombinant form, and two patient isolates, namely Subtype B, C, F, CRF01_A/E, and V6 and MDR769. However, this research should be expanded to include additional subtypes and patient isolates including but not limited to Subtype D, G, H, J, and K. Additionally, DEER experiments should be repeated using active constructs for comparison to in active construct data. CW EPR experiments showed that if the active protease is purified quickly with prompt addition

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262 of inhibitor, autoproteolysis is slowed sufficiently to perform analyses. Relevant single point mutations should be probed to determine the effect of secondary polymorphisms on the conformational ensemble of the protease and the development of drug resistance. Each of these results should be compared with results from other techniques, including isothermal titration calorimetry, different ial scanning calorimetry, and nuclear magnetic resonance. A crucial step in marking the significance of this work would be to correlate results on the conformation ensemble, or more specifically the relative percentage closed conformation, to in vivo wor k, particularly IC 50 values, viral fitness, and drug resistance. IC 50 is defined as the half maximal inhibitory concentration and is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. Viral fitness refers to th e relative replication competence of a virus. Viral fitness can be assessed directly, by several methods, in tissue culture systems. Recombinant Bacterial Expression and Biophysical Characterization of the Aspartic Acid Zymogen Prorenin After further op timization of the purification and subsequent liberation of prorenin from the fusion partner t hioredoxin, EPR and NMR techniques can be used to better understand the activation mechanism of prorenin by obtaining direct spectroscopic evidence of conformatio nal changes of the pro segment. The conformations and/or conformational changes of the pro segment of prorenin can be characterized in both the active and inactive states, and the reversibility of the acid induced activation can be studied As mentioned previously, no crystal structure of prorenin currently exists. Thus, a fut ure aim focuses on obtaining a high resolution X ray crystal structure of prorenin in its inactive state. X Ray crystallography is capable of obtaining atomic resolution structure o f proteins, though it requires relatively high amounts of protein. Current yields will likely provide sufficient amounts

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263 of protein for SDSL EPR investigations of prorenin; however a continuing goal is to optimize our method of protein expression to impro ve yield for X Ray crystallography and NMR investigations.

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264 APPENDIX A PRORENIN DNA AND AMI NO ACID SEQUEN CE Appendix A provides the deoxyribonucleic acid ( DNA ) and amino acid sequences for the prorenin construct produced for th e research in this disse rtation, as well as each of the mutant prorenin constructs successfully created via site directed mutagenesis (T7C, F8C, L13C, V28C, and G35C). pET32 a(+) expression vector. The DNA for the E scherichia coli ( E. coli ) codon optimized prorenin was synthesiz ed by DNA2.0. The DNA sequence is flanked with an Nco I restriction site (yellow) and a FactorXa cutsite (teal) on the N terminal end and two stop codons (magenta) followed by a BamHI restriction site (green) on the C terminal end for sub cloning into the pET32a vector. The start codon (ATG) is located upstream of the sequence encoding for t hioredoxin which is found within the sequence of the pET32a vector (Figure 5 x). Residue one of prorenin is underlined for clarity. Sites of engineered CYS residues are highlighted in grey. Table A 1. E. coli codon optimized prorenin sequence with Factor Xa cutsite ccatgg atcgatggtcgc ctg ccaaccgatactactacttttaaacgcatcttcctgaaa P W I E G R L P T D T T T F K R I F L K cgtatgccttccatccgtgaatctctga aagagcgtggtgtagatatggcacgtctgggt R M P S I R E S L K E R G V D M A R L G cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc P E W S Q P M K R L T L G N T T S S V I ctgactaactacatggacacgcaatattacggcgaaattg gcattggtaccccgccgcag L T N Y M D T Q Y Y G E I G I G T P P Q accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt T F K V V F D T G S S N V W V P S S K C tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgact cctctagc S R L Y T A C V Y H K L F D A S D S S S tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc Y K H N G T E L T L R Y S T G T V S G F ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt L S Q D I I T V G G I T V T Q M F G E V accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt T E M P A L P F L A E F D G V V G M G F attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt I E Q A I G R V T P I F D N I I S Q G V ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg L K E D V F S F Y Y N R D S E N S Q S L

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265 Table A 1. Continued. g gcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaac tttcactac G G Q I V L G G S D P Q H Y E G N F H Y atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct I N L I K T G V W Q I Q M K G V S V G S tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc S T L L C E D G C L A L V D T G A S Y I tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg S G S T S S I E K L M E A L G A K K R L ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg F D Y V V K C N E G P T L P D I S F H L ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc G G K E Y T L T S A D Y V F Q E S Y S S aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc K K L C T L A I H A M D I P P P T G P T tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac W A L G A T F I R K F Y T E F D R R N N cgcatcggtttcgctctggcgcgtggttcc taataa ggatcc R I G F A L A R G S G S Table A 2 E. coli codon optimized prorenin T7C sequence with Factor Xa cutsite ccatgg atcgatggtcgc ctg ccaaccgatact act tgt tttaaacgcatcttcctgaaa P W I E G R L P T D T T C F K R I F L K cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtct gggt R M P S I R E S L K E R G V D M A R L G cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc P E W S Q P M K R L T L G N T T S S V I ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag L T N Y M D T Q Y Y G E I G I G T P P Q accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt T F K V V F D T G S S N V W V P S S K C tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc S R L Y T A C V Y H K L F D A S D S S S tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc Y K H N G T E L T L R Y S T G T V S G F ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt L S Q D I I T V G G I T V T Q M F G E V accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt T E M P A L P F L A E F D G V V G M G F attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt I E Q A I G R V T P I F D N I I S Q G V ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg L K E D V F S F Y Y N R D S E N S Q S L ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac G G Q I V L G G S D P Q H Y E G N F H Y atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct I N L I K T G V W Q I Q M K G V S V G S

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266 Table A 2. Continued. tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc S T L L C E D G C L A L V D T G A S Y I tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg S G S T S S I E K L M E A L G A K K R L ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg F D Y V V K C N E G P T L P D I S F H L ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc G G K E Y T L T S A D Y V F Q E S Y S S aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc K K L C T L A I H A M D I P P P T G P T tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac W A L G A T F I R K F Y T E F D R R N N cgcatcggtttcgctctggcgcgtggttcc taataa ggatcc R I G F A L A R G S G S Table A 3 E. coli codon optimized pr orenin F8C sequence with Factor Xa cutsite ccatgg atc gatggtcgc ctg ccaaccgatactactact tg t aaacgcatcttcctgaaa P W I E G R L P T D T T T C K R I F L K cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggt R M P S I R E S L K E R G V D M A R L G cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc P E W S Q P M K R L T L G N T T S S V I ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag L T N Y M D T Q Y Y G E I G I G T P P Q accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt T F K V V F D T G S S N V W V P S S K C tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc S R L Y T A C V Y H K L F D A S D S S S tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc Y K H N G T E L T L R Y S T G T V S G F ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt L S Q D I I T V G G I T V T Q M F G E V ac cgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt T E M P A L P F L A E F D G V V G M G F attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt I E Q A I G R V T P I F D N I I S Q G V ctgaaagaagatgt gttctctttttactataaccgtgattctgaaaactcccaatctctg L K E D V F S F Y Y N R D S E N S Q S L ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac G G Q I V L G G S D P Q H Y E G N F H Y atcaacctgattaaaaccggcgtgtg gcagatccagatgaaaggcgtttctgttggctct I N L I K T G V W Q I Q M K G V S V G S tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc S T L L C E D G C L A L V D T G A S Y I tccggttccacctctagcattgagaaactgatggaagc tctgggcgccaagaaacgtctg S G S T S S I E K L M E A L G A K K R L

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267 Table A 3. Continued. ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg F D Y V V K C N E G P T L P D I S F H L ggtggtaaagaata tactctgacctccgccgactacgttttccaggaatcttattcttcc G G K E Y T L T S A D Y V F Q E S Y S S aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc K K L C T L A I H A M D I P P P T G P T tgggcgctgggcgctaccttcatccg taaattctataccgaattcgaccgtcgcaataac W A L G A T F I R K F Y T E F D R R N N cgcatcggtttcgctctggcgcgtggttcc taataa ggatcc R I G F A L A R G S G S Table A 4 E. coli codon optimized prorenin L13C sequence with Factor Xa cutsite ccatgg atcgatggtcgc ctg ccaaccgatactactacttttaaacgcatcttc tgc aaa P W I E G R L P T D T T T F K R I F C K cgtatgccttccatccgtgaatctctgaaagagcgtggtgtagatatggcacgtctgggt R M P S I R E S L K E R G V D M A R L G cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtgatc P E W S Q P M K R L T L G N T T S S V I ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag L T N Y M D T Q Y Y G E I G I G T P P Q accttcaag gttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt T F K V V F D T G S S N V W V P S S K C tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc S R L Y T A C V Y H K L F D A S D S S S tacaaacacaatggtaccgaa ctgaccctgcgttattctaccggtaccgtttctggtttc Y K H N G T E L T L R Y S T G T V S G F ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt L S Q D I I T V G G I T V T Q M F G E V accgaaatgccagcgctgccgttcctggctgaa ttcgacggtgttgtaggtatgggtttt T E M P A L P F L A E F D G V V G M G F attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt I E Q A I G R V T P I F D N I I S Q G V ctgaaagaagatgtgttctctttttactataaccgtgattctgaa aactcccaatctctg L K E D V F S F Y Y N R D S E N S Q S L ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac G G Q I V L G G S D P Q H Y E G N F H Y atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggc tct I N L I K T G V W Q I Q M K G V S V G S tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc S T L L C E D G C L A L V D T G A S Y I tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg S G S T S S I E K L M E A L G A K K R L ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg F D Y V V K C N E G P T L P D I S F H L ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc G G K E Y T L T S A D Y V F Q E S Y S S

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268 Table A 4. Continued. aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc K K L C T L A I H A M D I P P P T G P T tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaat aac W A L G A T F I R K F Y T E F D R R N N cgcatcggtttcgctctggcgcgtggttcc taataa ggatcc R I G F A L A R G S G S Table A 5 E. coli codon optimized prorenin V28C sequence with Factor Xa cutsite ccatgg atcgatggtc gc ctg ccaaccgatactactacttttaaacgcatcttcctgaaa P W I E G R L P T D T T T F K R I F L K cgtatgccttccatccgtgaatctctgaaagagcgtggt tgt gatatggcacgtctgggt R M P S I R E S L K E R G C D M A R L G cctgaatggagccaaccgatgaaacgcc tgaccctgggcaacactacctcttctgtgatc P E W S Q P M K R L T L G N T T S S V I ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag L T N Y M D T Q Y Y G E I G I G T P P Q accttcaaggttgtttttgacaccggctctagcaacgtat gggtgccttcttccaagtgt T F K V V F D T G S S N V W V P S S K C tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc S R L Y T A C V Y H K L F D A S D S S S tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgttt ctggtttc Y K H N G T E L T L R Y S T G T V S G F ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt L S Q D I I T V G G I T V T Q M F G E V accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt T E M P A L P F L A E F D G V V G M G F attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt I E Q A I G R V T P I F D N I I S Q G V ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg L K E D V F S F Y Y N R D S E N S Q S L ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac G G Q I V L G G S D P Q H Y E G N F H Y atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct I N L I K T G V W Q I Q M K G V S V G S tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc S T L L C E D G C L A L V D T G A S Y I tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg S G S T S S I E K L M E A L G A K K R L ttcgattatgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg F D Y V V K C N E G P T L P D I S F H L ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc G G K E Y T L T S A D Y V F Q E S Y S S aaaaagctgtgtactctggcaattcatgctatggacatcccgccgccgaccggtccgacc K K L C T L A I H A M D I P P P T G P T tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac W A L G A T F I R K F Y T E F D R R N N

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269 Table A 5. Continued. cgcatcggtttcgctctggcgcgtggttcc taataa ggatcc R I G F A L A R G S G S Table A 6 E. coli codon optimized prorenin G35C sequence with Factor Xa cutsite ccatgg atcgatggtcgc ctg ccaaccgatactactacttttaaac gcatcttcctgaaa P W I E G R L P T D T T T F K R I F L K cgtatgccttccatccgtgaatctct gaaagagcgtggtgtagatatggcacgtctg t gt R M P S I R E S L K E R G V D M A R L C cctgaatggagccaaccgatgaaacgcctgaccctgggcaacactacctcttctgtga tc P E W S Q P M K R L T L G N T T S S V I ctgactaactacatggacacgcaatattacggcgaaattggcattggtaccccgccgcag L T N Y M D T Q Y Y G E I G I G T P P Q accttcaaggttgtttttgacaccggctctagcaacgtatgggtgccttcttccaagtgt T F K V V F D T G S S N V W V P S S K C tctcgtctgtacactgcatgcgtttaccacaaactgtttgatgcgtctgactcctctagc S R L Y T A C V Y H K L F D A S D S S S tacaaacacaatggtaccgaactgaccctgcgttattctaccggtaccgtttctggtttc Y K H N G T E L T L R Y S T G T V S G F ctgagccaagatatcattactgttggcggtatcaccgtaacgcagatgttcggcgaagtt L S Q D I I T V G G I T V T Q M F G E V accgaaatgccagcgctgccgttcctggctgaattcgacggtgttgtaggtatgggtttt T E M P A L P F L A E F D G V V G M G F attgaacaagcgatcggtcgtgtaactccgatcttcgacaacattattagccagggtgtt I E Q A I G R V T P I F D N I I S Q G V ctgaaagaagatgtgttctctttttactataaccgtgattctgaaaactcccaatctctg L K E D V F S F Y Y N R D S E N S Q S L ggcggccagatcgtgctgggtggctctgatccgcagcactacgagggcaactttcactac G G Q I V L G G S D P Q H Y E G N F H Y atcaacctgattaaaaccggcgtgtggcagatccagatgaaaggcgtttctgttggctct I N L I K T G V W Q I Q M K G V S V G S tctaccctgctgtgcgaagacggctgtctggcgctggtcgataccggtgctagctacatc S T L L C E D G C L A L V D T G A S Y I tccggttccacctctagcattgagaaactgatggaagctctgggcgccaagaaacgtctg S G S T S S I E K L M E A L G A K K R L ttcgatta tgtggttaaatgcaacgaaggtccgacgctgccggacattagcttccacctg F D Y V V K C N E G P T L P D I S F H L ggtggtaaagaatatactctgacctccgccgactacgttttccaggaatcttattcttcc G G K E Y T L T S A D Y V F Q E S Y S S aaaaagctgtgtactctggc aattcatgctatggacatcccgccgccgaccggtccgacc K K L C T L A I H A M D I P P P T G P T tgggcgctgggcgctaccttcatccgtaaattctataccgaattcgaccgtcgcaataac W A L G A T F I R K F Y T E F D R R N N cgcatcggtttcgctctggcgcgtggttcc ta ataa ggatcc R I G F A L A R G S G S

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270 APPENDIX B YEAST PROTEINASE A D NA AND AMINO ACID SE QUENCES Soluble expression and purification methods were employed with Yeast Proteinase A (data not included in dissertation). Appendix B provide s the DNA and amino acid sequences for the inactive (D215N) pro yeast proteinase A ( pro YPRA) construct as well as the vector map. DNA for the E. coli codon optimized pro YPRA was synthesized by DNA2.0. The DNA sequence is flanked with an NcoI restricti on site (yellow) and a FactorXa cutsite (teal) on the N terminal end and two stop codons (magenta) followed by a BamHI restriction site (green) on the C terminal end for subcloning into the pET32a vector. The start codon (ATG) is located upstream of the s equence encoding for thioredoxin, which is found within the sequence of the pET32a vector (Figure 5 x). Residue one of prorenin is underlined for clarity. Sites of engineered CYS residues are highlighted in grey. Mutated active site reside D215N is high lighted in dark yellow. Table B 1. E. coli codon optimized Pro YPRA D215N sequence with Factor Xa cutsite ccatgg ctatcgatggtc gca aggttcacaaggcaaagatttacaaacatgaactgagcgat M A I D G R K V H K A K I Y K H E L S D gaaatgaaagaggtcaccttc gagcagcacctggcgcatttgggtcaaaaatacctgacc E M K E V T F E Q H L A H L G Q K Y L T cagttcgagaaagctaatccggaggtcgttttcagccgcgagcacccgtttttcacggaa Q F E K A N P E V V F S R E H P F F T E ggcggtcacgatgttccgctgaccaattatctg aatgcccaatactataccgacatcacg G G H D V P L T N Y L N A Q Y Y T D I T ttgggcaccccaccgcaaaactttaaggttatcctggacacgggtagcagcaatttgtgg L G T P P Q N F K V I L D T G S S N L W gttcctagcaacgaatgtggtagcttggcctgctttctgcactcc aaatatgaccatgag V P S N E C G S L A C F L H S K Y D H E gcgtcgagcagctacaaggccaacggtacggaatttgccatccagtacggcaccggtagc A S S S Y K A N G T E F A I Q Y G T G S ctggaaggctatatcagccaagatacgctgagcatcggcgatctgactatcccgaag cag L E G Y I S Q D T L S I G D L T I P K Q gatttcgcagaagccaccagcgagccgggtctgaccttcgctttcggtaaatttgatggt D F A E A T S E P G L T F A F G K F D G attctgggtctgggttacgacacgatcagcgtcgacaaagtggtcccaccgttttataac I L G L G Y D T I S V D K V V P P F Y N gcaattcagcaggacctgctggatgaaaaacgcttcgcgttctatctgggtgacacgtcg A I Q Q D L L D E K R F A F Y L G D T S

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271 Table B 1. Continued. aaggacaccgag aac ggtggtgaggccacctttggcggtatcga tgagagcaagtttaag K D T E N G G E A T F G G I D E S K F K ggcgacattacttggctgccggtccgccgtaaggcgtactgggaggtcaagttcgagggt G D I T W L P V R R K A Y W E V K F E G atcggcttgggtgatgagtacgccgagctggagtctcatggtgcagcgatcaacac cggc I G L G D E Y A E L E S H G A A I N T G acgagcctgatcacgctgccgtctggtttggccgagatgatcaacgcggagattggtgca T S L I T L P S G L A E M I N A E I G A aagaagggttggactggtcagtacacgctggattgtaatacccgtgataacttgccggat K K G W T G Q Y T L D C N T R D N L P D ctgatcttcaatttcaacggctacaatttcaccatcggcccgtacgactacacgttggag L I F N F N G Y N F T I G P Y D Y T L E gtgagcggcagctgtatcagcgcgatcaccccgatggacttcccggagccggttggcccg V S G S C I S A I T P M D F P E P V G P ctggcgattgttggtgatgcgtttctgcgcaaatactacagcatttatgatctgggtaac L A I V G D A F L R K Y Y S I Y D L G N aatgccgtgggcctggccaaagcgatc taataa ggatcc N A V G L A K A I G S Figu re B 1 Map of pJ201:19864 with D215N pro y east proteinase A insert (red).

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272 APPENDIX C HIV 1 PROTEASE DNA AND A MINO ACID SEQUENCES Appendix C provides the DNA and amino acid sequences for the HIV 1 Protease constructs produced for the dissertation researc h The DNA for the E. coli codon optimized HIV 1 Protease (HIV 1PR) subtype F si K45C and E. coli codon optimized CRF01_A/E si K55C was synthesized and ordered by DNA2.0. Chapter 1 provided a more detailed discussion on construct nomenclature and amino aci d substitution code. In our naming scheme, a refers to sequences that have incorporated the Q7K, L33I, and L63I substitutions that stabilize against autoproteolysis. The ( D25N ). Each of the other c onstructs listed in this appendix were produced via site directed spin labeling using the primers listed in Appendix E. All DNA sequences are flanked with an NdeI restriction site (yellow) on the N terminal end and two stop codons (magenta) followed by a BamHI restriction site (green) on the C terminal end for subcloning into the pET23 expression vector. The start codon (ATG) is located, in frame, within the NdeI restriction site. Residue one of HIV 1PR is underlined for clarity. Sites of engineered CYS residues are highlighted in grey. Active site residue D25 (for active constructs) or D25N (for inactive constructs) is highlighted in dark yellow. Subtype F C onstruct S equences Table C 1. E. coli c odon optimized HIV 1 P R subtype F si K45C sequence c atatg ccg cagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg aac accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L N T G A D D T V I E D M N L ccgggtaagtgga aaccg tgc atgattggcggtattggtggtttcatcaaagtcaagcaa P G K W K P C M I G G I G G F I K V K Q tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc Y D Q I I I E I A G H K A I G T V L V G ccaaccccggtgaatatcattggtc gcaacttgctgacgcagattggtgcaacgctgaac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggatcc F G S

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273 Table C 2. E. coli c odon optimized HIV 1P R subtype F s K45C sequ ence catatg ccg cagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg gat accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L D T G A D D T V I E D M N L ccg ggtaagtggaaaccg tgc atgattggcggtattggtggtttcatcaaagtcaagcaa P G K W K P C M I G G I G G F I K V K Q tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc Y D Q I I I E I A G H K A I G T V L V G ccaaccccggtgaat atcattggtcgcaacttgctgacgcagattggtgcaacgctgaac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggatcc F G S Table C 3. E. coli c odon optimized HIV 1P R subtype F si K55C sequence catatg ccg cagattaccctgtggaagcgtccg ctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg aac accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L N T G A D D T V I E D M N L ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttc atc tgc gtcaagcaa P G K W K P K M I G G I G G F I C V K Q tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc Y D Q I I I E I A G H K A I G T V L V G ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctg aac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggatcc F G S Table C 4. E. coli c odon optimized HIV 1P R subtype F s K55C sequence catatg ccg cagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg gat accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L D T G A D D T V I E D M N L ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatc tgc gtcaagcaa P G K W K P K M I G G I G G F I C V K Q tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc Y D Q I I I E I A G H K A I G T V L V G ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggatcc F G S Table C 5. E. coli c odon optimized HIV 1P R subtype F si T74C sequence catatg ccg cagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg aac accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L N T G A D D T V I E D M N L

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274 Table C 5. Continued. ccgggtaagtggaaaccga aaatgattggcggtattggtggtttcatcaaa gtcaagcaa P G K W K P K M I G G I G G F I K V K Q tacgatcagattatcatcgaaatcgctggccacaaagcgatcggt tgc gttctggttggc Y D Q I I I E I A G H K A I G C V L V G ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggatcc F G S Table C 6. E. coli c odon optimized HIV 1P R subtype F s T74C sequence catatg ccg cagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg gat accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L D T G A D D T V I E D M N L ccgggtaagtggaaaccga aaatgattggcggtattggtggtttcatcaaa gtcaagcaa P G K W K P K M I G G I G G F I K V K Q t acgatcagattatcatcgaaatcgctggccacaaagcgatcggt tgc gttctggttggc Y D Q I I I E I A G H K A I G C V L V G ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggat cc F G S Table C 7. E. coli c odon optimized HIV 1P R subtype F si sequen ce catatg ccg cagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg a ac accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L N T G A D D T V I E D M N L ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatcaaagtcaagcaa P G K W K P K M I G G I G G F I K V K Q tacgatcagattatcatcgaaatcgctggcc acaaagcgatcggtactgttctggttggc Y D Q I I I E I A G H K A I G T V L V G ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggatcc F G S Tab le C 8. E. coli c odon optimized HIV 1P R subtype F s sequence catatg ccg cagattaccctgtggaagcgtccgctggtcacgatcaaagttggcggccaa H M P Q I T L W K R P L V T I K V G G Q ttgaaggaggccctgctg gat accggtgcggacgataccgtgattgaggacatgaatctg L K E A L L D T G A D D T V I E D M N L ccgggtaagtggaaaccgaaaatgattggcggtattggtggtttcatcaaagtcaagcaa P G K W K P K M I G G I G G F I K V K Q tacgatcagattatcatcgaaatcgctggccacaaagcgatcggtactgttctggttggc Y D Q I I I E I A G H K A I G T V L V G

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275 Table C 8. Continued. ccaaccccggtgaatatcattggtcgcaacttgctgacgcagattggtgcaacgctgaac P T P V N I I G R N L L T Q I G A T L N ttc taataa ggatcc F G S CRF01_A/E C onstru ct S equences Table C 9. E. coli c odon optimized HIV 1P R subtype A/E si K55C sequence catatg ccg cagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa H M P Q I T L W K R P L V T V K I G G Q ctgaaa gaagcgctgctg aac accggtgcggatgatacggtcattg aggacatcaatctg L K E A L L N T G A D D T V I E D I N L ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatc tgc gtgcgccaa P G K W K P K M I G G I G G F I C V R Q tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttg gt Y D Q I I I E I A G K K A I G T V L V G ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac P T P V N I I G R N M L T Q I G A T L N ttc taataa ggatcc F G S Table C 10. E. coli c odon o ptimized HIV 1P R subtype A/E s PMPR K 55C sequence catatg ccg cagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa H M P Q I T L W K R P L V T V K I G G Q ctgaaagaagcgctgctg gat accggtgcggatgatacggtcattgaggacatcaatctg L K E A L L D T G A D D T V I E D I N L ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatc tgc gtgcgccaa P G K W K P K M I G G I G G F I C V R Q tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttggt Y D Q I I I E I A G K K A I G T V L V G ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac P T P V N I I G R N M L T Q I G A T L N ttc taataa ggatcc F G S Table C 11. E. coli c odon optimized HIV 1P R subtype A/E si T74C s equence catatg ccg cagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa H M P Q I T L W K R P L V T V K I G G Q ctgaaagaagcgctgctg aac accggtgcggatgatacggtcattgaggacatcaatctg L K E A L L N T G A D D T V I E D I N L c cgggtaagtggaaaccga aaatgattggcggcatcggcggctttatcaaa gtgcgccaa P G K W K P K M I G G I G G F I K V R Q tacgaccagatcatt atcgagattgctggtaagaaggcaattggc gtc gtcttggttggt Y D Q I I I E I A G K K A I G C V L V G ccgaccccggtga atatcatcggtcgtaacatgctgactcagattggtgccacgctgaac P T P V N I I G R N M L T Q I G A T L N

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276 Table C 11. Continued. ttc taataa ggatcc F G S Table C 12. E. coli c odon optimized HIV 1P R subtype A/E s T74C sequence catatg ccg cagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa H M P Q I T L W K R P L V T V K I G G Q ctgaaagaagcgctgctg gat accggtgcggatgatacggtcattgaggacatcaatctg L K E A L L D T G A D D T V I E D I N L ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatcaaagtgcgccaa P G K W K P K M I G G I G G F I K V R Q tacgaccagatcattatcgagattgctggtaagaaggcaattggc tgc gtcttggttggt Y D Q I I I E I A G K K A I G C V L V G ccgacc ccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac P T P V N I I G R N M L T Q I G A T L N ttc taataa ggatcc F G S Table C 13. E. coli c odon optimized HIV 1P R subtype A/E si sequen ce catatg ccg cagatcacgctgtggaaacgtccactggttaccgttaagattggtggtcaa H M P Q I T L W K R P L V T V K I G G Q ctgaaagaagcgctgctg aac accggtgcggatgatacggtcattgaggacatcaatctg L K E A L L N T G A D D T V I E D I N L ccgggtaagtggaaaccgaaa atgattggcggcatcggcggctttatcaaagtgcgccaa P G K W K P K M I G G I G G F I K V R Q tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttggt Y D Q I I I E I A G K K A I G T V L V G ccgaccccggtgaatatcatcggtcgtaacatg ctgactcagattggtgccacgctgaac P T P V N I I G R N M L T Q I G A T L N ttc taataa ggatcc F G S Table C 14. E. coli c odon optimized HIV 1P R subtype A/E s sequence catatg ccg cagatcacgctgtggaaacgtccactggttaccgttaagattggtg gtcaa H M P Q I T L W K R P L V T V K I G G Q ctgaaagaagcgctgctg gat accggtgcggatgatacggtcattgaggacatcaatctg L K E A L L D T G A D D T V I E D I N L ccgggtaagtggaaaccgaaaatgattggcggcatcggcggctttatcaaagtgcgccaa P G K W K P K M I G G I G G F I K V R Q tacgaccagatcattatcgagattgctggtaagaaggcaattggcaccgtcttggttggt Y D Q I I I E I A G K K A I G T V L V G ccgaccccggtgaatatcatcggtcgtaacatgctgactcagattggtgccacgctgaac P T P V N I I G R N M L T Q I G A T L N ttc taataa ggatcc F G S

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277 APPENEDIX D A SOLUBLE EXPRESSION SYSTEM FOR GM2 A CTIVATOR P RO TEIN The thioredoxin fusion methodology was employed for GM2 activator protein (GM2 AP ) in an attempt to find a better, cheaper, less time consuming purification scheme. The overall results of tha t project will be discussed here in Appendix D. The gene for GM2AP was removed from pET16 expression vector and ligated into pET32a to facilitate thioredoxin fusi on expression via standard restriction digestion and ligation procedures. Figure D 1 shows the results of a pilot expression of the GM2AP thioredoxin fusion construct in OrigamiB(DE3) E. coli cells and subsequently purified according to the procedure desc ribed in Chapter 5 A typical affinity c olumn chromatogram is shown in F igure D 2. Figure D 3 shows a sodium dodecyl sulfate polyacrylamide gel electrophorsis (SDS PAGE) gel with > 90% pure GM2AP trx, and the results of enzymatic cleavage of GM2AP from t hioredoxin. Figure D 1. SDS PAGE gel showing p ilot expression of prorenin GM2AP thioredoxin fusion construct in OrigamiB(DE3) strain E. coli cells. Lanes 1 8: NOT INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 6, 8, 12, and 24 hours, respect ively, Lane 9: broad range protein marker, Lanes 10 17: INDUCED WITH IPTG, time points taken at t = 0, 1, 2, 4, 6, 8, 12, and 24 hours, respectively.

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278 Figure D 2 Typical chromatogram for GM2AP trx fus ion construct using 5 mL n i ckel charged HiTr ap HP affinity column. Figure D 3 SDS PAGE gel showing in purity of GM2AP trx fusion construct following senquential affinity and anion exchange chromatography steps and succe ssful cleavage of GM2AP trx fusion construct into GM2AP and thioredoxin (t he third band is likely the FactorXa enzyme )

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279 Additionally, a fluorescence resonance energy transfer ( FRET ) based assay was performed in order to assess the functionality of the protein. Shown in Figure D 4 are the results of a lipid vesicle binding assay w ith 3 mg/mL GM2AP trx fusion construct at pH 8.0 and pH 4.5, respectively. Vesicle composition was 10 M POPC:dansyl acetate buffer. Sample was excited at 280 nm with a slit width of 2 nm. GM2AP is known to bind to membranes at pH 4.5. At pH 4.5 an increase in fluorescence is seen around 510nm. That increase in the fluorescence signal is indicative of GM2AP function At pH 8.0, where GM2AP does not bind to membranes, no increase in fluorescence is seen. Thus, the GM2AP trx fusion construct is binding to lipid vesicles. Figure D 4 Results of a FRET based functional assa y, where the increase in fluorescence signal is indicative of GM2AP function.

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291 BIOGRAPHICAL SKETCH Jamie Laura Kear was born in 1982 in Carbondale, Illinois, and mov ed to Bloomington, Illinois in 1989 where she lived until 2002. She graduated from Bloomington High School in 2000, and subsequently attended Illinois State University until December 2001. She transferred to Southern Illinois University in January 2002, where she graduated with honors with both a bachelors of science (B.S) degree in c hemistry and a bachelors of scien ce (B.S.) degree in biological s ciences. She was admitted to the Department of Chemistry graduate program at the University of Florida in 20 05, where she joined the research group of Dr. Gail E. Fanucci. She defended her dissertation in April 2010 and obtained her Ph.D. in Biochemistry in A ugust 2010.