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Biophysical Characterization of Peptide Mimics of Lung Surfactant Protein-B

Permanent Link: http://ufdc.ufl.edu/UFE0021688/00001

Material Information

Title: Biophysical Characterization of Peptide Mimics of Lung Surfactant Protein-B
Physical Description: 1 online resource (205 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: calorimetry, circular, deuterium, dichroism, distress, kl4, lung, nmr, peptide, phospholipid, phosphorous, respiratory, solid, state, structure, surfactant, syndrome
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Lung surfactant is a lipid rich fluid that coats the inner surface of the alveoli, the primary units of respiration where oxygen diffuses into the bloodstream. Due to the small radii and high curvature of the alveoli, significant pressure is required to overcome surface tension and to inflate the lung. Lung surfactant is a biological coating that lowers surface tension and minimizes the work required for breathing. It is primarily composed of two phospholipids, the zwitterionic 1,2-dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and the anionic 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phospho-rac-(1-glycerol) (POPG). The 79-amino acid surfactant protein B (SP-B), however, is an absolutely essential component of lung surfactant. Although present at low levels (approximately 1% by weight), mutation or loss of SP-B results in respiratory distress syndrome (RDS). The design of simple peptides to mimic the charge distribution and the hydrophobic characteristics of SP-B was investigated in the early 1990s. A peptide composed of only the basic residue lysine and hydrophobic leucine systematically repeated (KLLLLKLLLLKLLLLKLLLLK) has demonstrated clinical efficacy in the treatment of RDS when administered with DPPC, POPG and palmitic acid. In this dissertation, solid-state NMR spectroscopy (ssNMR) was used to examine in detail the phase properties of these lipids and the effects of KL4 on lipid organization and dynamics. Structural measurements were also performed to determine the secondary structure of KL4. These studies were carried out using two model lipid systems: 1) 3:1 POPC: POPG, a bilayer system used in many studies probing peptide:lipid interactions, including amphipathic antibiotics of similar size and hydrophobicity to KL4, and 2) 4:1 DPPC:POPG, which is similar to the lipid composition in the lung. Complementary studies using differential scanning calorimetry (DSC) and circular dichroism (CD) were also carried out. Comparative experiments were also performed on residues 59-80 of SP-B (SP-B59-80), the C-terminal region upon which the sequence of KL4 was derived. Based on these studies, a molecular model incorporating the structure of KL4 and its interactions with 3:1 POPC:POPG bilayers and 4:1 DPPC:POPG bilayers was developed. This model can serve as a guide for understanding how proteins modulate surface tension and for developing more effective peptide mimetics for clinical use.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Long, Joanna R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021688:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021688/00001

Material Information

Title: Biophysical Characterization of Peptide Mimics of Lung Surfactant Protein-B
Physical Description: 1 online resource (205 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: calorimetry, circular, deuterium, dichroism, distress, kl4, lung, nmr, peptide, phospholipid, phosphorous, respiratory, solid, state, structure, surfactant, syndrome
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Lung surfactant is a lipid rich fluid that coats the inner surface of the alveoli, the primary units of respiration where oxygen diffuses into the bloodstream. Due to the small radii and high curvature of the alveoli, significant pressure is required to overcome surface tension and to inflate the lung. Lung surfactant is a biological coating that lowers surface tension and minimizes the work required for breathing. It is primarily composed of two phospholipids, the zwitterionic 1,2-dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and the anionic 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phospho-rac-(1-glycerol) (POPG). The 79-amino acid surfactant protein B (SP-B), however, is an absolutely essential component of lung surfactant. Although present at low levels (approximately 1% by weight), mutation or loss of SP-B results in respiratory distress syndrome (RDS). The design of simple peptides to mimic the charge distribution and the hydrophobic characteristics of SP-B was investigated in the early 1990s. A peptide composed of only the basic residue lysine and hydrophobic leucine systematically repeated (KLLLLKLLLLKLLLLKLLLLK) has demonstrated clinical efficacy in the treatment of RDS when administered with DPPC, POPG and palmitic acid. In this dissertation, solid-state NMR spectroscopy (ssNMR) was used to examine in detail the phase properties of these lipids and the effects of KL4 on lipid organization and dynamics. Structural measurements were also performed to determine the secondary structure of KL4. These studies were carried out using two model lipid systems: 1) 3:1 POPC: POPG, a bilayer system used in many studies probing peptide:lipid interactions, including amphipathic antibiotics of similar size and hydrophobicity to KL4, and 2) 4:1 DPPC:POPG, which is similar to the lipid composition in the lung. Complementary studies using differential scanning calorimetry (DSC) and circular dichroism (CD) were also carried out. Comparative experiments were also performed on residues 59-80 of SP-B (SP-B59-80), the C-terminal region upon which the sequence of KL4 was derived. Based on these studies, a molecular model incorporating the structure of KL4 and its interactions with 3:1 POPC:POPG bilayers and 4:1 DPPC:POPG bilayers was developed. This model can serve as a guide for understanding how proteins modulate surface tension and for developing more effective peptide mimetics for clinical use.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Long, Joanna R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021688:00001


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BIOPHYSICAL CHARACTERIZATION OF PEPTIDE MIMICS OF LUNG SURFACTANT
PROTEIN-B























By

VIJAY C. ANTHARAM


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

2008

































O 2008 Vijay C. Antharam



































To my Mom.









ACKNOWLEDGMENTS

I thank my mentor Dr. Joanna Long for providing me the opportunity for working in her

laboratory. I thank her for her support, patience, sense of humor, and kindness. I hope to

acquire some of those qualities that she has as I continue to mature and develop. I would also

like to offer my gratitude to members of my committee: Dr. Arthur Edison, Dr. Robert

McKenna, Dr. Susan Frost and Dr. Ron Castellano. I appreciate their support and their help. I

am particularly grateful for Dr. Susan Frost for giving me some extra support with regards to my

presentation and for her willingness to afford some of her time to look over my dissertation.

I want to extend my heartfelt gratitude and sincere thanks to members of the laboratory

throughout these years. These include Dr. Frank Mills, Dr. Mini-Samuel Landtiser, Dr. Doug

Elliott, Seth McNeill, Suzanne Farver, David Fleishmann, Julie Vanni, Joanne Anderson, and

Jonathan Lane. I also want to thank some very special people I have met: Sonny Flores, Nelson

Klahr, Sauray Chandra, Karen vonDeneen and Mariam Rahmani. I am grateful and fortunate to

have known Sonny, Nelson, Jonathan, and especially Mariam--all of them are very special to me.

Lastly, I thank my family members, mainly my mother Urmila Antharam. I feel very

blessed and grateful for all that they have done for me.











TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ............ _...... ._ ...............8....

LI ST OF FIGURE S .............. ...............9.....


LI ST OF AB BREVIAT IONS ............_ ..... ..__ ............... 14..

AB S TRAC T ........._. ............ ..............._ 17...

CHAPTER

1 DISCOVERY AND HISTORY OF LUNG SURFACTANT ................. .......................19

The Alveoli and Surface Tension .............. ......... ....... ...........2
Chemical Composition and Origin of Lung Surfactant ....._.__._ ..... ... .__. ................. 22
Cellular Biology of Lung Surfactant ........._.__........_.__._ ...............24...
Important Surface-Acting Proteins of Lung Surfactant ......__............... .... .........._.25
Lung Surfactant Protein B (SP-B) and RDS............... ...............27..
Mammalian Lung Surfactant Cycle ................. ...............31................
Exogenous and Artificial Lung Surfactants ....__ ......_____ .......___ ...........3

2 BIOPHYSICAL TECHNIQUES TO PROBE PEPTIDE STRUCTURE AND
PEPTIDE-LIPID INTERACTIONS ............ .....___ .....__ ............4

Circular Dichroism .............. .. .... .... ............4
Differential Scanning Calorimetry (DSC) ................ ...............47................
Solid state N M R Spectroscopy ................... ........... ...............50......
Spin Interactions Commonly Seen in ssNMR ................. ...............50...............
Chemical shift............... ...............50.
Dipole-dipole couplings .............. ...............51....
Quadrupole couplings ................... ..... ....... ..... ............ .............5
Applications and Methodologies in Solid-State NMR ......____ ..... ... ._ .............. ..52
Pake Powder Pattern .........._............____ ...............52.....
Chemical Shift Anisotropy (CSA)............... ...............53.
Magic Angle Spinning NMR. ............ ..... .__ ...............54..
Range of NMR Time Scales.......................... ...............5
NMR-Active Nuclei and Importance to Biological Molecules .............. .....................5
3P (Phosphorus) NM R............... ...............57..
2H (Deuterium) NMR ............ ..... ._ ...............60...
D eP aki ng ........._.._... ......____ ...............62....
M agnetic Field Orientation ........._... ...... ..... ...............63....











3 SURFACTANT PEPTIDE KL4 DIFFERENTIALLY MODULATES LIPID
COOPERATIVITY AND ORDER INT DPPC: POPG AND POPC: POPG LIPID
VESICLES................ ...............8

Relevance of KL4 to Lung Surfactant Biology ................. ...............81........... ..
Methodology to Study KL4 with Lipids .............. ...............83....
KL4 Affects Lipid Phase Behavior .............. ... ... ......... .. ..... ........ ... .. ........8
3 P NMR: Addition of KL4 Leads to Changes in Orientation of the PG Headgroups. ...........87
KL4 Effects on Lipid Acyl Chain Ordering Dependent on the Saturation of the Acyl
C hains............... ... ... .......... .... .. .... .. ..........8
KL4 in Relation to other Peptides of Similar Size, Composition and Length ........................91
KL4 Shares Many Properties with Cholesterol and Transmembrane Helices in DPPC.........94
Molecular Model of KL4 with POPC:POPG and DPPC:POPG ........._._. ...... .._.._..........94

4 STRUCTURAL STUDIES OF KL4 ........._._.._......_.. ...............125....

Characterization of KL4 Secondary Structure ................. ........._.._.......125_._....
M materials and M ethodology ................. ...............127..............
CD shows KL4 to be helical....................... .. ..... ...............12
Different Types of Helices KL4 May Adapt in a Lipid Bilayer ........._.._... ........._..........133
Solid-State NMR Studies of KL4 in POPC:POPG and DPPC:POPG ................. ............... 134

5 COMPARATIVE BIOPHYSICAL STUDIES OF SP-B59-80........ ...............15215

Fragments of SP-B have biophysical activity............... ...............15
M materials and M ethodology ................... ........... __... .......... ............15
DSC Studies on SP-B59-80 with DPPC:POPG LUVs ................... .. ......... ................... ...156
3P NMR of Lipid MLVs Containing SP-B59-80 Show POPG Interacting with the Peptide .158
2H N\MR Indicates that the Properties of KL4 are Similar to SP-B59-80........ ............... 160
Evidence of Alternative Dynamics at High Concentrations of SP-B59-80? ........................... 163
Comparisons of SP-B59-80 and KL4 ........._...... .... ... .._ ... ...............164...
Preliminary Molecular Model of SP-B59-80 with Lipids .........._.... ....___.. ........._...166

6 CONCLUSIONS AND FUTURE EXPERIMENTS ....._____ .........__ ...........__....184

APPENDIX

A CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS 4: 1 DPPC(D-62):POPG WITH KL4 (MOLAR
PERCENTAGES)...............................8

B CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS 4: 1 DPPC:POPG(D-31) WITH KL4 (MOLAR
PERCENTAGES)...............................9










C CALCULATED 2H ORDER PARAMETERS (TO TWO SIGNIFICANT FIGURES)
FOR DEUTERATED LIPIDS 3:1 POPC(D-31):POPG WITH KL4 (MOLAR
PERCENT AGE S) .............................1

D CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS INT 3:1 POPC:POPG(d-31) WITH KL4 (MOLAR
PERCENTAGES)...............................9

E CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS INT 4: 1 DPPC(d-62):POPG WITH SP-B(59-80) (MOLAR
PERCENTAGES)...............................9

F CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS INT 3:1 POPC(D-31):POPG WITH SP-B(59-80) (MOLAR
PERCENTAGE S)..............................9

LIST OF REFERENCES .........._ _... .... ._ ...............195..

BIOGRAPHICAL SKETCH ............. ..............205.....










LIST OF TABLES


Table page

1-1 Approximate weight percentages of lipids in mammalian lung surfactant. ................... ....39

1-2 Table of artificial lung surfactants used clinically.. ............. ...............43.....

3-1 Thermodynamic parameters obtained from DSC thermograms ................... ...............99

3-2 CSA span for phosphate headgroup in 3:1 POPC:POPG and 4: 1 DPPC:POPG MLVs
with and without KL4 ........._.___..... .___ ...............100...

4-1 Ellipticity (in mdeg) of KL4 at 222nm and 208nm and ratio of helical signatures
222nm and 208nm............... ...............145.

4-2 Ellipticity (in mdeg) of 40C1M KL4 TOCOnstituted in LUVs from organic solvent. ..........146

5-1 Thermodynamic parameters derived from DSC on residues on 4:1 DPPC (d-
62):P OPG LUVs with SP-B 59-80 ............. ...............170

A-1 Order Parameters for sn-1 and sn-2 chain of DPPC(d-62) ................. ......................189

B-1 Order Parameters for deuterated sn-1 chain of POPG ................. .........................190

C-1 Order Parameters for deuterated sn-1 chain of POPC .......... ................ ............... 191

D-1 Order Parameters for deuterated sn-1 chain of POPG ................. .........................192

E-1 Order parameters for sn-1 and sn-2 chain of DPPC with SP-B(59-80) ................... ........ 193

F-1 Order Parameters for deuterated sn-1 chain of POPC with SP-B(59-80)........................194










LIST OF FIGURES


Figure page

1-1 Cartoon illustration of surface tension. .............. ...............37....

1-2 The alveolar environment. ............. ...............38.....

1-3 Hypothesized interactions of SP-B and SP-C with lipid lamellae ................. ................40

1-4 Histology of SP-B mutation ................. ...............41........... ...

1-5 Mammalian lung surfactant homeostasis............... ..............4

1-6 Primary amino acid sequence of SP-B and KL4. ............. ...............44.....

2-1 CD spectra from various secondary structure elements............... ...............64

2-2 Schematic of a differential scanning calorimeter (DSC). ................. .................6

2-3 The gel to liquid-crystalline phase transition of phospholipids bilayers. ........._.._.............66

2-4 A) DSC thermogram of 2mM DPPC large unilamellar vesicles (LUVs) dispensed in
5mM HEPES pH 7.4. .............. ...............67___......

2-5 Time scale of motional processes for nuclear spins in NMR. ................ .....................68

2-6 31P NMR static spectrum of DPPC hydrated vesicles (approximately 60mg). The
spectrum was taken at 44 degrees on a 600MHz Bruker instrument with 1024 scans......69

2-7 31P NMR of static hydrated DPPC vesicles (approximately 60mg) taken on a 600
MHz Bruker instrument (1024 scans) at 240C, well below its phase transition
tem perature, .............. ...............70....

2-8 Graphical depiction of shielding tensor for a nucleus with an axis of symmetry..........._...71

2-9 Phosphorous NMR lineshape patterns for lipid mesophases.............___ ........._ ......72

2-10 Magic angle spinning (MAS) spectra of the 13C I HUClOUS in glycine............._.._ ..............73

2-11 DRAWS pulse sequence of DRAWS employed during MAS for dipolar recoupling.. ....74

2-12 Collective motions of each methylene position along the acyl chain roughly averages
out to a conicall shape. ........._.__ .... ..__ ...............75..

2-13 Deuterium spectra of (4: 1) DPPC:POPG(d-31) (mol/mol) with POPG fully
deuterated on the sn-1 acyl chain. ........._. ...... .___ ...............76..










2-14 Example of order parameter profile generated from dePaking 2H NMR spectra, for
perdeuterated acyl chains. .............. ...............77....

2-15 Example of 31P NMR (blue) and the dePaked result (red)............... .................7

2-16 Lipid vesicles when placed in a magnetic field can deform to form an ellipsoidal
shape. ............. ...............79.....

3-1 Differential scanning calorimetry on KL4 with 4:1 DPPC:POPG vesicles with KL4 at
the indicated molar percentages ................. ...............98................

3-2 Static 31P NMR spectra of 3:1 POPC(d-31):POPG MLVs with the increasing
addition of K L4. ............. ...............101....

3-3 Static 31P dePaked NMR spectra of 3:1 POPC(d-31):POPG with increasing amounts
of KL4 ................. ...............102................

3-4 Static 31P NMR spectra of 4: 1 DPPC(d-62):POPG MLVs with increasing addition of
KL4 ................. ...............103................

3-5 Static 31P dePaked NMR spectra of 4:1 DPPC(d-62):POPG with increasing amounts
of KL4 ................. ...............104................

3-6 Static 31P NMR spectra of 3:1 POPC:POPG(d-31) MLVs with increasing amounts of
K L4 ................ ...............105__._.......

3-7 DePaked 31P NMR spectra of 3:1 POPC:POPG(d-31) with increasing amounts of
K L4 ................ ...............106._.._._ ......

3-8 Static 31P NMR spectra of 4: 1 DPPC:POPG(d-3 1) MLVs with increasing amounts of
KL4 ........._..... ...__. ...............107...

3-9 DePaked 31P NMR spectra of 4: 1 DPPC:POPG(d-3 1) with increasing amounts of
KL4 ........._.___..... ._ __ ...............108....

3-10 The shift in dePaked frequency in ppm (Ac) for DPPC and POPG (in ppm) by KL4
in 4: 1 DPPC:POPG(d-31) ................. ...............109.............

3-11 Static 31P NMR spectra of single lipid and lipid with 1.5 mol% KL4 A: DPPC(d-62),
B: POPC(d-31) and C: POPG(d-31) ................. ...............110.............

3-12 DePaked 31P spectra for (Top) DPPC(d-62), (Middle) POPC(d-31) and (Bottom)
POPG(d-31) with and without 1.5mol% KL4. --------------... ---------------111.......... ..

3-13 2H spectra of 3:1 POPC(d-3 1):POPG MLVs with increasing amounts of KL4...............1 12

3-14 2H NMR spectra of 4: 1 DPPC(d-62):POPG with increasing amounts of KL4................ 113










3-15 DePaked 2H spectra for (A) 4: 1 DPPC(d-62):POPG MLVs and (B) 3:1 POPC(d-
3 1):POPG MLVs with increasing amounts of KL4 ................. ........_ ................1 14

3-16 Deuterium NMR spectra for (A) 3:1 POPC:POPG(d-31) MLVs and (B) DePaked
spectra with increasing amounts of KL4. ................ .........__. ......115__. ...

3-17 Static 2H spectra for (A) 4: 1 DPPC:POPG(d-31) MLVs and (B) DePaked spectra
with increasing amounts of KL4. ........... ......_. ...............116..

3-18 Static 2H NMR spectra of single lipid MLVs with and without 1.5mol% KL4. ............117

3-19 Order parameter profiles for (4:1) DPPC(d-62):POPG MLVs with and without KL4.
Top:sn-1 chain Bottom: sn-2 chain............... ...............118.

3-20 Order parameter profile for the sn-1 chain of 3:1 POPC(d-31):POPG MLVs with and
without KL4 ........... __..... ._ ...............119....

3-21 Three dimensional plot of change in order parameter for DPPC(d-62):POPG MLVs
as a function of mole percentage of KL4. ......... .............._ ........_ .........12

3 -22 Three dimensional plot of change in time averaged order parameter for 3:1 POPC(d-
3 1):POPG MLVs as a function of mole percentage of KL4. ........_.._....... .......... ..... 121

3-23 Order parameter profile for A) 4: 1 DPPC:POPG(d-31) MLVs and B) 3:1
POPC: POPG(d-3 1) M LVs ................. ................ 122........ ...

3 -24 Change in order parameter values on (A) POPC:POPG(d-31) and (B)
DPPC:POPG(d-3 1) upon addition of KL4 ...._._ .....__.___ .......____ .........12

3-25 Model of KL4 penetration in two lipid environments. .....__.___ ........___ ...............124

4-1 CD Spectra of KL4 in 5mM HEPES at pH 7.4. Below is a spectrum of 1 50C1M KL4
indicating potential aggregation. ........._._._........ ...............138...

4-2 CD spectra of KL4 in Organic solvents. Red is a spectrum of peptide in 150C1M
hexafluroisopropanol and black is a spectrum in 50:50 MeOH:dH20. ............................139

4-3 CD Spectra of 40C1M KL4 added to 1.33mM LUVs. DPPC containing samples were
run at 45oC .............. ...............140....

4-4 CD spectra of 40C1M KL4 TOCOnstituted in 4mM 4: 1 DPPC:POPG and 3:1
POPC:POPG LUVs. ............. .....................141

4-5 CD spectra of 40C1M KL4 TOCOnstituted in 2mM 4: 1 DPPC:POPG and 3:1
POPC:POPG LUVs. ............. .....................142

4-6 CD spectra of 40C1M KL4 reconstituted in 1.33mM 4: 1 DPPC:POPG and 3:1
POPC:POPG LUVs. ............. .....................143










4-7 CD Spectra of (Top) POPC:POPG LUVs and (Bottom) DPPC:POPG LUVs with
increasing mol% of KL4 ........._._._..... ..... ...............144...

4-8 Helical wheel proj sections of KL4 aS: (left) 3 10 helix, (middle) standard a-helix, and
(right) xn helix. ............. ...............147....

4-9 DQ-DRAWS buildup curves generated from spectra on 13C labeled KL4 with
POPC:POPG (3:1).. ................ ...............148..............

4-10 Ramachandran plot showing a X2 minimum at cp=-105 and uy=-26 for KL4 in 3:1
POPC:POPG. .............. ...............149....

4-11 Model of KL4 based on torsion angles obtained from the 2D-DRAWS experiments.....150

4-12 KL4 with torsion angles of cp = -65, uy= -78 obtained from ssNMR studies of KL4 in
a DPPC:POPG lipid environment. ........... _......__ ...............151

5-1 Putative helical wheel for each type of helix rendered for the C-terminal residues 59-
80 of SP-B. ........... ..... ._ ...............168..

5-3 31P NMR data of 4: 1 DPPC:POPG MLVs with varying molar percentages of SP-
B59-80............... ...............171

5-4 DePaked 31P NMR data of 4: 1 DPPC:POPG MLVs with varying molar percentages
o f S P -B 59-80............... .. .. 7

5-5 Change in 31P CSAs for 4: 1 DPPC:POPG MLVs as a result of increasing molar
percentages of SP-B 59-80 ........ ...............173

5-6 Phosphorous NMR data of 3:1 POPC:POPG MLVs with varying molar percentages
o f S P -B 59-80............... .. .. 7

5-7 DePaked 31PNMR data of 3:1 POPC:POPG MLVs with varying molar percentages
of SP-B59-80 .............. ...............175....

5-8 Change in 31P CSAs for 3:1 POPC:POPG MLVs as a result of increasing molar
percentages of SP-B 59-80 ........ ...............176

5-9 Deuterium NMR spectra of 4:1 DPPC(d-62):POPG MLVs with SP-B 59-80. ................177

5-10 Stacked dePaked spectra of 4: 1 DPPC(d-62):POPG MLVs with SP-B59-80....................178

5-12 2H N\MR spectra of 3:1 POPC(d-31):POPG MLVs with SP-B59-80............... .........10

5-13 Stacked dePaked spectra of 3:1 POPC(d-31):POPG MLVs with SP-B59-80....................181s

5-14 Percent change in time averaged order parameter for POPC (d-31) in 3:1
POPC(d-31):POPG MLVs on addition of SP-B59-80. ................... ............... 182









5-15 Hypothesized orientational model of SP-B59-80 in two different lipid MLV systems......183









LIST OF ABBREVIATIONS

AAA amino acid analysis

AP alveolar proteinosis

BAL bronchoalveolar lavage

Bo external magnetic field

C-D carbon-deuterium bond

Cpmax maximum heat capacity

CD circular dichroism

CPAP continuous positive airway pressure

CPMAS cross polarization with magic angle spinning

CSA chemical shift anisotropy

CU cooperativity unit

D or d deuterium atom (2H)

DPPC 1 ,2-Dipalmitoyl-sn-Glycero-3 -Phosphocholine

DRAWS dipolar recoupling with a windowless sequence

DSC differential scanning calorimetry

Eo electric field vector

FTIR Fourier-transform infrared spectroscopy

P pressure across alveoli

Ym gyromagnetic ratio of a NMR nucleus

AGo change in Gibbs free energy (standard state)

A~ealchange in calorimetric enthalpy

AHyH change in van Hoft enthalpy

As change in shift in ppm

HFOV high frequency oscillatory ventilation









HPLC high performance liquid chormatography

ITC isothermal titration calorimetry

KL4 KLLLLKLLLLKLLLLKLLLLK (sinapultide)

LUVs large unilamellar vesicles

L, gel phase of phospholipid bilayer

Lp liquid-crystalline phase of phospholipid bilayer

MLVs multilamellar vesicles

NMR nuclear magnetic resonance

Pp ripple phase of a phospholipid bilayer

PA palmitic acid

PDB protein data bank

POPC 1 -Palmitoyl-2-Oleoyl-sn-Glycero-3 -Phosphocholine

POPG 1 -Palmitoyl-2-Oleoyl-sn-Glycero-3 -[Phospho-rac-(1 -glycerol)]

R universal gas constant (1.987 cal K^1 mol l)

rad radians

RDS respiratory distress syndrome

time averaged order parameter of a C-D bond at position i

ssNMR solid-state NMR

SP-A Lung surfactant protein A

SP-B Lung surfactant protein B

SP-C Lung surfactant protein C

SP-D Lung surfactant protein D

SUVs small unilamellar vesicles

AS change in entropy

YP surface tension









Tm phase transition temperature

AT peak-width at half height in a DSC trace









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


BIOPHYSICAL CHARACTERIZATION OF PEPTIDE MIMICS OF LUNG SURFACTANT
PROTEIN-B

By

Vijay C. Antharam

May 2008

Chair: Joanna RLong
Maj or: Medical Sciences-Biochemi stry and Molecular Biology

Lung surfactant is a lipid rich fluid that coats the inner surface of the alveoli, the primary

units of respiration where oxygen diffuses into the bloodstream. Due to the small radii and high

curvature of the alveoli, significant pressure is required to overcome surface tension and to

inflate the lung. Lung surfactant is a biological coating that lowers surface tension and

minimizes the work required for breathing. It is primarily composed of two phospholipids, the

zwitterionic 1 ,2-dipalmitoyl-sn-Glycero-3 -Phosphocholine (DPPC) and the anionic 1-Palmitoyl-

2-Oleoyl-sn-Glycero-3 -[Phospho-rac-(1 -glycerol)] (POPG). The 79-amino acid surfactant

protein B (SP-B), however, is an absolutely essential component of lung surfactant. Although

present at low levels (approximately 1% by weight), mutation or loss of SP-B results in

respiratory distress syndrome (RDS).

The design of simple peptides to mimic the charge distribution and the hydrophobic

characteristics of SP-B was investigated in the early 1990s. A peptide composed of only the

basic residue lysine and hydrophobic leucine systematically repeated

(KLLLLKLLLLKLLLLKLLLLK) has demonstrated clinical efficacy in the treatment ofRDS

when administered with DPPC, POPG and palmitic acid.









In this dissertation, solid-state NMR spectroscopy (ssNMR) was used to examine in detail

the phase properties of these lipids and the effects of KL4 On lipid organization and dynamics.

Structural measurements were also performed to determine the secondary structure of KL4.

These studies were carried out using two model lipid systems: 1) 3:1 POPC: POPG, a bilayer

system used in many studies probing peptide:1ipid interactions, including amphipathic antibiotics

of similar size and hydrophobicity to KL4, and 2) 4: 1 DPPC:POPG, which is similar to the lipid

composition in the lung. Complementary studies using differential scanning calorimetry (DSC)

and circular dichroism (CD) were also carried out. Comparative experiments were also

performed on residues 59-80 of SP-B (SP-B59-80), the C-terminal region upon which the sequence

of KL4 WAS derived.

Based on these studies, a molecular model incorporating the structure of KL4 and its

interactions with 3:1 POPC:POPG bilayers and 4: 1 DPPC:POPG bilayers was developed. This

model can serve as a guide for understanding how proteins modulate surface tension and for

developing more effective peptide mimetics for clinical use.









CHAPTER 1
DISCOVERY AND HISTORY OF LUNG SURFACTANT

In 1929, Swiss physiologist Kurt von Neergard discovered that the pressure required to fill

the lungs with air was much greater than the pressure required to fill the lungs with water. He

hypothesized that surface tension was the force responsible for inhibiting the expansion of the

lung with air (1-6). It was not until two decades later that an absolutely essential surface active

material was found in the lungs for overcoming this force and reducing the work of breathing.

Research describing the difficulties the lung undergoes during alveolar expansion and

contraction can be traced back to 1854, including contributions made by Alexander Graham Bell

before he shifted interests and help invent the telephone (1). The discovery of lung surfactant as

a material that minimizes alveolar surface tension, and its pivotal role in respiratory distress

syndrome (RDS) has a history that contains contributions from physicists, physiologists,

biochemists, and medical doctors. The knowledge generated by this diverse community has led

to an understanding of the mechanics involved in lung function as well as better diagnoses and

treatments of RDS.

RDS is a disease prevalent amongst premature infants, but it also occurs in adults and

children affected by significant injury or inflammation to their lungs. General treatment of the

disease involves mechanical ventilation, a procedure that forces air into the lungs by positive

pressure. Epidemiologic estimates indicate that onset of adult respiratory distress occurs in 75 of

100,000 individuals per year (7); and the incidence of respiratory distress after delivery is 1% of

all newborns. The advent of preventative surfactant instillation therapies has greatly improved

survival outcomes, especially in premature infants where mortality decreased by 6% from 1989

to 1990 (8). Recent estimates indicate less than a thousand infants annually are diagnosed with

RDS as compared to 10,000-15,000 in the 1950s and 1960s (9). However, improvements need









to be made in terms of therapy, and basic clinical and scientific questions remain to be answered

which can aid in the development of therapeutic agents.

Originally, RDS in infants was incorrectly diagnosed as hyaline membrane disease, based

on the mistaken assumption that the strain and grunting noises that occurred when premature

infants took their first breath was caused by the inspiration of amniotic fluid or milk, leading to

the appearance of glassy membranes found during lung autopsy. In the mid-1950s, however,

physicians Dr. Mary Ellen Avery and Dr. Jere Mead found that babies who died from RDS had

no residual air in their lungs implying that premature lungs were unable to retain air. At the

same time, Dr. John Clements, a physiologist working at the United States Army Chemical

Center, designed a surface balance where a movable barrier was allowed to cyclically compress

and expand a shallow trough containing minced lung tissue while forces were measured. Such

an apparatus, akin to a conventional Langmuir-Wilhelmy balance (6), allowed Clements to

monitor surface tension as surface area was varied. He found that surface tension rose as the

surface area containing lung extracts was expanded, but dropped from 45 to 10mN/m when the

layer was compressed without any collapse (6, 10). The evidence pointed to a fluid that

modulated surface tension in the lung that Clements first referred to as an "anti-atelectasis"

factor but was then later renamed (by Clements) as pulmonary surfactant.

Countless research by innumerable investigators contributed to the surmounting evidence

of the importance of this surface active material. This has lead to the current and almost

indisputable claim that an absence or delayed appearance of pulmonary surfactant material is the

prevailing cause ofRDS in premature infants.

The Alveoli and Surface Tension

The alveoli are the respiratory units of the lung and contain an air/water interface for

oxygen diffusion into the bloodstream. It is estimated that human lungs contain approximately









350 million alveoli with diameters ranging from 75 to 300 microns (11). Unique molecular

forces arise at interfaces compared to molecules in solution or in a bulk phase. Surfactants are

substances that have an energetic preference for interfacial regions and affect intermolecular

forces there. A critical force that exists at the alveolar interfacial region is surface tension.

Surface tension arises due to unbalanced forces that exist for molecules residing at the surface,

particularly since these molecules have diminished interaction with the bulk fluid phase (Figure

1-1). As a result of this imbalance, the surface tends to minimize in area.

Surface tension is the work needed to expand the surface area of a system, in respiration

this is the work from expanding the alveolar air sacs. Surface tension is defined as the change in

free energy per unit surface area, or equivalently it can be interpreted as work done on a surface,

with units of force per distance (mN/m) (ll).

In the lung, amphipathic phospholipid molecules serve as the surface film that separates

the air volume of the alveoli from the fluid layer (Figure 1-2). These lipid surface fi1ms have

been found to lower surface tension values during in vitro dynamic compression experiments.

The most abundant lipid in lung surfactant, 1 ,2-Dipalmitoyl-sn-Glycero-3 -Phosphocholine

(common name and abbreviated DPPC) contains two saturated 16-carbon fatty acid chains

esterfied to a zwitterionic phosphatidylcholine headgroup. The rigid nature of the disaturated

fatty acyl chains allows DPPC to form tightly packed surface fi1ms that can lower surface tension

to values of less than 1mN/m (ll). Hence, the addition of a phospholipid film found in the lung

greatly alleviates the surface tension problem faced by the lung.

The essential need of the alveolus to have an inner coating of surfactant becomes clearer

when considering the law of Young and Laplace This law states that the pressure difference

normal to surface must be greater than twice (or equal to at equilibrium) the surface tension









divided by the radius of the obj ect: Ap=2 y /r (where y represents surface tension) for inflation to

occur (4). In terms of expansion and contraction of pulmonary alveoli, the Young-Laplace

equation suggests considerable force is required to overcome the surface tension in the highly-

curved alveoli. Theoretically, y values fall to values near 0 upon expiration in the lung, and is

estimated to be roughly 30mN/m in the trachea, and about 15mN/m in the airways; consequently,

a surface tension gradient exists during exhalation (12). Methods to study surface tension in-

vitro include the Langmuir-Wilhelmy balance, captive bubble surfactometer, and pulsating

bubble surfactometer (13). Such methods for measuring the properties of surfactant are highly

dependant on lung surfactant composition and the buffer salts present in the hypophase chosen to

simulate an air/water interface.

Chemical Composition and Origin of Lung Surfactant

Knowing the exact chemical constitution of lung surfactant is important to understanding

how it lowers surface tension. Initial analyses using material lavaged from bovine lung

indicated the dominant fraction was lecithin (14), commonly known as phosphatidylcholine. Of

phosphatidylcholine, the primary fraction was the dipalmitoylated form, or DPPC. However, the

chemical composition of lung surfactant can best be described as heterogeneous mixture of

proteins and lipids with the protein fraction constituting ~8% of the total dry weight and the lipid

constituting ~92% of the total dry weight. In the lipid fraction, DPPC is predominant,

comprising up to 70%, followed by phosphatidylglycerols, comprising about 8% of the lipid

mass (5, 9). However, variations amongst species do exist (15). In humans, the second most

prominent lipid in lung surfactant is palmitoyloleoylphosphatidylglycerol (POPG). With the

advent of mass spectrometry and other analytical techniques, quantification of surfactant material

after lavage treatment has revealed a far more assorted mixture of lipids (16). Table 1 lists the

most common lipid and protein species found in lung surfactant. Other surfactant components









include phosphatidylinositols, phosphatidylethanolamines, sphingomyelins,

lysophosphatidylcholines, cholesterol, Ca+2, and free fatty acids (5, 15, 17). Despite the

heterogeneity present in lung surfactant, most artificial formulations used as candidates for

clinical treatments contain only a few components, particularly DPPC and a monounsaturated

phospholipid (usually POPG) (18).

The lipid and protein components of lung surfactant are also evolutionary conserved.

Surfactant has been discovered in the Australian lungfish Neoceratodus forsteri, which dates

back more than 300 million years, raising the question of whether the anti-adhesive properties of

surfactant may have served in the primitive respiratory system and led to the evolution of gas-

containing organs (19). Surfactant still remains one of the most highly conserved systems in

vertebrate biology (17).

Lung surfactant contains four proteins named in chronology of their discovery: (surfactant

protein A) SP-A, SP-B, SP-C and SP-D. Both SP-A and SP-D are large multi-component

proteins with an extensive collagenous structure and carbohydrate recognition domains. These

two proteins have been implicated in immune surveillance of the alveolar structure of the lung,

although SP-A has been found to also have roles in surface activity as well as intracellular

surfactant processing (20). The presence of SP-A and SP-D and their close structural

resemblance to components of the complement system (a glycoprotein cascade found in the

blood for clearance of viral and bacterial pathogens) raises interesting questions regarding

divergent evolution of the immune system and lung surfactant (20). While SP-A is critical to

surfactant homeostasis, and SP-D is important to immunological recognition, the focus of this

thesis is on factors modulating surface tension, particularly the molecular basis for SP-B

changing the phase behavior of lipids in the lung.









Cellular Biology of Lung Surfactant

Pulmonary lung surfactant is synthesized by the highly differentiated type II alveolar

epithelial cell derived from the alveolar epithelial lining (11, 15, 21-23). These cells are

distinguished from type I cells by their flat appearance, apical microvilli, and the presence of

lamellar bodies in the cytoplasm. Type II epithelial cells comprise 5% of total alveolar cell

surface area, account for 15% of peripheral lung cells, and have a surface area of 250Clm2 per cel

(21). In addition to producing the four maj or surfactant proteins, type II alveolar epithelial cells

have recently been demonstrated to be an important line of defense in innate immunity. Various

anti-microbial and anti-inflammatory substances have been found to be secreted by type II cells,

including lysozyme, defensins, cathelicidin, lipocalin 2, as well as SP-A and SP-D which

agglutinate fungi, bacteria, and viruses (21). Also, since lung surfactant consists of primarily

lipids, a high level of lipogenesis occurs in these cells. The upregulation of fatty acid

biosynthetic enzymes such as fatty acid synthase, acetyl-CoA carboxylase, ATP citrate lyase,

and stearoyl CoA desaturase is stimulated by transcription factors SREBP-1c and C/EBP alpha

delta. These transcription factors are in turn upregulated in response to various growth factors,

most prominently keratinocyte growth factor (KGF) (21). Due to the critical role of the type II

cell, it has often been dubbed as the "great pneumocyte" and the "defender of the alveolus" (22),

since it not only produces the necessary lung surfactant lining the alveoli, but also functions in

maintaining sterility within the lung.

Lung surfactant is synthesized in utero beginning at 28-32 weeks of gestation and babies

born before 35 weeks of gestational age typically suffer from RDS (11, 24) due to inadequate

levels of lung surfactant. The phospholipid profile in amniotic fluid has historically been used as

a diagnostic marker for the onset of lung surfactant synthesis. Tests for predicting the likelihood

of RDS typically analyze amniotic fluid for the presence of phosphatidylglycerol, the second









most abundant phospholipids in lung surfactant; its absence is a highly predictive marker for

RDS. Historically, preventive measures include stimulation of lung surfactant production by

type II cells through treatment with corticosteroids or glucocorticoids, which increase the

enzymes necessary for lipogenesis (15). Lung surfactant production has been found to be

regulated by hormonal agents (25) and metabolism of the lipid/protein species are under the

control of granulocyte-macrophage colony stimulating factor (GM-CSF) (26).

Prior to the development of lung surfactant replacement therapy, the clinical course for the

management of newborns with RDS was mechanical ventilation. Variations in ventilatory

management give rise to differing outcomes of the disease, and treatments included continuous-

positive airway pressure (CPAP) and high-frequency oscillatory ventilation (HFOV). Both

methods involve heavy oxygenation of the lung but high frequency-ventilation has become the

preferred course due to its advantages of reducing lung distension and minimizing shear forces.

In this procedure small volumes airway of gas are pumped into the airways of the lung at rates of

15 Hz (27).

Important Surface-Acting Proteins of Lung Surfactant

A fundamental problem associated with air-breathing stems from the dynamic nature of the

lung: the need to reduce surface tension in small, gas-containing, aqueous lined structures which

are constantly changing in volume. For most mammals, the task of reducing this force is

achieved by the proteins SP-B and SP-C, which directly interact with lipids specific to the air-

water interface. Initial sequencing of isolated protein showed SP-B to be extraordinarily

hydrophobic (~41% to ~52%) with a high relative fraction of the amino acids valine, leucine,

isoleucine, alanine, phenylalanine, and tryptophan (12, 23, 28). Later studies found the protein

to also have a net cationic charge > +6 at physiologic pH (29, 30). Based on electrophoretic

mobility in sodium-dodecyl polyacrylamide gels, SP-B has a molecular weight of approximately









5-8 kDa (31). The native form contains 6 cysteines that form intramolecular disulfide bonds and

a seventh cysteine residue that forms an intermolecular disulfide bond (32). In the lung, SP-B

exists primarily as a 15-18 kDa homodimer with the monomers connected via a disulfide bond at

cysteine residue 48 (23, 30, 31). The conservation of the 6 cysteines forming intramolecular

disulfide bridges, the pattern of disulfide formation, and the high number of hydrophobic

residues place SP-B as the most hydrophobic member in the saposin family of proteins. Other

proteins in the saposin family include amoebapores from Entttttttttttttttttttaoeb histolytica, acid

sphingomyelinase, acyloxyacyl hydrolase, and sphingolipid activator proteins A-D

(saposins A-D). Saposin family members are important to both lipid homeostasis and lipid

physical properties as they can facilitate lipid fusion or bind lipids to increase the activity of lipid

modifying enzymes. Saposins have been characterized as being heat-stable, with a helical

secondary structure (33). What differentiates SP-B from other saposin family members is its

hydrophobicity, rendering the protein insoluble in aqueous solutions, and the seventh cysteine

residue, used in forming the homodimer, is unique to SP-B (33).

The second surface-acting protein in lung surfactant is SP-C. SP-C is a 35 amino acid

peptide of molecular weight 4.2 kDa. Its amino acid sequence lacks any known homologs and

possesses a high valine, leucine, and isoleucine content. An NMR structure of porcine SP-C

determined in organic solvent reveals a helical secondary structure (29). Two cysteine residues

near the N-terminus are palmitoylated in human SP-C. The protein has an overall

hydrophobicity of ~66% and is considered the most hydrophobic protein ever to be found in a

biological system (11, 28, 29). While SP-C has been deemed essential in respiratory dynamics,

mutations in, or of knock-out of, the gene have not been proven to be lethal (34). SP-C null mice

show normal respiratory kinetics. Nonetheless, mutations in SP-C have been linked to lung









disease and susceptibility to infection (34). It has also been shown that genetic knockout of SP-

B causes aberrant processing and misfolding of SP-C (35) and can lead to structures reminiscent

of amyloid fibrils causing pulmonary alveolar proteinosis, a condition in which pathologically

high protein levels are found in the airspaces (36). Models of the structure and interaction of SP-

B and SP-C with lipids are shown in Figure 1-3.

Lung Surfactant Protein B (SP-B) and RDS

The critical role of SP-B has been highlighted by targeted disruption of the gene in mice,

as well as the identification of genetic mutations and polymorphisms in humans correlated with

respiratory distress (23, 37, 38). The difficulty in breathing associated with respiratory distress

stems from an altered histopathology seen in the lungs of patients who have inadequate levels of

mutated SP-B. Altered morphology of the lung air spaces as well as perturbations in the normal

surfactant processing, secretion and storage are seen (23, 38). SP-B mutations or deletions lead

to disorganization of lamellar bodies and alveolar type II cells, and alterations of the lung

surfactant cycle leading to severe disruption of respiratory mechanics. (30, 39). Besides the

general abnormal morphology of type II cells lacking SP-B, an accumulation of distorted, or

irregularly shaped multivesicular bodies in these cells suggests an impairment in lipid packaging

and secretion as seen in SP-B(-) mice (Figure 1-4) (40). Depletion of SP-B also causes distortion

or disorganization of tubular myelin (23), resulting in lower lung compliance resulting from the

pathological organization of lamellar bodies in the Type II cell. The most prevalent nucleotide

mutation of SP-B linked to RDS is an insertion of 2 bases at position 121 leading to a frameshift

in the open reading frame (23, 41).

The above findings clearly indicate the central importance of SP-B in lipid packaging,

cycling and the lung surfactant cycle. Knockout studies and genetic analysis clearly indicate that

SP-B is essential in the reorganization and restructuring of lipids at the air/water interface to









allow for respiratory function. While inadequate levels of functional SP-B is linked to

respiratory distress, an excess of lung surfactant proteins also leads to the clinical condition

known as alveolar proteinosis (AP). Patients afflicted with AP often have 100-fold higher ratios

of SP-B to disaturated lipids compared to normal subj ects (42) and tend to suffer from dyspnea.

Typically 75% of individuals with AP are smokers.

To date, only saposin-B of the saposin family has had its structure solved via x-ray

crystallography. Recombinant expression of saposin B in Escherichia coli gave rise to crystals

diffracting to 2.2 1 (43, 44). The structure of saposin B revealed an a-helical rich dimer

enclosing a hydrophobic cavity (45). Structures of saposin-A, saposin-B, and saposin-D have

been characterized by 15N solution NMR and also show an a-helical rich fold (46). Modeling of

one subunit of SP-B based on the sequence ofNK-lysin has also been reported (47). While these

structures provide insight for modeling the conformation of SP-B, its hydrophobicity and tight

association with lipids make it an unlikely candidate for either x-ray crystallography or solution

(high resolution) NMR, which can yield a more definitive structure as compared to modeling.

Efforts to recombinantly express SP-B have been met with limited success (48).

SP-B was first isolated in 1986 (49). The initially discovered 6 kDa protein fragment,

based on Edman sequencing of protein isolated from pig lung lavage, was found to lower surface

tension when reconstituted in lipids (50). Studies directly linking SP-B to RDS were done in the

mid-1990s (3 7 39, 51). A structural model of how protein reduces surface tension was first

predicted in 1993 by Longo (52). Isolation and purification procedures for SP-B are rather

extensive and time-consuming with low yields; new methods were developed to simplify

purification, but many different protocols for SP-B isolation exist in the literature (53-56).

Isolation of SP-B generally includes a low speed centrifugation of lung lavage fluid to remove









cells, followed by a high speed centrifugation to pellet large surfactant aggregates. The

hydrophobic constituents are extracted into chloroform, the lipids are removed using a Sephadex

LH-20 column, and a final reverse phase C18 column chromatography step can be used to ensure

highest purity (23, 56).

Human SP-B is encoded on the short arm of chromosome 2 and the gene has 9500 base

pairs and 11 exons. Translation of the protein results in the expression of a precursor protein of

381 amino acids containing both N-terminal and C-terminal signal sequences. The N-terminal

signal sequence directs the precursor to the endoplasmic reticulum (ER) while the C-terminal

signal, containing a glycosylated asparagine, appears to have no role in the trafficking of the

protein (23). Cleavage of the precursor protein by proteases in the ER results in mature protein

which transits from the ER to the Golgi apparatus where it associates with lipids that self-

assemble to form a multi-lamellar concentric structure, called a lamellar body, containing SP-B,

SP-C and SP-A (3), which act as a functional storage unit of lung surfactant containing both the

essential lipids and proteins. Exocytosis from the type II pneumocyte results in unraveling of the

lamellar bodies into a meshwork film known as tubular myelin. Electron microscopy images of

tubular myelin show an interlocking lattice of lipids (1 7). Tubular myelin is the lipid reservoir

from which adsorption and resorption occur at the air/water interface (23). Mutations in or

deletions of SP-B have been demonstrated to lead to breakdown of the multi-lattice organization

of tubular myelin which is essential for the maintenance of this interface (23, 39). Functions

ascribed to SP-B include the promotion of rapid phospholipid insertion at the air-liquid interface

during lung expression, promotion of tubular myelin formation, and an impact on the overall

ordering and organization of phospholipids (5).










Lipids cannot account for the near zero surface tension seen in lung tissue during

exhalation or its surface compression characteristics; the presence at low molar ratios of SP-B

and SP-C is required. Direct atomic level measurements on how surfactant proteins associate

with lipids to achieve surface tension minimization has previously not been accomplished.

Preliminary models of SP-B and SP-C structure and orientation in phospholipid environments

are based on examining charge distributions and presumed secondary structures based on FTIR

and Raman measurements (6, 23, 57). An important consideration in determining structure and

interactions of SP-B and SP-C with lipids is the cycle that occurs at the air-water interface during

compression and expansion of the alveoli. The interface separating the air space of the alveoli

from the adj acent liquid lining is a lipid film that is enriched in DPPC, but its physical

composition must be under constant flux between a liquid-expanded phase and a condensed film

and there may also be cyclic changes in lipid composition (58). The saturated acyl chains of

DPPC provide a surface film that can withstand compression of the surface, as would be the case

during exhalation, but due to the molecules' poor respreading qualities, requires the assistance of

unsaturated lipids to aid in surface expansion that is required during the subsequent inhalation.

Western blots probing for hydrophobic surfactant proteins often show no presence of SP-B

from BAL (bronchoalveolar lavage) samples from premature infants with RDS. Some infants

with chronic cases of RDS have been found positive for antibody to an aberrant 40-42kDa pro-

form of SP-B indicating inadequate proteolytic processing, not just genetic mutation, as a

contributor to disease (41). A highly sensitive technique employing HPLC in conjunction with

Western blotting and light scattering detection on human BAL fluid showed SP-B from normal

individuals to be on the order of 740+85 ng/mL. Premature infants with respiratory tract

infections or chronic bronchitis have SP-B levels 10-15% of normal (59).









Mammalian Lung Surfactant Cycle

A cycle exists that allows for a change of film composition when the surface film in the

alveoli expands (inhalation) and contracts (exhalation). The "classical hypothesis" of the lung

surfactant cycle has been described in a review by Weaver and Conkright (1991) as well as

Veldhuizen and Hagsman (2000) and will be briefly described here: (1) after transit from the ER

to the Golgi, lung surfactant proteins SP-A, SP-B and SP-C are packaged with associated lipids

to form a concentric organization of lipids known as lamellar bodies, (2) once secreted from type

II pneumocytes, lamellar bodies unravel to form an extended mesh structure known as tubular

myelin, (3) tubular myelin interacts with the lipid monolayer film at the air/water interface by

aiding in the adsorption of DPPC enriched lipids during film expansion, and promoting

"selective squeeze out" of non-DPPC lipids occurs during film compression, thus allowing for

DPPC enrichment leading to a more stable compressed film, (4) the residual surfactant particle

that is "squeezed out" can then (5) undergo catabolism by nearby macrophages, or (6) be taken

up by a type II pneumocyte and recycled (Figure 1-5). Data disagreeing with this classical

hypothesis exist, most notable is the appearance of lipids undergoing a "buckled structure" at the

air/water interface, rather than a complete "selective squeeze out"(58). Furthermore, recent

fluorescence and Brewster angle microscopy data also seem to contradict the classical hypothesis

and support the notion that the interface remains compositionally heterogeneous during film

compression and not DPPC enriched as previously believed (58, 60).

Exogenous and Artificial Lung Surfactants

As the chemical composition of lung surfactant was elucidated, replacement therapy, or

instillation of the most critical components of lung surfactant become another mode of treatment

for RDS. Administration of micro-aerosolized DPPC resulted in negative results, while slight

changes in breathing profiles were seen by blowing DPPC and PG powders into the trachea of










premature infants (61). Better outcomes were observed for instillation of lung surfactant isolated

from BAL of animals or animal lung homogenates. The first clinical success using exogenous

lung surfactant was reported by Fujiwara, et al, who administered bovine lung homogenate via

an endotracheal tube. The result was improved oxygenation in 9 of the 10 infants in the trial (61,

62). Despite such procedural successes, concerns about viral and other pathogenic

contamination from these animal sources remained significant. Furthermore, development of

synthetic surfactants for clinical treatment overcomes the expense of isolation and permits better

levels of quality control (32).

Initial success obtained from administration of exogenous lung surfactant heralded a new

avenue of exploration using lung surfactant isolated either from lung lavage or tissue

homogenates for the treatment of premature newborns afflicted with respiratory distress.

Application of lung tissue homogenates from bovine, porcine sources, or lavage material are now

standard treatments for RDS, and formulations are currently marketed by several pharmaceutical

companies. A comprehensive list of available lung surfactant formulations used for the

treatment of respiratory distress syndrome is compiled in Table 1-2 and includes formulations

using derivatized compounds as well as peptide mimetics not found biologically.

The first concept of testing the "bare minimum" components for surface activity came in

1981. Tanaka, et al, tested the surface properties of 25 lipid mixtures in Langmuir trough assays

as well as in rabbits to examine the minimum composition necessary to minimize surface

tension. His findings concluded that the best and most minimal requirements for a successful

administration of artificial lung surfactant (in rabbits and in vitro) were DPPC, a saturated fatty

acid (either palmitic or stearic), an anionic phospholipid (phosphatidylglycerol or

phosphatidylserine), and a yet to be discovered "lipid bound protein" (18). The protein referred









to by Tanaka was a not yet fully characterized form of SP-B. Subsequent work has shown that in

the presence of DPPC, palmitic acid acts as a "spreading agent" facilitating monolayer expansion

as the surface area increases (63, 64). The need to assist in monolayer expansion is critical since

DPPC itself has poor respreadability and adsorptive characteristics (60, 63, 65).

Artificial lung surfactants greatly simplify the problem of studying the underlying

biophysical aspects of lung surfactant function against the complicated, heterogeneous

background existing in vivo and allow the possibility of designing simpler replacements of SP-B

with peptide mimetics (66). Non-natural peptide analogs of SP-B that exploit the molecule's

helical and amphipathic quality, such as KL4, can circumvent the need to isolate SP-B to

sufficient purity. Oligo N-substituted glycines (phenylethyl, 2-butyl, or 4-aminobutyl

substituents attached to the nitrogen of glycine), or peptoids, have also been employed to mimic

the helicity of SP-B and show potential (67). Most lung surfactant formulations have in common

DPPC and a chemical agent that facilitates interfacial spreading such as palmitic acid or

tyloxepol. The role of palmitic acid in spreading has been investigated by grazing incidence x-

ray diffraction and by fluorescence microscopy on DPPC:PA monolayers. It has been found PA

enhances membrane fluidity and causes the DPPC chains to be more rigid and ordered. Higher

surface tensions of greater than 40mN/m cause phase segregation of PA into condensed domains

(64).

KL4 is a 21 amino acid peptide designed to mimic the activities of SP-B. To date, KL4 has

enjoyed the most research focus and clinical success as a replacement for SP-B (61, 68, 69). The

primary amino acid sequence of KL4 is KLLLLKLLLLKLLLLKLLLLK with a pattern of

hydrophobic leucines punctuated by the basic, hydrophilic lysines. KL4-based surfactant therapy

relies on a formulation of the peptide with DPPC, POPG, and a spreading agent palmiticc acid).









Indicators used to measure improvements in oxygenation after administration of lung surfactant

include assaying the ratio of arterial/alveolar partial pressure of oxygen (a/AO2) (which must not

fall below 0.22), the fraction of inspired oxygen (FiO2), and mean airway pressure (MAP).

Declines in MAP, and FiO2, and an increase in a/AO2 are markers for improvement in the

respiratory status of a premature infant (69). KL4 improves respiratory status and has been found

to prevent the appearance of diffuse, granular radiopacity seen in chest radiographs typical of

RDS and meconium aspiration syndrome (69, 70). A recent antimicrobial function of KL4 has

also been suggested, in line with some other recent reports concerning the anti-bacterial

properties of SP-B (71, 72). However, the biophysical properties of KL4 and their relation to

alleviating RDS remains the focal point of current investigations, particularly in regard to its

structure and organization in lipid environments.

Sections of SP-B have also been assayed for their surface active properties including the

C-terminal 21 amino acids. These peptides are amenable to solid-phase peptide synthesis and can

circumvent some of the problems associated with isolation of the full-length mature protein; the

C-terminal segment has demonstrated surface activity and an effect on surface tension values in

phospholipids films (67, 73). Figure 1-6 shows the primary amino acid sequence of SP-B and

KL4 which was designed based on the charge distribution in SP-B59-80.

In this thesis, the effects of KL4 and SP-B59-80 On the biophysical properties of two lipid

systems, DPPC:POPG MLVs and POPC:POPG MLVs, are described based on an array of

techniques for biophysical characterization of both the peptides and the lipids. DPPC:POPG in

a 4: 1 molar ratio was chosen as a model lipid system because it represents the native lipid profile

in lung surfactant and artificial lung surfactants. POPC:POPG in a 3:1 molar ratio was chosen

as a second model system because it has convenient physical properties and has been used in









characterizing other amphipathic peptides. The POPC:POPG phase transition temperature is

below OoC allowing for easy manipulation of samples at room temperature; furthermore, the

composition of PC and PG headgroups in this ratio is typically found in eukaryotic and

prokaryotic membranes. Differential scanning calorimetry (DSC), circular dichroism (CD), and

a variety of solid-state NMR (ssNMR) techniques were employed to assess the structure of these

peptides in solution and bound to lipids, and their impact on lipid self-assembly. A model of

these peptides interacting with both these lipid systems is presented and described in the

following chapters. The integration of the structure of these peptides with their orientations in

the lipid bilayer is a critical first step towards obtaining an atomic level understanding of how

lung surfactant functions and may lead to development of more potent peptide mimetics. The

power of ssNMR in examining biomolecules in heterogeneous environments was critical in the

development of this model. Such measurements were previously unattainable using x-ray

crystallography, where peptide:1ipid complexes are typically not amenable to the crystallization

process. Furthermore, solution state NMR, where purity and size of the complex place

limitations on samples, was unsuitable.

Chapter 2 discusses background and theory behind the biophysical techniques used in

these experiments. Chapter 3 describeS 2H NMR, 31P NMR and DSC studies of the lipid systems

and the effects of KL4 On their properties. Chapter 4 discusses structural studies of KL4

interacting with phospholipids by applying ssNMR techniques to isotopically 13C labeled KL4

peptides, as well as circular dichroism. The results discussed in Chapter 4 were done in

collaboration with Professor Joanna Long and Dr. Douglas Elliott. Chapter 5 introduces DSC,

2H and 31P NMR data taken on SP-B59-80, the C-terminal 21 amino acids of SP-B from which

KL4 WAS derived, and compares these results to results for KL4. Finally, Chapter 6 provides










conclusions regarding this work, thoughts on future experiments, and questions that stem from

these studies.







*


O


0
0
O


0 0 0


O O


0
O


0
O


0


O


O
O


0000000
Figure 1-1. Cartoon illustration of surface tension. Molecules at the surface experience an
enhanced attraction (shown as the darkened arrows near molecule 2) The result is net
attractive inward force causing the surface to curve seeking a minimal area, thus
generating surface tension.


Gas


Liquid












Type Ipneurnocyte


~ur;ClaCB


Type II
pnreumocyte

Ulrrent OPulloian m 17tichllrl Biology


Figure 1-2. The alveolar environment. In the alveoli, fluid coats the inner surface of the alveoli.
The air/fluid interface is demarcated by a monolayer enriched in DPPC as shown in
the inset. The periphery is composed of 95% Type I pneumocytes and 5% Type II
pneumocytes. The type II pneumocytes secrete lung surfactant. Reproduced with
permission from Current Opinion in Structural Biology 2002 Aug, 12(4):487-94.










Table 1-1. Approximate weight percentages of lipids in mammalian lung surfactant (Adapted
from Lung Surfactants: Basic Science and Clinical Applications by Robert H. Notter)
Lipid^ Lavaged lung surfactant
Phosphatidylcholine 80.0 10.9
Phosphatidylglycerol 6.811.4
Phosphatidylinositol & Phosphatidylserine 5.411.3
Pho sphati dyl ethanol ami ne 3 70.4
Sphingomyelin 2.010.3
Other 2.010.3
Protein*
SP-A 5%
SP-B 1-2%
SP-C 1-2%
^` Percent weight of total lipid *Percent weight of total lung surfactant. Lung surfactant proteins
constitute approximately 10% by weight of lung surfactant






















C


Figure 1-3. Hypothesized interactions of SP-B and SP-C with lipid lamellae. Left: SP-B is a
homodimer with the two monomers lining adjacent lamellae. Right: SP-C is believed
to exist as a transmembrane helix within DPPC bilayers. SP-C is palmitoylated at
cysteine residues near the N-terminus.















(- -


C D


Figure 1-4 Histology of SP-B mutation. Figure taken with permission from JC Clark, originally
appearing in PNAS 92(17):7794-8. Lung surfactant from mice heterozygous (+/-) for
SP-B is shown in the left column while lung surfactant from SP-B knockout mice (-/-)
is shown to the right. Clearly seen is the loss of lamellar bodies and tubular myelin in
homozygous (-/-) knock-out mice.


SP-B (+/-)









3. Interfacial adsorption/
~200pM resorption aided by
SP-B and SP-C

Ai-ae r interface

SExpansion Compression







O-

1.Lamllar Body4. Recycling of Lung

2. nrvelig .O Surfactant Particle to~lcohg
off lamellar body to 5. Recycled
form Tubular Myelin Lung surfactant
Particle into Type II cell

Type II cell
Figure 1-5. Mammalian lung surfactant homeostasis: (1) Lung surfactant is packaged into its the
functional storage unit, lamellar bodies, in Type II pneumocytes, (2) lamellar bodies
are excreted and unravel to form a network of lipids known as tubular myelin, (3)
lipids adsorb to the air-fluid interface, (4) non-DPPC lipids resorb into the hypophase
to be degraded by macrophages or (5) type II cells recycle lung surfactant material to
reinitiate lamellar body formation.










Table 1-2..Table of artificial lung surfactants used clinically. With permission from TA Merritt.
Reproduced from Acta Paediatr. 2006 Sep;95(9):1036-48.


Brand Name

Protein
containing
Alveofact
BLES

Curosurf

HL-10
Human
amniotic
fluid
surfactant
Infasurf

Newfacten
Surfacten

Surfaxin

Survanta
Venticute

Non-protein
containing
Adsurf



Exosurf


Generic Name


Source/constituents


Company


SF-RI 1
Bovine lipid
extract surfactant
Poractant alfa


Bovine-lung lavage
Bovine-lung lavage

Porcine-lung lavage

Porcine-lung tissue
Human amniotic fluid at
term


Bovine-lung (calf)
lavage
Bovine-lung
Bovine-lung
homogenate
Synthetic (DPPC,
POPG, PA, KL4 peptide)
Bovine-lung tissue
Synthetic (DPPC,
POPG, PA, rSP-C)


Synthetic (DPPC, PG)



Synthetic (DPPC,
hexadecanol, tyloxepol)


Boehringer (Germany)
BLES Biochem (Canada)

Chiesi Pharmaceuticals (Italy)
Dey, LP (USA)
Leo Pharmaceutical (Denmark)
University of California, San
Diego, USA, Univesity of
Helsinki, Finland

Forest Pharmaceuticals (USA)

Yuhan (Korea)
Mitsubishi Pharma (Japan,
Korea)
Discovery Laboratories (USA)

Abbot Laboratories (USA)
Altana Pharmaceuticals (UK)



Britannia Pharmaceuticals (UK)



GlaxoSmithKline (UK)


NA


Calfactant CLSE


Surfactant-TA

Lucinactant

Beractant
rSP-C surfactant


Pumactant (ALEC:
artificial lung-
expanding
compound)
Colfosceril
palmitate










SP-B
FPI PLPY CWLCRALI KRIQAMI PKGALAVAVAQVCRVVPLVAGGI
CQC LAE RY SVI LLD T LL GRML PQLVC RLVLRC SMD


FPI PLPYCWLCRALI KRIQAMI PKG
D TLL GRML PQLVCRLVLRC SMD
KLLLLKLLLLKLLLLKLLLLK


SP-B (1-25)
SP-B (59-80)
KL4 (sinapultide)


Figure 1-6. Primary amino acid sequence of SP-B and KL4. KL4 is modeled after the charge
distribution in residues 59-80 of SP-B. KL4, SP-B(1-25), and SP-B(59-80) affect the
surface activity in lipid monolayers at air/water interfaces. Basic residues are
highlighted in blue and acidic residues are highlighted in red.









CHAPTER 2
BIOPHYSICAL TECHNIQUES TO PROBE PEPTIDE STRUCTURE AND PEPTIDE-LIPID
INTERACTIONS

This chapter details biophysical techniques used in this thesis to study peptide-lipid

interactions. These techniques have also been applied in the study of membrane proteins,

aggregated proteins (amyloid fibrils), and other complex biomolecular systems. The following

descriptions are by no means comprehensive treatments on the rich and detailed history,

intricacies, and science behind each technique, but are intended as primers on the information

obtainable from each technique in its application to understanding molecular interactions and

structure in lung surfactant; references are included for the reader wishing to obtain a more

thorough understanding.

Circular Dichroism

Circular dichroism (CD) represents a straightforward method to assess global secondary

structure in proteins via detecting the interaction of polarized electromagnetic waves (light) with

the chiral protein backbone. Typically, the light source is circularly polarized light generated by

a xenon or a helium lamp and filtered to select a particular circular polarization. Light waves are

composed of electric and magnetic field waves propagating perpendicularly to each other.

Circularly polarized light requires two of these waves (of equal magnitude and frequency)

traveling together but 900 out of plane, allowing the magnitude of the electric field to remain

constant but its direction to trace the path of a circle. This polarized light differentially excites

electronic transitions in proteins leading to CD spectra that are reflective of these excitation

energies with both positive and negative absorption. When light passes through an optically

active chiral solution, such as amino acids in a protein, the right handed and left handed

circularly polarized light are absorbed to different levels at each wavelength.









The differential absorption of left handed and right handed circular polarized light is

characterized by

AE = EL -R (2-1)

and gives rise to the CD signal (74-77) The raw CD signal is denoted as ellipticity (in units of

millidegrees). The CD signal normalized to the protein concentration is denoted as molar

ellipticity me and is given by the following equation:

m = mdeg (2-2)
#AA -[M] -1

where #AA is the number of amino acids in the protein, [M] is the molar concentration of the

protein, mdeg is the raw CD signal in millidegrees, and I is the path length of the sample cell

cuvette (in centimeters).

Protein secondary structures such as a-helices, P-sheets, and turns have spectra of

distinctive shape and magnitude in the far-UV region. The overall shape of the spectra is due to

the periodicity of the amide bonds inherent in each type of secondary structure. For instance, the

CD spectrum for an alpha helix has a minimum absorption at ~190nm representing the

excitation of 2 (nb) to 2*" as well as double minima at ~205 to ~220nm reflecting the excitation

of a lone pair oxygen electron to the 2*" state (76). Typical signature CD spectra found for a

helix, P-sheet and random coil conformations are shown in Figure 2-1.

CD spectra have been used extensively to monitor secondary structure in proteins free in

solution as well as for proteins bound to lipid vesicles at lipid concentrations of 2-10mM (78-

80). Factors such as the concentration of the protein, path length, buffer composition, and

dielectric constant of the buffer, temperature and pH play a role in determining the resultant CD

spectra obtained. Helical secondary structure can often be induced in unfolded or random coil









protein by addition of "helix-enhancing" solvents such as fluoro or alkyl alcohols including

trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) (81).

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) is a technique used to study the temperature

dependent phase behavior of lipids, due to their tendency to self-assemble in both model and

biological systems (82). The technique has been used to examine the thermally induced

transition of lipids from an ordered crystalline state (Lp) to the disordered liquid-crystalline state

(L,), which occurs at a characteristic temperature Tm for a particular lipid or lipid mixture (82,

83). Such a transition is characterized by an increase in the trans-gauche isomerization rates

along the fatty acyl chains, a decrease in bilayer thickness, the onset of axial diffusion, and an

increase in the lateral cross section occupied by the phospholipid molecules (82, 84). Pure

phospholipids have sharp symmetric phase transitions indicating the gel-liquid crystalline

transition is a first order process; the Tm at which this transition occurs depends on the headgroup

composition, fatty acyl chain lengths, and the degree of saturation in the fatty acyl chains.

Broad, asymmetric DSC peaks are commonly seen in biological membranes where appreciable

headgroup and acyl chain heterogeneity is present. While the Lp to L, transition is the most

common transition measured by DSC, other phase changes such as pre-melting transitions,

liquid-liquid phase separations, and domain formations have been seen and characterized by this

technique (15, 82, 85, 86).

The principle of DSC is relatively straightforward (Figure 2-2). A sample cell (containing

sample) and a reference cell (containing solvent) are heated at very controlled rates. As the

temperature is increased linearly, the temperature difference between the reference and sample

cell is kept at zero via a feedback loop. This is done by heaters connected to the sample and

reference cell. When the sample undergoes a thermally-induced event, such as a phase










transition, a temperature differential is sensed between the reference and sample cell and

corrected by varying the power input to the individual cells. The power required to maintain

both cells at the same temperature scan rate is measured. This raw differential power signal is

converted to heat capacity at constant pressure (C,) and graphed versus the sample temperature

(82, 87, 88). More accurately denoted, the DSC instrument measures the excess specific heat,

the amount by which the apparent specific heat of a particular solute transition exceeds the

baseline specific heat required for heating the reference cell. Figure 2-3 illustrates the output

generated after the excess specific heat is converted to heat capacity by the DSC instrument.

DSC thermograms can be analyzed to extract the thermodynamic parameters for a lipid

phase transition. The calorimetric enthalpy AHeal of a transition is obtained by integration of the

peak related to the transition peak: (84)





The van't Hoff enthalpy is the enthalpy calculated as a function of Tm; assuming a two-

state process, the formula for AHVH iS: (89)


AH, = 4RT2 24
TH ni cal

where R is the universal gas constant and C,"mis the peak heat capacity measured for the

transition.

Another parameter which can be extracted is the cooperativity of the transition between

phases. In some cases the phase transition can be instantaneous and the DSC thermogram will

be highly dependant on the scan rate. In other scenarios, the process might first be initiated by

domains or "islands" of lipids and gradually spread throughout the sample as the temperature is

changed. The cooperativity can be assayed by comparing the values of the derived enthalpies









AHeal and AHyH (89) and evaluating their ratio AHyH / AHeal. When this ratio is less than unity,

intermolecular cooperation dominates the transition mechanism; however, when the ratio is

greater than unity, intermediate states are significantly populated.

The effects that exogenous agents such as cholesterol, proteins and antimicrobial peptides

have on the Tm of model lipid systems have been extensively investigated using DSC (79, 90-

92). DSC has also been used to delineate peptide induction of alternative lipid polymorphisms,

such as the HII phase, which has been shown to occur when transmembrane a-helices enriched in

the amino acid leucine are added to lipids (93). (This is of particular importance since the

peptide under study (KL4) has a high percentage of leucines). Thus, DSC is a powerful tool to

study the effects peptides have on the thermotropic phase behavior of lipid systems.

It should be noted that the "pre-transition" peak often seen in DSC on pure lipids (such as

that seen in Figure 2-4) has been fully evaluated in terms of the structural changes within the

bilayers. For phosphatidylcholines, the pretransition occurs in the range of ~5o below the main

phase transition temperature and has been denoted the Pp or "ripple" phase. Since it is due to a

transformation from a pure lamellar phase to a two-dimensional monoclinic lattice, in which the

bilayer contains undulations (86, 88, 94). Addition of peptides or other exogenous agents have

been shown to abolish or modulate the appearance of this pre-transition phase. Also shown in

Figure 2-4a is the information content yielded from a DSC thermogram. The Tm corresponds to

the temperature where the maximal heat capacity Cmax of the system is observed for each

transition; the integration of any peak reflects AHeal, and the PWHH or AT1/2 meaSures how

broad the phase transition is. A broad peak (which implies a large PWHH and hence a greater

temperature span or AT1/2) indicates less lipid cooperativity during the phase transition. Adding









monounsaturated lipids to pure DPPC has consequences in terms of thermodynamics, lipid

cooperativity and phase transition temperature (Figure 2-4b).

Solid state NMR Spectroscopy

Solid state NMR spectroscopy (ssNMR) has advanced significantly as a tool to study the

structure and dynamics of solid systems ranging from glasses to polymers to catalysts. Advances

and progress in the field have now been increasingly focused on developing methodologies for

complicated biomolecules such as membrane proteins (95, 96). Unlike solution NMR, where

narrow resonances result due to rapid reorientation of molecules and averaging of anisotropic

interactions, ssNMR must deal with unaveraged interactions (97, 98).

On the NMR time scale (Figure 2-5), interactions such as chemical shielding, dipole-dipole

coupling, and quadrupole coupling are time averaged in the solution state by the rapid

reorientation of the molecule. Slower motions of molecules in the solid state give rise to a

superposition of many overlapping resonances representing the many possible orientations of the

molecules with respect to the magnetic Hield; the resulting broad spectrum can be referred to as a

powder pattern (96, 98). These powder patterns observed in ssNMR spectra suffer from low

resolution, low sensitivity, and (for the most part) very little extractable structural information.

However, magic angle spinning (MAS), described briefly below, can yield liquid-like spectra

from solid-state experiments; and clever pulse sequences during MAS can be used to re-

introduce and to measure anisotropic information (99). A discussion of time scales used to

measure molecular processes by NMR is detailed later in this chapter.

Spin Interactions Commonly Seen in ssNMR

Chemical shift

When placed in a strong magnetic Hield, nuclei in different parts of a molecule experience

varying Hields due to their electronic environment. This phenomenon is referred to as chemical









shielding and is caused by the external magnetic field (Bo) generating currents in the molecule's

electron cloud, which in turn generates induced Hields. The magnitude of this induced Hield at a

given nuclear site depends on the orientation of the molecule with respect to Bo and on the

location of the nuclear spin within the molecule. This variation in chemical shielding leads to

different nuclei of the same isotope resonating at different frequencies or chemical shifts.

In liquids the chemical shielding is averaged and is referred to as the isotropic chemical shift.

This chemical shift depends upon the gyromagnetic ratio of the nuclear spin, the shielding by

local electron currents, and the influence of local low-lying electronic ground states (100).

For a single crystal sample, the NMR spectrum looks similar to a solution NMR spectrum in

resolution (neglecting dipole-dipole couplings) but chemical shifts depend on the orientation of

the crystal with respect to Bo. Therefore, by rotating the crystal relative to the magnetic Hield, the

resonance positions change. In powder samples, where all such orientations are present, the

chemical shift spectrum is broad reflecting that it is a superposition of spectra from each

crystallite. This is described in more detail below.

Dipole-dipole couplings

The interaction of nuclear spins with one another also affects NMR spectra. Since each NMR

active nucleus has a magnetic moment, each spin experiences the Hield generated by other

nuclear spins nearby. This interaction is called the (direct) dipolar coupling.

Dipolar couplings (d) are through-space interactions and provide important structural

information. The dipolar coupling is directly proportional to the gyromagnetic ratio of each

participating spins (yl and y2) and inversely proportional to the cube of the distance between the

spins (r) (100). The spatial dependence of the dipolar coupling with respect to the external

3 cos 2
magnetic field is accounted for in the angular term










d=l 1 yzy (3cos' 0-1)


(2-13)


2 r

The gyromagnetic ratio describes the size of the Zeeman interaction for a spin with respect to the

magnetic field and determines its Larmor frequency. For example, the y value for a H spin is

2.68x10s rad/sec/Tesla (101), corresponding to 600MHz in a 14.1T field. In a powdered NMR

samples, dipolar, quadrupolar, and (in part) chemical shift interactions scale by a factor of -0.5 to

1 based on the angular dependence of these interactions on the term (3cos26-1)/2 (102, 103).

Quadrupole couplings

Quadrupole couplings represent the interactions of spin > nuclei with the electric fields


from their non-spherical nuclear charge distribution. The measure of a nucleus's effective

ellipsoidal shape is called its quadrupole moment (Q, often reported as eQ). The strength of the

quadrupole coupling is a good measure of a nucleus's mobility. For example, in this study, spin


1 deuteriumn (2H) has a quadrupolar coupling constant: ey used to assess the mobility of lipid


acyl chains where e is the charge of an electron, q is the principle component of the electric field

gradient tensor, and h is Plank's constant.

Applications and Methodologies in Solid-State NMR

Pake Powder Pattern

Large biomolecular systems such as membrane proteins, polymers, or inorganic complexes

give rise to difficulties in structure determination because the orientation dependencies of the

NMR interactions described above are not time averaged. Resultant NMR spectra yield broad

lineshapes known as PakePPP~~~~~PPPPP~~~~ powder patterns or powder spectra (104), after George Pake who

derived the equations for these patterns in the late 1940s. A typical 31P chemical shift spectrum

for the phospholipid DPPC above it' s Tn, is shown in Figure 2-6 and shows a powder pattern









over a range of frequencies covered due to multiple orientations of the lipids with respect to the

external magnetic Hield (98, 105). Below a phospholipids Tm, the motion of the phospholipid is

non-axial and results in a spectrum shown in Figure 2-7. In lipid dispersions taken above the

phase transition temperature the onset of motion about an axis of symmetry leads to a lineshape

that has distinct parallel edge and perpendicular edges relative to the static magnetic Hield. All

other intermediate orientations of the symmetry axis relative to magnetic Hield are also enveloped

in the powder pattern. The relative intensity at each point in the pattern is based on the

probability distribution of that orientation. For a phospholipid dispersion, the powder pattern can

take different lineshapes, not only dependant on Tm, but also dependent on the dynamics present

for various lipid polymorphisms, such as hexagonal or inverted hexagonal phases (Figure 2-9).

Chemical Shift Anisotropy (CSA)

In an unoriented sample, many orientations of a particular molecule to the magnetic Hield are

present. The chemical shifts seen are dependent on the distribution of orientations in the sample.

It is the chemical shift anisotropy that determines the 31P spectra in this work.

The chemical shift anisotropy for a spin V/2 HUClOUS (such as 31P) can be described in its

principle axis system by three values, designated as oil, o22, and (533 (Figure 2-7). By

convention, (533 has the largest difference from the isotropic value and lower intensity and

corresponds to the parallel edge in the powder pattern, (522 corresponds to the highest intensity of

the powder pattern, and oil has an intermediate intensity and corresponds to the perpendicular

edge in a powder pattern. The isotropic peak, oso, seen in MAS spectra and solution NMR, is

equal to the average of the principal values. These values are dependent on the electronic field

surrounding the nucleus, which shields the nucleus from the external magnetic Hield. Since the

shielding is three dimensional, it is represented by a second rank tensor where the principal

values correspond to the diagonal elements in a principal axis system (PAS), where this 3x3









matrix is diagonalized. This interaction tensor can be visualized as an ellipsoid whose center is

the active NMR nucleus of interest and the axes of the ellipsoid coincide with the principal

values of the CSA tensor in Cartesian space. If the molecular orientation changes with respect to

the magnetic Hield, then so does the nucleus and its corresponding CSA tensor (98) (Figure 2-8).

For phospholipid bilayer assemblies, a symmetry axis due to rotational averaging around the

director axis perpendicular to the plane of the bilayer, (or the "bilayer normal") averages the 31P

CSA leading to axial symmetry where 011 = (522 as shown in Figure 2-5 (94). This onset of

motion causes rotational averaging of the 31P CSA tensor around the director axis and as a result,

the z-axis of the time averaged CSA, or o33PAS, coincides with the bilayer normal and remains

unchanged. In such a scenario, . The isotropic chemical shift

oiso is defined as the average of the three main components of the tensor

orso = 1 (11 + 22 + 33) (94, 98) (2-14)

and corresponds to the frequency if the sample were tumbling isotropically in solution. The

lower than usual signal at ai seen in Figure 2-6 is due to the fact that DPPC lamellae orient in

high magnetic Hields which is addressed below.

Magic Angle Spinning NMR

If the NMR sample is spun at a "magic" angle (54.74o) relative to the magnetic Hield, the

orientation dependent term, (3cos28-1), iS zero. At fast enough spinning speeds, dipolar

couplings and chemical shift anisotropies (CSA) can be completely averaged, thus removing

their anisotropic effects in resulting NMR spectra (95). MAS thus improves resolution and

subsequently the applicability of ssNMR to complicated biomolecular spectra (99).

Figure 2-10 illustrates the phenomenon of MAS and its effects on a chemical shift spectra (106).

However, with the averaging of dipolar couplings, important structural information is lost. But









dipolar couplings that are averaged under MAS conditions can still be observed by the

introduction of radiofrequency pulses synchronized with the spinning of the sample (dipolar

recoupling). Pulse sequences such as DRAMA (dipolar recovery at the magic angle) use n/2 RF

pulses applied twice per rotation period to interrupt the averaging of the dipolar coupling caused

by MAS (95). A variation of the DRAMA sequence called DRAWS (dipolar recoupling with a

windowless sequence) uses additional pulses to retain more of the dipolar interactions, while still

averaging CSA interactions. DRAWS is therefore more effective for nuclei with large CSAs

such as carbonyls (Figure 2-11).

Structural aspects of the molecule in question can also be determined independent of the

application of external RF pulses, simply by modulating the speed of the spinning rotor while the

sample is under MAS conditions. The presence of spinning side bands at spin rates of 3000-

4000 Hz allows the determination of CSAs. Thus observation of any molecular motions of

peptides in lipid MLVs as a function of sample composition is possible.

Range of NMR Time Scales

While solid state NMR is used to look at molecules that are slowly tumbling on the NMR

time scale, it is important to realize that NMR can probe molecular motion over a wide array of

time scales ranging from picoseconds to several seconds. The following section serves as a brief

primer on the vast ranges of frequencies that an NMR experiment can detect.

The internal dynamics of molecules allow nuclei to oscillate to a net average position. These

molecular vibrations are on the order of picoseconds or shorter. Internal dynamics within the

molecule allow for large rotation of substituents within the molecule as well, such as a methyl

group or an amine. If the substituent has an axis of symmetry such as around a local three-fold

axis existing in a -CH3 grOup, then such motion can be on the order of internal molecular

vibrations (picoseconds), but if the same -CH3 grOup is hindered by another nearby substituent,









as can be expected during the folding of a protein or in a lipid bilayer environment, then such

rotational motion can occur on the timescale of milliseconds to seconds. Hence, the boon of

NMR stems from its ability to detect molecular motions as well as the number of molecules in

different states (100).

Time scales which can be probed using NMR experiments are vast--from hundreds of

picoseconds (molecular rotations), to many seconds macroscopicc diffusion and chemical

exchange processes) (Figure 2-5). Depending on the sampling time during signal acquisition,

one can discriminate between types of motion being measured. Very fast motions in the

picosecond to nanosecond timescale are on the order of the Larmor frequency representing the

shortest time available to specifically encode information-anything faster is encoded as an

average. However, NMR is not generally sampled this quickly and the totality of different

actions must also be considered. For example, vibrational motion and molecular rotation can

average out direct dipole-dipole couplings (often in the millisecond domain) in liquids (100).

Motions on this timescale often form the basis for T1 relaxation measurements.

Motional time ranges on the order of microsecond to milliseconds affect the typical spectral

timescale strongly. As a result, lineshape perturbations such as a broadening, are seen. An

example of the changes in NMR spectra within this regime is hindered rotation. In this type of

experiment, a molecule has two (or more) non-equivalent conformations with a significant

energy barrier between them. The first NMR experiment is run "cold" and shows each

conformation as a distinct resonance-indicating that their intraconversion is slow on the

timescale. As the temperature is raised, the spectra first broaden and then coalesce into a single

peak--indicating that their intraconversion is fast on the timescale.









Lastly, molecular motion that ranges from milliseconds to seconds can also be probed from

NMR. Macroscopic diffusion or flow occurs via the transport of molecules from one region of

space to another occurs on this timescale. Such a phenomenon forms the basis of diffusion

NMR, whereby an inhomogenous magnetic field is generated and used to quantitate molecular

diffusion and flow.

Depending on what frequency of motion one is interested in examining, the NMR experiment

can be correspondingly devised. For natural abundance 31P NMR experiments, the timescale of

motion being probed are on the order of 10-9 to 10-"1 seconds. The frequency range of a 31P CSA

span is 50 ppm or approximately 30,000 Hz for an NMR experiment run at 600MHz.

Headgroup vibrations, lateral diffusion, and rotational motion along the lipid axis are faster than

this, so their effects are seen as primarily narrowing the spectra to an average value. Likewise,

the frequency range of quadrupolar interaction are on the order of approximately 167,000 Hz.

The 2H NMR experiments, looking at the dynamics of the acyl chain also involve motions that

are faster than the spectral frequency so again, averaging of the spectra components are seen.

Finally, in solids there is incomplete motional averaging of the internal spin interactions, so both

intramolecular and intermolecular spin interactions retain their dependence on the orientation of

the sample with respect to the magnetic field (100).

NMR-Active Nuclei and Importance to Biological Molecules

31P (Phosphorus) NMR

Phosphorous is the spin V/2 NMR-active nucleus that gives rise to the powder lineshape

mentioned above. 31P NMR is one of the tools that can be used as a non-perturbing probe to

measure the orientation and conformation of the phosphate headgroup in a lipid molecule (94).

Another advantage of 31P NMR is that no isotopic incorporation is necessary since the target

nucleus is 100% naturally abundant. Furthermore, dipole-dipole interactions between 31P and 1H









in phosphates are very small, and can easily be decoupled. As detailed above, lipid geometries

important in membrane-membrane mediated events such as the lamellar and hexagonal phase,

are sensitive to the onset of axial diffusion and molecular motion. These can be discriminated by

31P NMR; also, the technique is sensitive to headgroup orientation and charge interaction effects.

31P NMR spectra of small, sonicated vesicles have a single resonance at their isotropic chemical

shift. This is due to the vesicles being small enough to tumble rapidly on the NMR timescale.

The tumbling rate of 4000 lipid molecules in a 30-50nm small unilamellar vesicle of radius 250A

has been estimated to be ~1MHz. This is in stark contrast to lamellae which have a tumbling

frequency estimated at <1Hz (107). Sharp peaks at the isotropic chemical shift seen in NMR

spectra of vesicles are also due to a small radius of curvature and lateral diffusion (107). The

types of lipid polymorphisms described above have had their spectra distinguished and

characterized by 31P NMR (108). Lipid phases seen by 31P NMR are essentially field

independent; however, orientation of lipid assemblies can cause variations in the relative

intensities of oi and cl edges with higher Bo fields (94).

While 31P NMR of phospholipids ensembles yield specific lineshapes due to CSA interactions, it

is also critical to mention that sharp, nearly Lorentzian lineshapes at a particular resonance

frequency can occur on hydrated phospholipids if the assemblies are highly ordered with respect

to the magnetic field. Similarly, single crystals of phospholipids produce single Lorenztian lines,

which when the crystal is rotated with respect to the magnetic field, change their resonance

position. In fact, this has been exploited by many research labs that create oriented bilayers on

glass plates, giving rise to a single orientation of the bilayers. The glass plates can then be

placed in the magnet with the director axis perpendicular to the Bo yielding a single resonance at

the perpendicular edge of the oriented lineshape. This approach has also been pivotal in using









15N spectroscopy to determine the orientation of 1N backbone labeled peptides in bilayers (106,

109-111).

The potential detergent and lytic properties of antibiotics and anti-microbial peptides have

also been examined by 31P NMR. Addition of molar percentages of down to 4 mol% of these

molecules have been found to lead to an appearance of an isotropic peak in 31P spectra indicating

significant membrane degradation or perturbation (112, 113). In addition to assessing the

geometry and polymorphisms of phospholipids phases, 31P NMR has found use in a multitude of

applications including muscle and tissue research diagnosing epilepsy, and in specific sectors of

the food industry (114-116).

An unoriented static 31P NMR spectrum of a phospholipid is due to the CSA of the 31P

nucleus in the phosphate headgroup. CSA values have been measured for various phospholipid

headgroups in crystallized lipids (94). Furthermore, the size and sign of the phosphate CSAs can

differentiate between different lipid geometries. As discussed above, lamellar phase lipids have

asymmetric spectra below their Tm due to restricted motion, and above their Tm, have axially

symmetric lineshapes which are approximately 50 ppm broad. Another lipid mesophase, the

hexagonal phase, has an additional axis about which the averaging of the CSA occurs.

The hexagonal phase describes lipids packed as elongated cylindrical structures. It is divided

into two types: the HI and HII phase. The HI phase contains a cylindrical array of lipid molecules

with the polar head group facing an aqueous exterior and the acyl chains facing inward. In the

HII phase, or inverted hexagonal phase, the headgroup face an inner aqueous cavity of water and

the acyl chains face outward stacked in a tubular form (15, 94). Figure 2-9 shows lipids in

lamellar and HII phases and their resulting 31P NMR spectra. Lipids arranged in a hexagonal

phase have the same motions as lamellar phases, and an additional degree of motional averaging









occurs due to lateral diffusion of the lipids about the cylindrical axes (94). The spectral

lineshape for a hexagonal phase is half the width and opposite in sign to the corresponding

lamellar phase since this motion is perpendicular to the axial diffusion of the individual

molecules.

2H (Deuterium) NMR

Deuterium (2H or D) is a, spin 1 nucleus with a natural abundance of 0.016%. Hence, any

NMR signal obtained from sample unambiguously belongs to deuterons specifically incorporated

into the molecule of interest provided deuterium free solvents were used (107, 11 7). Due to

the spin 1 nature of the deuterium nucleus, 2H NMR powder spectra are characterized by two

overlapping and opposite in sign lineshapes, due to the two possible transitions -1 & 0 and

0 + 1. These lead to characteristically symmetric perpendicular peaks, often referred to as the

"Pake" doublet (102). Deuterium spectra of acyl chain deuterated lipids provide structural

information since averaging of the Pake powder patterns depends on motions of the individual

methylene segments (118).

The time averaged order parameter can be assigned for each C-D bond along the

length of the acyl chain. This order parameter is a discrete value assigned for the collective

ensemble motions that occur for each C-D bond and average the powder spectrum for that

position. Such a value provides information on the internal motions of the fatty acyl chain.

Motions of phospholipids pertinent to the NMR time scale (10-5 seconds) which average the

quadrupolar interaction include oscillatory motions of bond lengths, bond angles, and torsion

angles (10-12 Seconds), gauche/trans isomerizations (10-10 seconds), axial diffusion (10-s to 10-9

seconds), fluctuations of the director axis known as "wobble" (10-s seconds), and translational

diffusion along bilayer surfaces (10-7 seconds) (84, 107). The motion of a phospholipid molecule

can be approximated as a cylinder that axially rotates (119) with each C-D bond wobbling as a









cone along the length of the acyl chain (Figure 2-12) and determines overall averaging of the 2H

patterns at all positions along the acyl chain. Additional gauche/trans isomerization motions

affect the averaging at each C-D along the acyl chain differently, since there is less steric

hindrance encountered by methylene segments going toward the methyl terminus. The order

parameter for a specific methylene position labeled i is defined with a second order Legendre

polynomial


S=2 <3o ,->(2-15)


where 6i is the angle between the symmetry axis of the molecule and the C-D chemical bond.

The brackets indicate a time-average ensemble of all molecular motions of the C-D bond that are

fast on the NMR time scale (107, 119-122). The bilayer normal is coincident with the symmetry

axis for lipids in a fluid lipid bilayer due to the fast axial rotation of the individual molecules

(84).

The deuterium NMR spectra of lipids with perdeuterated acyl chains contain powder patterns

from all the positions in the acyl chains. Full deuteration of both the sn-1 and sn-2 acyl chains of

DPPC results in 30 2H NMR lineshapes correlating to each methylene position in each of the

acyl chains. Each lineshape is also symmetric, with a Pake doublet or quadrupolar splitting (Avg,

measured in k
position (Figure 2-13).

The frequency splitting Av, for each C-D position is used to calculate the time averaged

order parameter for the position according to the following simple relation:

Ar = < S,3 >~H (2-16)









The quadrupolar coupling constant expected for a deuterium atom in a saturated C-D bond in the

static limit is 167k
coupling constants on deuterated paraffins such as ethane and butane at low temperatures (123).

A numerical procedure, termed dePaking, is used to deconvolute the spectra. The methylene

positions can then be easily assigned since they give rise to single resonances rather than

lineshapes. These assignments can then be used to create an order parameter profile. Resonance

assignments for each position were initially done by selectively deuterating individual positions

along the acyl chain and measuring the NMR spectra (117, 118). From the dePaked 2H NMR

spectra, a characteristic order parameter profile is generated by graphing the calculated
values against the methylene positions in the acyl chain (Figure 2-14). The order parameters

decrease as the carbon number of the acyl chain is increased reflecting the increased motional

freedom of the acyl chains near the center of the bilayer (121, 124).

DePaking

As stated above, dePaking is a numerical deconvolution procedure that takes the lineshape

arising from randomly oriented molecules and converts it to an

"oriented spectrum". The procedure can be applied to any lineshape resulting from the spatial

dependence of the spectrum having the form of a second order Legendre polynomial, i.e.

3 cos 2
( ).Through a mathematical transform, the spatial component is removed resulting


in an "oriented spectrum" in which the signal is refocused at the frequency corresponding to the

parallel edge of the powder lineshape (125). For our work, the dePaking procedure employs an

algorithm provided by Edward Sternin and colleagues at Brock University, which has been used

to generate both prOfiles and determined the extent of spontaneous magnetic field

orientation of lipids (102, 103). The result of the transform is a frequency spectrum









corresponding to when the lipid bilayer normal is parallel to Bo (126, 127). This is the

equivalent to the parallel edge for each C-D powder pattern in 2H NMR and likewise the parallel

edge for each lipid phosphorous moiety in 31P NMR. An example of dePaked 2H and 31P spectra

are shown for 4: 1 DPPC(d-62):POPG in Figure 2-13b and Figure 2-15 respectively. By

measuring the dePaked frequency as a function of added agent such as a peptide, the extent and

level of interaction of the lipids with the additive can be probed.

Magnetic Field Orientation

An additional complexity arising when interpreting NMR spectra for lipids being placed in large

magnetic fields is macroscopic lipid alignment, particularly for lamellar phase lipids. The

macroscopic orientation is due to the negative diamagnetic susceptibility (Ay<1) of phospholipid

molecule assemblies (128). The phenomenon of a preferred orientational alignment has been

well documented in the literature for many phospholipids classes, ranging from synthetic to

biological extracts from E.coli and has been observed at field strengths as low as 7T (300 MHz

for 1H) (103, 129-132). This complicates analysis ofNMR spectra, particularly dePaking

algorithms, since the typical powder distribution of angles no longer holds. The formulae and

mathematical procedures involved when the powder pattern is distorted by lipid alignment are

discussed in Chapter 3. When multilamellar vesicles orient, the spherical shape becomes

distorted to a geometry that is ellipsoidal (Figure 2-16). The dePaking algorithms used take into

account the fact that the probability distribution of the bilayer normal vector is no longer

proportional to sin(6), as it would be for a totally random distribution of MLVs. To account for

this discrepancy, the dePaking algorithm written by Professor Edward Sternin generates a

orientation parameter, K,, which accounts for the extent of spontaneous orientation of the lipid

vesicles in the magnetic field Bo, assuming the MLVs deform to more ellipsoidal geometries.









































190 210 23~0 250
wavlenth (nm)


Figure 2-1 CD spectra from various secondary structure elements. Reproduced with permission
from Omjoy K. Ganesh.





Electronics to regulate heat
to sample and reference cells


Heate r


Computer
Interface


Data Output .


Sample Cell
Tem peratu re
Sensor


\LAdiabatic
shield


Reference Cell


_ __ __


Figure 2-2. Schematic of a differential scanning calorimeter (DSC). Information pertaining to its
operation and output are described in Chapter 2.


Heater





















Figure 2-3. The gel to liquid-crystalline phase transition of phospholipids bilayers. When the
temperature of a phospholipid dispersion is above its characteristic main phase
transition temperature, Tm, the acyl chains acquire additional degrees of freedom
causing the individual molecules to freely rotate and an expansion of the bilayer
volume occurs.


Gel (Lp)


Liquid-crystalline (L,)











(A)


*m
41.6oC








AT,,


CP -
m ax


4





E 2


0o


Temperature (oC)


(B)


DPPC
4:1 DPPC:POPG


5000-

4000


3000-


2000-


1000-


20 30 40
Temperature(oC)


50 60


Figure 2-4 A) DSC thermogram of 2mM DPPC large unilamellar vesicles (LUVs) dispensed in
5mM HEPES pH 7.4. Scan rate was at 1 degree per minute and temperature scans
were from 10 to 70 degrees Celsius. The phase transition at 41oC indicating the Lp to
L, is clearly shown. The pre-transition or the P'P phase is also shown. B) The effect
on the DSC thermogram by the addition of monounsaturated POPG to DPPC LUVs.










very slow slow


fast


very fast


ultra-fast


S LOW


FAST


MIOL
ROT


DULAR
TIONS


Timescale:


Relaxation


Spectral


Larmor


Figure 2-5. Time scale of motional processes for nuclear spins in NMR.













Perpendicular edge
of powder pattern


022 ~11 ~1


o


Parallel edge
of powder pattern


I l l ii l i l ii l i
-40 -30 -20 -10 0 10 20 30 40

Chemical Shift (ppm)

Figure 2-6. 31P NMR static spectrum of DPPC hydrated vesicles (approximately 60mg). The
spectrum was taken at 44 degrees on a 600MHz Bruker instrument with 1024 scans.
Above its phase transition, Tm, the powder pattern takes on a shape with axial
symmetry. A clear perpendicular and parallel edge are seen.









~22
~111


~iso =( 11 + 22 + 33)



~33







I I I I I I I
-75 -50 -25 0 25 50 75
Chemical Shift (ppm)
Figure 2-7. 31P NMR of static hydrated DPPC vesicles (approximately 60mg) taken on a 600
MHz Bruker instrument (1024 scans) at 240C, well below its phase transition
temperature, Tm. Below Tm, the powder pattern reflects incomplete averaging of the
asymmetric 31P CSA tensor. Shown are the principal values of the CSA.









Bo


Bo


Figure 2-8. Graphical depiction of shielding tensor for a nucleus with an axis of symmetry. The
CSA tensor is illustrated as an ellipsoid with the center being at the nucleus of
interest, in this case the phosphate headgroup of a phospholipid. When the molecular
orientation changes, so does the orientation of the interaction tensor with respect to
the magnetic field. For liquid-crystalline phospholipids in a lamellar phase, the
symmetry axis is the bilayer normal. Shown in orange and green are principal axes of
the ellipsoid, while the dark black line is the axis of rotation. The phosphate
headgroup is shown as a red sphere and wavy lines represent the acyl chains.


Phospholipid
molecule


Nucleus
at site
of symm





















-50 0 50
Chemical Shift (ppm)


Bilayer











-50 0 50
Chemical Shift (ppm)


Figure 2-9. Phosphorous NMR lineshape patterns for lipid mesophases. Shown above is the
lineshape for an inverted HII hexagonal phase common in many lipid-lipid mediated
events. The isotropic peak at 0 frequency represents partial degradation of the
sample. Below is the standard powder pattern seen in the bilayer lamellar phase.


H ,phase























S6 kHz



8 kHz

250 200 150 100 ppm


.. ..1 kHz


1.


1 I


3 kHz


Figure 2-10. Magic angle spinning (MAS) spectra of the 13C I HUClOUS in glycine. Sample was
packed in a rotor and spun at the magic angle of 54.74 degrees at spin speeds between
1 and 8k the spin speed becomes comparable with the CSA; the isotropic peak is seen in the
center. Data provided with permission from Dr. Manish Mehta.



















13C


Figure 2-11i. DRAWS pulse sequence of DRAWS employed during MAS for dipolar recoupling.
Each rectangle represents a RF pulse that rotates the magnetization either 90 or 360
degrees. CP is cross-polarization, the transfer of magnetization from protons to lower
gamma 13C HUClei. TR and R corresponds to a MAS rotor period.


DRAWS Pulse Sequence









C




D


D1


g~t g


Tail


g' t g+


All trans


Figure 2-12. Collective motions of each methylene position along the acyl chain roughly
averages out to a conicall shape. The methylenes near the headgroup trace out a cone
of smaller radius while due to greater motional freedom and lack of steric
interference, the acyl chains at the terminal end trace out a cone of larger radius.
Shown on the right are the difference orientations of one C-D bond. With respect to
other carbons in the chain the C-D bonds can be all trans or gauche-trans-gauche.


Headg roup












(A)










-20 -15 -10 -5 0 5 10 15 20
Frequency (kHz)



(B)












-30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30
Frequency (kHz)
Figure 2-13. Deuterium spectra of (4: 1) DPPC:POPG(d-31) (mol/mol) with POPG fully
deuterated on the sn-1 acyl chain. Spectrum was taken on a 600 MHz Bruker
instrument with 1024 scans. Each pair of peaks corresponds to the perpendicular
edges of a powder pattern for a methylene position along the acyl chain. The most
intense peaks near 0 frequency correspond to the terminal methyl group while
overlapping peaks with the largest quadrupolar splitting corresponds to C-D bonds
near the headgroup region. B) The corresponding dePaked spectrum allowing clear
determination of the order parameter at each methylene position.











4:1 DPPC(d-62):POPG

0.25-



h, 0.15


0.1 -



0.05-
0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
carbon number

3:1 POPC(d-31):POPG

0.25-


^ 0.2-

$ 0.15-




8 0.05-


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
carbon number


Figure 2-14. Example of order parameter profile generated from dePaking 2H NMR spectra, for
perdeuterated acyl chains. The time averaged order parameter for each methylene
along the fatty acyl chains were calculated from spectra of 4:1 DPPC(d-62):POPG
(above) and 3:1 POPC(d-31):POPG) (below). The order parameter profile for the sn-2
chain of DPPC is shown although both chains were deuterated. Only the sn-1 chain
of POPC shown was deuterated.














- DPPC(d-62)
-DePaked


-60 -50 -40


-30 -20 -10 0 10 20 30 40 50 60


Chemical Shift (ppm)
Figure 2-15. Example of 31P NMR (blue) and the dePaked result (red). From the dePaking, a
single frequency, corresponding to the parallel edge of the powder pattern, is
generated. This spectrum was collected on 25mg of DPPC(d-62) on a 500MHz
spectrometer with 3072 scans at 44 degrees.

































Figure 2-16. Lipid vesicles when placed in a magnetic field can deform to form an ellipsoidal
shape. An orientational order parameter designated K, is the square of the ratio of the
minor to maj or axis of the ellipsoid and yields information pertaining to the extent of
magnetic field alignment by phospholipid molecules. A random distribution, as seen
in a classic powder pattern, gives rise to intensities proportional to sin(6), where 6 is
the angle between the bilayer normal and Bo. When lipids align, the probability of
finding a particular bilayer normal orientation relative to Bo changes and is
proportional to the orientational order parameter, according to the equation shown at
the bottom. Equations taken from Sternin E in J Magn Reson. 2001 Mar; 149(1): 110-


Bo


p(0) oc sin(0)[1- (1-KE)COS2 8-2


p(0) oc sin(0)









CHAPTER 3
SURFACTANT PEPTIDE KL4 DIFFERENTIALLY MODULATES LIPID COOPERATIVITY
AND ORDER INT DPPC: POPG AND POPC: POPG LIPID VESICLES

The following is a manuscript in preparation to be submitted to the journal Biochemistry.

The final version submitted may differ from what is presented in this dissertation due to

revisions and corrections during the peer review process and j journal format specifications.

KL4 is a 21-residue peptide employed as a functional mimic of lung surfactant protein

SP-B, an essential protein which lowers surface tension in the alveoli. In this study, 31P and 2H

NMR were utilized to study the effects of KL4 On lipid organization in 3:1 POPC: POPG and 4: 1

DPPC: POPG MLVs. NMR spectra recorded at 14.1T indicate a high degree of lipid alignment,

particularly for DPPC: POPG MLVs. The addition of KL4 decreases this alignment in a

concentration dependent manner. 31P NMR spectra of the phospholipids clearly indicate KL4

affects the orientation of the anionic headgroups in a concentration dependant manner. 2H NMR

spectra of the deuterated lipid acyl chains clearly show KL4 effects the ordering of the bilayer

interior in a manner which is dependant on the degree of saturation in the fatty acid tails.

Substantial increases in the acyl chain order parameters were observed in 2H NMR spectra of

DPPC(d-62):POPG MLVs with increasing levels of KL4. The largest changes in order occur at

carbon acyl position 9-15 suggesting the peptide deeply penetrates into DPPC:POPG bilayers.

Conversely, 2H spectra of POPC(d-3 1):POPG, DPPC:POPG(d-3 1), and POPC:POPG(d-3 1)

MLVs showed smaller but measurable decreases in the acyl chain order parameter on addition of

KL4. Thus, fatty acid saturation has a marked effect on the insertion depth of KL4 into

phospholipid MLVs and lipid miscibility. The effects are seen to be approximately linear with

KL4 COncentrations up to 3mol% peptide, the highest percentage studied. The influence of KL4

on lipid phase transitions was also monitored for 4: 1 DPPC:POPG MLVs via DSC. Addition of

KL4 led to slightly higher lipid phase transition temperatures, supporting NMR data suggesting









the peptide causes lipid domain separation in a concentration dependant manner. Based on these

findings, a model of how KL4 interacts with these lipids is presented.

Relevance of KL4 to Lung Surfactant Biology

Lung surfactant is a lipid-rich substance containing key proteins that lines the inner layer

of the alveoli. The primary functions of lung surfactant are to minimize surface tension at the

alveolar air-fluid interface and to provide a barrier against disease (2, 15, 60, 133-135).

Inadequate protein levels are a leading cause of respiratory distress syndrome (RDS) in

premature infants as well as in adults and children experiencing lung trauma or respiratory

infections (39, 51, 66). Current therapies for RDS primarily rely on administration of lung

surfactant from exogenous sources (1, 136). This reliance on xenogenic surfactant is due to the

critical role of SP-B, a highly hydrophobic, 79 residue protein which functions as a homodimer

containing 7 disulfide bridges (29). In mice, deletion of SP-B at the genetic level is lethal (16)

and disruption via mutation causes respiratory failure (39, 137). Peptide-based lung surfactant

replacements designed to replicate the properties of SP-B have received noticeable attention (67,

138) as the use of synthetic analogs would remove the immunologic risks associated with

animal-derived surfactant and allow for greater therapeutic consistency (136).

The lipid constitution in mammalian lung surfactant is heterogeneous, but is dominated by

zwitterionic DPPC (dipalmitoylphosphatidylcholine) (~60%) and anionic POPG

(palmitoyloleoylphosphatidylglycerol) (~10%). Levels of the lipids are relatively conserved in

the lungs of vertebrates (4, 16, 29). SP-B is present at low levels, 0.7-1.0% by weight or a molar

percentage of <0.2% relative to the lipids (16). A multitude of roles for SP-B in surface tension

minimization, intracellular and extracellular surfactant trafficking and respiratory dynamics in

general have been proposed and experimentally established (23). Lung surfactant undergoes a

cycle of lipid adsorption and resorption at the air-fluid interface which is postulated to be









facilitated by SP-B (23). Lipid polymorphisms that change the geometry and arrangement of

lipid headgroups have also been posited as critical for the functional properties of lung

surfactant, as well as in other membrane-membrane mediated events (15, 108, 139). Molecular

level information on how synthetic peptides, unrelated at the primary amino acid level to SP-B,

can modulate surface tension in alveolar compartments is lacking, yet KL4 in COmbination with

POPG, DPPC, and PA palmiticc acid) has been approved as an agent for treatment of RDS due to

its efficacy (136). In-vitro assays as well as animal studies have shown the ability ofKL4 to

lower surface tension in different lipid systems (133, 134). Clinically, administration of a KL4

surfactant preparation (lucinactant) to very premature infants was markedly more effective in

treating RDS than other commercially available formulations (140). Thus, understanding how

KL4 affects the molecular and biophysical properties of the lipids is of particular relevance to the

treatment of various forms of RDS.

Peptide mimics which rely on a presumption of helicity and amphipathicity have been

pursued both therapeutically and as model systems for understanding the unique properties of

SP-B. For example, KL4 WAS designed based on the charge distribution of the C-terminal

residues 59-80 (133, 134). Of particular interest is that KL4 retains many of the macroscopic and

biophysical properties of SP-B despite bearing little resemblance to the primary sequence of the

protein other than a similarity in charge distribution at the C-terminus.

The use of differential scanning calorimetry in combination with NMR can provide insight

into how KL4 induces changes in lipid phase properties and/or geometric arrangement. In this

study, 2H NMR, 31P NMR, and DSC were employed to investigate the properties of 4:1

DPPC:POPG and 3:1 POPC:POPG lipid vesicles on addition of KL4. The former composition

was selected to mimic the composition of lung surfactant while the latter composition is similar









to formulations used in numerous NMR studies of membrane-active antimicrobial peptides,

allowing us to compare the properties of KL4 to other amphipathic, helical peptides.

Methodology to Study KL4 with Lipids

Synthesis of KL4: KL4 (KLLLLKLLLLKLLLLKLLLLK) was synthesized via

automated solid-phase peptide synthesis on a Wang resin (ABI 430, ICBR, UF). The peptide

was cleaved from the resin with 90% TFA/5% triisopropyl-silane/5% water and ether

precipitated. The crude product was purified by RP-HPLC using an acetonitrile/water gradient

and purity was verified by mass spectrometry.

Dried peptide was weighed and dissolved in methanol to a stock concentration of

approximately 1mM. Aliquots were analyzed by amino acid analysis to allow for a more

accurate determination of concentration (Molecular Structure Facility, UC Davis).

Preparation of Peptide:Lipid Samples for DSC: DPPC and POPG were purchased

(Avanti Polar Lipids, Alabaster, AL) as chloroform solutions and concentrations were verified by

phosphate analysis. The lipids were mixed at a molar ratio of 4:1 DPPC:POPG in chloroform,

aliquoted, and a methanol solution of KL4 WAS added as needed to make samples with

peptide:1ipid ratios ranging from <1:1000 to >1:50. The peptide-lipid samples were dried,

dissolved in cyclohexane and freeze-dried overnight to remove residual solvent. Each resultant

peptide-lipid powder was solubilized in 5mM HEPES buffer at pH 7.4, with 140mM NaC1, ImM

EDTA, and 5mM CaCl2 to a Einal lipid concentration of approximately 3mM. Samples were

placed in a 50oC water bath to facilitate solubilization accompanies by 3-5 freeze thaw cycles to

facilitate solubilization and equilibration. Peptide:1ipid MLVs were extruded through 100nm

fi1ters (AvantiPolar Lipids, Alabaster, AL) to form LUVs and degassed just prior to DSC. DSC

experiments were conducted over a range of 10-70oC at a scan rate of loC/min and run in

triplicate. Following DSC, aliquots of each sample were removed and assayed by phosphate









analysis to determine final phospholipid concentration. All assayed concentrations were within

10% of initial estimated concentrations.

Data analysis was performed with Origin v6.0. For heat capacity measurements, the molar

lipid concentration in the DSC sample cell was based on inorganic phosphate assays. AHeal was

determined by integration of the phase transition peaks in the DSC thermograms after baseline

correction. The van't Hoff derived enthalpy (AHVH), an enthalpic parameter utilizing the phase

transition temperature (Tm) of the lipid, was determined by equation (3-1) (86):


AH, = 4R7' 'P (3-1)
SHcal

Lipid cooperativity was assessed by both measuring the peak width at half height (ATy2) and by

calculating the cooperativity unit based on the ratio of the van't Hoff enthalpy to the

calorimetric enthalpy.

Solid state NMR sample preparation: A methanol solution of KL4 WAS added to

chloroform solutions of 4: 1 DPPC(d-62):POPG, 4: 1 DPPC:POPG(d-31), 3:1 POPC(d-

31):POPG, 3:1 POPC:POPG(d-31), DPPC(d-62), POPC(d-31), and POPG(d-31) to make a

series of samples with final peptide to lipid ratios ranging from <1:1000 to >1:50. The MeOH

and CHCl3 were evaporated under nitrogen and the peptide/lipid film was dissolved in

cyclohexane and lyophilized overnight to remove residual solvent. Approximately 25mg of each

sample was then placed in a 5mm diameter NMR tube and 300C1L of buffer containing 5mM

HEPES buffer at pH 7.4,140mM NaC1, and ImM EDTA in 2H depleted water (Cambridge

Isotope Laboratories, Andover MA) was added. NMR samples were then subj ected to 3-5

freeze-thaw cycles to form MLVs.









Phosphorous (31P) NMR: 31P NMR data were collected on a 600 MHz Bruker Avance

system (Billerica, MA) using a standard 5mm BBO probe and a standard pulse-acquire sequence

with 25 k
44 and 490C) with 1024-2048 scans for each spectrum and a 5 second recycle delay between

scans to minimize RF heating of the samples.

The transform of a powder lineshape to an oriented spectrum at a single frequency is

termed dePaking. The dePaking of NMR spectra was performed using an algorithm provided by

Professor Edward Sternin. The algorithm employed a Tikhonov regularization method (102,

103, 105) to simultaneously determine the extent of macroscopic ordering in partially aligned

lipid spectra and the dePaked frequencies (103).

Deuterium (2H) NMR: 2H NMR data were collected on a 500MHz Bruker Avance

System (Billerica, MA) using a standard 5mm BBO probe and quad echo sequence with a B1

field of 40 k
scans and 0.5 second recycle delay.

The dePaking of 2H NMR data was performed using the same algorithms as for the 31P NMR

spectra. Individual assignments of peaks were made based on work by Petrache, et al (104, 11 7,

124) and Seelig (104).

KL4 Affects Lipid Phase Behavior

The dynamic air-fluid interface in the lung requires lung surfactant to possess specific

attributes to lower surface tension and allow rapid respreading. Lipid-rich surfactant relies on

relatively low levels of lung surfactant proteins B and C (<0.2mol%) to alter the structure,

dynamics, and phase properties of the lipids to achieve the characteristic properties of lung

surfactant. The peptide KL4 Similarly affects the macroscopic properties of the lipids and thus

has been pursued as a replacement for SP-B in therapeutic formulations (137, 140).









The size and shape of the 31P chemical shift anisotropy in phospholipid headgroups can

identify the existence of hexagonal, lamellar, or other lipid phases (108), and in lamellar phases

the size of the anisotropy is primarily dependant on the average orientation of the phosphate

headgroups relative to the bilayer normal. Furthermore, 2H NMR spectra of lipid systems where

the palmitoyl chains of either the phosphatidylcholines or the phosphatidylglycerol are

deuterated can determine the effects and insertion depth of KL4. The role of KL4 in lipid phase

behavior can also be observed using 31P NMR.

Calorimetric data at increasing concentrations of KL4 alSo assesses the effect of the peptide

on the thermodynamics and cooperativity of the Lp to L, lipid phase transition in DPPC:POPG

vesicles. The DSC data for 4: 1 DPPC:POPG vesicles containing KL4 Show a concentration

dependent effect of the peptide on the main phase transition (Figure 3-1). Adding POPG to

DPPC in a 1:4 ratio shifts the Tm for DPPC from 42oC to below 36oC due to the

monounsaturated fatty acid group in POPG. Addition of the peptide shifts the Tm back to higher

temperatures in a concentration dependant manner. Small amounts of peptide increase the Tm

slightly by 2-3oC; AHeal, and AHyH remain relatively the same except for a slight increase at

0.75 mol% of peptide. At 0.75 mol % KL4, the shape of the thermogram noticeably bifurcates,

providing indirect evidence of lipid domain separation. This bifurcation becomes more

pronounced at higher concentrations of peptide, most noticeably 1.5 and 2.2 mol%, agreeing well

with the observation of domain formation via Langmuir trough studies of KL4 interacting with

fluorescently labeled DPPC in a DPPC:POPG:PA system (141). This bifurcation has also been

seen by Saenz, et al, at a lower peptide ratio (0.5 mol%) in 3:1 (w/w) DPPC:POPG MLVs (142).

This correlates well with postulated domain formation mediated via electrostatic interactions

between anionic phospholipids (such as POPG) and cationic peptides (such as KL4) (85). This









behavior may be important to the function of the peptide in vivo during the lipid

resorption/adsorption cycle (23). Table 3-1 summarizes the thermodynamic behavior of

DPPC:POPG as a function of peptide concentration.

3P NMR: Addition of KL4 Leads to Changes in Orientation of the PG Headgroups

31P NMR spectra of 3:1 POPC:POPG and 4: 1 DPPC:POPG preparations show orientation

of the lipid bilayers in the magnetic field leading to distorted axially symmetric spectra with

higher than expected intensities at the perpendicular edges (Figure 3-2 and Figure 3-4).

Overlapping lineshapes resulting from the PC and PG headgroups are easily distinguished at this

field. Lipid alignment in high magnetic fields has been observed previously and is caused by the

negative diamagnetic susceptibility inherent to phospholipids (129, 130, 143). In lipid bilayers,

the cooperative alignment of the magnetic moments of individual lipid molecules leads to a large

bulk magnetic susceptibility and an overall macroscopic ordering of the sample within a

magnetic field (132). Increasing amounts of KL4 disrupts this alignment in a concentration

dependent manner, as evidenced by the gradual increase in the parallel edges of the 31P spectra.

The reduction in alignment of the lipid lamellae on addition of KL4 Suggests the peptide disrupts

lipid-lipid interactions and dissipates the ellipsoidal distortion of the vesicles in the magnetic

field; the degree of alignment seen is similar for all lipids within the sample. When dePaking the

NMR spectra the ellipsoidal parameter (K. ) provides a measure of decreases in alignment with

increasing KL4.

DePaking of the 31P NMR spectra for 3:1 POPC(d-3 1):POPG and 4:1 DPPC(d-62):POPG

clearly show changes in the PG headgroup orientations based on the shifts in the POPG peak on

addition of KL4 while the DPPC anisotropy remains constant (Figure 3-3 and Figure 3-5). When

the POPG acyl chains are deuterated, the results are the same as expected since the deuteration









should have little effect on the headgroup of the lipid. These results strongly support a

concentration dependant ionic interaction between the cationic KL4 peptide and electronegative

PG moiety. Table 3-2 lists the 31P CSA span for the headgroups and a general reduction in span

with increased KL4 COncentration is seen for POPG in both lipid systems. Figures 3-6 and

Figure 3-8 show the static 31P NMR spectra of lipids with deuterated POPG and Figure 3-7 and

3-9 show the corresponding dePaked spectra. The shift in the POPG parallel edge frequency is

consistently seen throughout all lipid systems studied regardless of which lipid was deuterated.

Figure 3-10 shows the Ao (change in CSA) for DPPC and POPG in DPPC:POPG(d-31).

To assess the underlying causes of the changes in POPG CSAs and the ordering of the acyl

chains in the binary lipid mixtures, 31P and 2H NMR spectra were collected on individual lipids

alone and with 1.5mol% KL4. 31P NMR spectra are shown in Figure 3-11 and the corresponding

dePaked spectra are shown in Figure 3-12. Only small changes in CSA are seen on addition of

KL4. However, the CSA for POPG alone is significantly smaller than observed for 4: 1

DPPC:POPG or 3:1 POPC:POPG mixtures prior to the addition of KL4. However, for the binary

lipid mixtures with higher amounts of KL4, the 31P CSAs are more similar to those expected

based on the spectra of the neat lipids. Thus, KL4 is clearly affecting the interactions of the PC

and PG lipids as well as their miscibility. This is in agreement with the DSC data showing phase

separation or lipid sequestration on addition of KL4. Hence one important role possibly

mediated by the peptide is lipid demixing via interactions of KL4 with the PG headgroups.

KL4 Effects on Lipid Acyl Chain Ordering Dependent on the Saturation of the Acyl Chains

Deuterium solid state NMR can serve as a sensitive non-perturbing probe for investigating

dynamic processes of proteins and lipids. The size of the 2H quadrupolar interaction is well-

matched to the time scale of many dynamic processes occurring in membranes and proteins,

particularly for fatty acid chains in lipid systems (144). The extent of peptide interaction with









the bilayer can easily be monitored by determining the time averaged order parameters,
for each position in the fatty acid chain and monitoring their changes with addition of peptide.

The order parameter encapsulates motions of the lipids, including axial rotation around the

lipid long axis, undulations or reorientations of the lipids with respect to the bilayer normal

known as "wobbling", and trans-gauche isomerization at individual methylene positions along

the acyl chains (119). The order parameter reflects the collective averaging of these motions on

the NMR timescale (105 seconds), which also includes lateral diffusion of the lipid molecule.

Typical ValUeS are on the order of 0.2 for fatty acyl chains in individual lipids near the

headgroup (145). The effect of KL4 On motions at each individual C-D bond methylene position,

and on lipid motions in general can thus be quantitatively determined.

NMR spectra taken for binary lipid systems with palmitoyl chains deuterated on different

lipids are shown in Figures 3-13 to 3-17 along with their dePaked spectra.

As with 31P NMR data, some lipid alignment in the static field is seen. Spectra taken with

different deuterated acyl chains were taken to determine whether and how the peptide interacts

with the acyl region of a particular lipid.

A recent reevaluation of deuterium order parameters on a series of disaturated PC

headgroups was performed by Petrache, et al at different temperatures (124) which were

extrapolated to 44oC to assign the sn-1 and sn-2 positions in DPPC spectra. Using this and the

acyl chain assignments from Seelig (104) as a guide, assignments and order parameter

calculations were made. The calculated order parameters (using Equation 2-14) from Avq values

for each lipid system studied can be found in the Appendices. Furthermore, 2H NMR spectra of

neat lipids DPPC(d-62), POPC(d-31), and POPG(d-31) with and without 1.5mol% KL4 were

also collected, dePaked and assigned (Figure 3-18) unfortunately, the poor signal to noise ratio









obtained from these measurements prevented clear assignments for each methylene position after

dePaking.

The order parameters profiles obtained for 4: 1 DPPC(d-62):POPG, 4: 1 DPPC:POPG

(d-31), 3:1 POPC(d-31):POPG and 3:1 POPC:POPG(d-31) demonstrate that the peptide interacts

with the two lipids in a manner that is dependant on the degree of saturation in the fatty acid

chains. Figure 3-19 shows the calculated order parameters as a function of carbon number for

the sn-1 and sn-2 chains of DPPC from spectra of 4:1 DPPC(d-62):POPG. KL4 clearly affects

the acyl chain dynamics of DPPC at positions 9-15 in a concentration dependant manner and

increases their ordering. Similar concentrations of peptide added to 3:1 POPC(d-3 1):POPG

MLVs lead to only small changes in order parameters (Figure 3-20). Smaller, negative changes

in order are seen for corresponding regions in the sn-1 chain of POPC in 3:1 POPC(d-3 1):POPG

bilayers suggesting a more peripheral interaction with the lipids or preferential interaction with

POPG as was found from 31P NMR. Hence, the degree of saturation and lipid system has an

effect on mode of binding of KL4.

The data shown above are more pronounced when the observations are viewed as the

percent change in order parameter 6 at each position along the acyl chain. Shown are the

changes in at a particular methylene i for 4: 1 DPPC(d-62):POPG (Figure 3-21) and

3:1 POPC(d-31):POPG (Figure 3-22). Changes in the order parameter, relative to lipids alone,

are shown for C-D bond representing the plateau region (carbon 3), middle of the acyl chain, and

the terminal ends. Significant changes in 8 Occur for both sn-1 and sn-2 chains ofDPPC,

with increases of up to 10% for carbon 10 and 12 and 8.5% for carbon 8. This high degree of

ordering suggests KL4 is penetrating deep into the bilayer and decreasing the mobility of the acyl

chains. This increase in ordering at the middle and ends of the acyl chains from deuterium data









for DPPC(d-62):POPG argue for a deep penetration of the peptide, but the lack of similar

changes in positions 2-3 suggest the peptide does not adopt a transmembrane orientation. Thus

KL4 lOdges into the hydrophobic region of the bilayer, while maintaining a perpendicular

orientation to the bilayer normal. Quantitatively, the calculated 8 for 3:1 POPC(d-

31):POPG, decrease in magnitude by up to 4-6% indicating the acyl chains have increased

mobility. The order parameter shifts are not obvious in the order parameter profiles shown in

Figure 3-22 because the difference in their order parameter values are very small, but their

percent changes make this distinction possible (Appendix A-D).

In experiments where the palmitoyl chain of POPG were deuterated; 3:1 POPC:POPG

(d-3 1) and 4: 1 DPPC:POPG(d-31i), show an overall decrease in ordering of the POPG acyl

chains on addition of KL4 (Figure 3-23 and Figure 3-24). Decreases in the order parameters are

seen at all the acyl positions in POPG. These results further corroborate 31P NMR findings that

show an association with KL4 and the PG headgroup.

KL4 in Relation to other Peptides of Similar Size, Composition and Length

A possible explanation for how KL4 inSerts into the bilayers is that it may form a structure

in which the lysines lie on one side of a helix. The "snorkeling" of these lysine sidechains would

allow an amphipathic helical conformation of KL4 to deeply penetrate the bilayer while still

having electrostatic interactions between the lysine sidechains and the lipid phosphate groups.

The snorkeling of lysine residues have previously been postulated to provide negative curvature

strain in the context of the transmembrane WALP and KALP peptides (146). Of particular

interest to the snorkeling model is a molecular simulation study of the 22 amino acid peptide

KKLLKLLLLLLLLLLKLLLLKK which was found to have a transmembrane orientation and

"snorkel" in POPC membranes (147). This peptide bears striking homology to KL4 in both









amino acid content and hydrophobic to hydrophilic ratio, but the lysines are distributed more

toward the ends of the peptide leading to a transmembrane configuration.

If KL4 adopts a helical structure in the lipid environment which is perpendicular to the

membrane normal, it cannot form a canonical a-helix as this would place the lysines evenly

around the helix rather than in an amphipathic configuration. Nonetheless, FT-IR measurements

indicate KL4 is helical in lipid bilayers, and sits on top of the membrane (148, 149). Lysine and

leucine rich peptides have been known to adapt to different amphiphathic secondary structures at

the air-water interface and on polymer surfaces based on the amino acid pattern of the residues

(149, 150). Similar driving forces would exist at the lipid bilayer surface and peptides of varying

ratios of lysine and leucine residues have been used to modulate the peptide helicity and

topology in bilayer systems (93). Amphipathic a-helices with specific ratios ofL to K that

generate a greater hydrophobic face to the helix have been demonstrated to perturb lipid bilayers

and aggregate, causing micelle formation (151). No indication of micelle formation by KL4

addition has been found again suggesting its structure and mechanism is different from

amphipathic a-helices. The structure of KL4 will be further discussed in Chapter 4.

The ability of peptides to affect lipid magnetic Hield alignment has previously been noted

(130, 132, 143, 152), particularly for amphipathic peptides. One example is the synthetic

alamethicin derivatives. In these studies 14 and 21 residue helical peptides with crown ether side

chains were studied by 31P NMR to probe peptide induced lipid polymorphism. These Eindings

indicate the peptide changing the elastic properties of the membrane and causing a deformation

of the bilayer. Synthetic peptides such KIGAKI, designed to be an amphiphillic beta-sheet, have

also been found to change the ratios of the perpendicular to parallel edges in static 31P spectra of

POPC:POPG MLVs (153). The opposite phenomenon--alignment of lipid bilayers on addition









of peptide--has also been observed by 31P NMR. Studies of the effect of melittin on

DPPC/cholesterol bilayers show the ordering of the lipids on addition of peptide (154), high

amounts of magainin antibiotics was found to magnetically orient POPC bilayers (112), and the

opioid peptide dynorphin was also demonstrated to increase alignment of DMPC MLVs (155).

Furthermore, the antimicrobial peptides found in Australian tree frogs, caerin 1.1 and

maculatin 1.1 have been demonstrated to increase molecular order in bicelles, but the shorter

peptides aurein 1.2 and citropin 1.1 do not (156). Hence, the influence of peptides on the

magnetic properties of lipids has been shown to exist across a broad spectrum of peptides of

similar size and length. Though the primary amino acid sequence of these peptides differs

markedly, the results indicate that the decrease in macroscopic lipid alignment mediated by KL4

may not be entirely unique. The differences in alignment properties are suggestive of different

effects on the lipid organizational properties. Unfortunately, the diversity in lipid composition,

peptide concentration and experimental conditions precludes a systematic evaluation of how

peptide sequences and length affects lipid organization.

A recent study does show antimicrobial activity for KL4 in hypoxic-injured mice infected

with lipopolysacharride (72). Cationic, leucine-rich amphipathic a-helical peptides have been

pursued as antimicrobial agents since their positive charge leads to preferential targeting of the

anionic rich membranes typical to prokaryotes (80, 146). These peptides are designed to be

amphipathic a-helices based on naturally occurring antimicrobial peptides which are thought to

disrupt the membranes on binding through the formation of toroidal or barrel stave pores

structure(79, 157). However, these peptides have significantly higher percentage of charged

residues (>50% compared to <25% for KL4) and in all probability a different secondary

structure.









KL,4 Shares Many Properties with Cholesterol and Transmembrane Helices in DPPC

Ordering of DPPC by KL4 interacting with bilayers is a phenomenon commonly associated

with molecules that penetrate the hydrophobic region of the bilayer, such as cholesterol, or span

it, such as transmembrane helices (127, 158-160). Much like KL4, cholesterol has also been

shown to decrease magnetic alignment of DMPC lipids at 20 mol% levels (159). 2H NMR

studies also indicate increased order of DPPC acyl chains, decreased area per lipid molecule, and

domain separation mediated by cholesterol on the range of roughly 15-25mol%, (161-163).

Raman spectroscopy studies reveal that higher amounts of cholesterol induce a "liquid-ordered"

(lo) phase on DPPC, a state which has limited acyl chain flexibility but a greater degree of lipid

mobility (164). This raises the interesting possibility of KL4 acting in a similar capacity to

cholesterol but at significantly lower mol%. The ValUeS for DPPC alone from the

literature or in DPPC:POPG vesicles are very similar (124); addition of KL4 inCreaSes SCD ValUeS

in a manner that has been seen for cholesterol. The resultant ordering seen could be the

consequence of phase separation as suggested by the DSC thermograms (Figure 3-1), but

complete segregation is unlikely given that SCD Of DPPC with KL4 >SCD Of neat DPPC. In

comparison to 4: 1 DPPC(d-62):POPG MLVs, the 2H spectra of 3:1 POPC(d-31):POPG MLVs

only show small downward shifts in the order parameter profiles on KL4 addition, suggesting

only an electrostatic interaction of KL4 with POPC and a more shallow interaction with the

bilayer.

Molecular Model of KL,4 with POPC:POPG and DPPC:POPG

Based on the assumption of helicity for KL4, with the lysines on one side of the helix, a

molecular model of its potential orientation and penetration based on our data is shown in Figure

3-25. The peptide was modeled based on data presented in Chapter 4 and PDB files to simulate

a lipid bilayer milieu were based on published values from Professor Scott Feller et al (71).









Based on the thickness of the bilayer and the length of the peptide, a transmembrane orientation

of the peptide is not likely due to the periodic placement of the lysine residues as well as the lack

of a shift in parameters for the lipid plateau region on addition of KL4 (146, 165). This

peripheral orientation has been verified by infrared reflection-absorption spectroscopy (IRRAS)

taken on KL4 with DPPC and 7/3 DPPC:POPG bilayers. In these measurements, a beta-sheet

structure at the membrane interface in a Langmuir trough arrangement was determined (166).

However, our data supports the conclusion that KL4 is helical with the lysine side chains aligned

to allow them to "snorkel" to the phosphate headgroups in the DPPC:POPG bilayer. Lysine

snorkeling is supported by the ValUeS we observed, particularly at acyl chain positions 8-

15. This maximizes electrostatic interactions between the amino groups and the phosphate

headgroups. Such snorkeling, which has been reported for many short, lysine capped peptides,

can allow for a much greater penetration of the peptide while allowing it to remain perpendicular

to the bilayer normal. The thermodynamic penalty imposed by placing KL4 in a transmembrane

orientation would be prohibitive since it would result in charged amino acids partitioned into the

hydrophobic core. Also, a transmembrane orientation of the peptide would cause more ordering

of the positions 2-8 in the acyl chains than we see, as has been shown for more hydrophobic a-

helical peptides with a high distribution of leucines (167). The binding of KL4 to model

POPC:POPG lipids are more peripheral based on 2H NMR derived ValUeS and 31P NMR.

Derivatives of magainin antibiotics, peptides which have an orientation perpendicular to the

bilayer normal, also cause a decrease in order parameters that decrease upon addition of 3mol%

of peptide, though the downward shifts in order parameter seen are significantly higher than

what is seen in this work (79). This is probably due to the toroidal pore mechanism of cell lysis

attributed to these peptide types. Order parameters seen for KL4 with POPC(d-31):POPG









similarly decrease indicating a mostly peripheral interaction with the lipid; however, the

comparatively smaller decrease in ValUeS presumably reflects KL4 facilitating lipid-lipid

interaction, and not cellular degradation as would be the case for the magainin family of

peptides.

The snorkeling model of KL4 within DPPC:POPG lipid multilamellar vesicles could have

important consequences for membrane structure and function. For the most part, this represents

a novel peptide-lipid interaction that is unique in comparison to what has been seen for

amphipathic a-helical antimicrobial peptides. Most antimicrobial peptides tend to bind

peripherally and destroy bacterial cell membranes via a toroidal pore or carpet mechanism (79,

113). While the binding of KL4 to POPC:POPG vesicles suggest it could bind to these lipids in a

manner similar to antibiotic peptides, its ability to snorkel in DPPC:POPG lipids would have a

different effect on lipid biophysics. Some of the thermodynamic costs of peptide penetration can

be relieved by the formation of alternative non-lamellar structures, such as hexagonal phases

(160, 165). The inverted HI phase, in which the headgroups invert and follow an aqueous

channel can form if peptide lipid hydrophobic mismatch occurs. Such inverted isotropic phases

have been shown to exist in phosphatidylcholine membranes at concentrations greater than

3mol% (168), the maximum concentration used in these studies. The formation of alternative

lipid geometries, specifically, the inverted HII phase, has been found to be important in the

proper formation lung surfactant (139). The model depicted suggests that KL4 COuld facilitate

formation of non-lamellar lipid phases and/or induce a curvature strain as its role in lung

surfactant. Alternatively, lipid geometry could be critical during fusion of lipids to the air-

surfactant interface and KL4 Snorkeling could be essential feature for the proper shuttling of

lipids.









In conclusion, DSC, 31P and 2H data indicate KL4 binds peripherally to 3:1 POPC:POPG

lipids through electrostatic interaction of the lipid phosphates with the positively charged lysines.

A different interaction is seen with 4: 1 DPPC:POPG lipids suggesting the peptide penetrates to a

far greater depth in the bilayer. Thus, both peptide structure and acyl chain saturation play an

important role in determining the insertion level of KL4. The DSC and 31P NMR results show

KL4 having similar attributes to cholesterol in terms of lipid ordering and DSC peak broadening,

as well the peptide displaying an electrostatic interaction with the PG headgroups when POPG is

added to DPPC and POPC lipid systems. 31P NMR spectra indicate spontaneous lipid alignment

to the magnetic field which is abrogated by the addition of increasing levels of peptide. A

decrease in lipid-lipid association is also seen from our 31P NMR data as peptide is added. With

the findings presented here, a more thorough structural model can be established for how this

small peptide mimics lung surfactant protein B and drive the development of future mimetics.


ACKNOWLEDGMENTS: The authors of this paper thank Dr. Alfred Chung for synthesis of

peptide KL4, and the Molecular Structure Facility at University of California, Davis for AAA

analysis. The research herein was funded by NIH 1R01HLO76586 awarded to Dr. Joanna R.

Long and UF, MBI.














2000 -1 15 mol% KL~

leco- -0.8 mol% KLI
0.4 mol% KL
Io -0.2 mol% KL:
1400-
a -01mol% K(L
12o- -noKL~


i3 800-

O 600-

aL 400-

200-



-200 llllIll
10 20 30 40 50 60 70

Tempera~ture ('C)


Figure 3-1. Differential scanning calorimetry on KL4 with 4: 1 DPPC:POPG vesicles with KL4 at
the indicated molar percentages. Shown is clear phase separation on addition of
peptide. DSC scans on each sample were performed in triplicate at a scan rate of 1
degree per from 10-70oC.












Table 3-1. Thermodynamic parameters obtained from DSC thermograms.


Cpmax
kcal/mol/oC

1.6 & 0.1

1.6 & 0.1
1.4 & 0.1
1.4 & 0.1
1.1
1.4 & 0.1
1.2 & 0.1


Tm
(oC)
63; 4 -e 02


A~cl HyH as
CU"
(kcal/mol) (energy/mol) (cal/mol/K) AT1/2
10.0 & 1.3 115 A 12 321 4 6.0 + 0.4 12 3

9.7 & 0.3 124 & 2 31 +1 5.2 & 0.1 13 1
8.0 10.9 133 A 1 26 & 3 5.5 & 0.1 17 1
9.0 10.7 121 & 2 29 & 2 5.5 & 0.2 14 1
6.7 113 &4 22 5.6 17
9.5 & 0.4 115 & 3 30 & 1 5.84 & 0.04 12 1
7.9 & 0.1 122 & 1 25 A 1 5.5 & 0.1 12 & 2
AHead ~ sample only run once in the DSC instrument


Main Transition


DPPC:POPG

0.1% KL4 36.9 & 0.1
0.2% KL4 36.710.2
0.4% KL436.8 & 0.2

0.8% KL4,, 37.1
1.5% KL4 39.20 + 0.03
2.3% KL4 39.50 + 0.04
*" cooperativity unit (AHyH/










Table 3-2. CSA span for phosphate headgroup in 3:1 POPC:POPG and 4: 1 DPPC:POPG MLVs
with and without KL4. The greatest change occurs in PG headgroups in 4:1
DPPC:POPG MLVs indicating a preferential interaction of this lipid with the peptide.
A change in the PG 31P CSA span is also seen in the 3:1 POPC:POPG MLVs on
addition of peptide.


4:1
DPPC:POPG
DPPC


POPC:POPG
POPC

-33.9
-35.1
-35.1
-28.2
-36.4
-35.8
-33.9


KL4
concentration
0
0.1
0.2
0.4
0.8
1.5
2.3


POPG

-26.9
-26.9
-27.6
-20.7
-27.6
-26.3
-23.2


POPG

-33.2
-32.3
-32.4
-32.4
-30.9
-28.6
-26.4


-42.2
-42.2
-41.4
-43.0
-43.7
-43.0
-42.4
































' '


S2.5% KL4
S1.6% KL4
S0.8% KL4
0.4% KL4
-0.2% KL4
-0.1% KL4
- % KL4


-40 -30 -20


I I I i i I
-10 0 10 20 30 40


Chemical Shift (ppm)

Figure 3-2. Static 31P NMR spectra of 3:1 POPC(d-31):POPG MLVs with the increasing
addition of KL4. As peptide levels increase, signal increases at the parallel edge
suggesting KL4 reduces macroscopic alignment of the lipids. Spectra taken with
1024 scans in a 600MHz Bruker instrument at 44oC.










PO PC


S2.5% KL4
-1.6% KL4
-0.8% KL4
-0.4% KL4
-0.2% KL4
-0. 1% KL4
-no KL4


PO PG


-60 -50 -40 -30 -20 -10 0 10 20 30

Chemical Shift (ppm)


Figure 3-3. Static 31P dePaked NMR spectra of 3:1 POPC(d-31):POPG with increasing amounts
of KL4. The movement of the POPG peak upon addition of peptide is clearly visible.











- 2.3%
- 1.6%
- 0.8%


KL,
4L
KL,


0.4% KL,
-0.2% KL,

-0.1% KL,
-no KL,


I
-40


-30


-20


-10


'I
0


10


20


'I
30


'I
40


Chemical Shift (ppm)


Figure 3-4. Static 31P NMR spectra of 4:1 DPPC(d-62):POPG MLVs with increasing addition of
KL4. As peptide levels increase, signal increases at the parallel edge as seen for
3:1 POPC:POPG MLVs.









DPPC

-- 2.5% KL4
1.6% KL4
O.8% KL4
O.4% KL4
0.2% KL4
-0 .1% KL4
no KL4




PO PG









-50 -40 -30 -20 -10 0 10 20 30

Chemical Shift (ppm)


Figure 3-5. Static 31P dePaked NMR spectra of 4:1 DPPC(d-62):POPG with increasing amounts
of KL4. The movement of the POPG peak upon addition of peptide is clearly visible.





























-40 -30 -20 -10 0 10 20 30
Chemical Shift (ppm)
Figure 3-6. Static 31P NMR spectra of 3:1 POPC:POPG(d-31) MLVs with increasing amounts of
KL4.


-3% K L
-1% KL,
- 0.2% KL,
3:1 POPC(d-31):POPG









-3% K L
-1% KL4
-0.2% KL,
3:1 POPC:POPG(d-31)


POPC


POPG


-60 -50 -40 -30 -20 -10


'I
0


10


20


'I
30


Chemical Shift (ppm)


Figure 3-7. DePaked 31P NMR spectra of 3:1 POPC:POPG(d-31) with increasing amounts of
KL4. The movement of the POPG peak upon addition of peptide is clearly visible.













_ .4


-3% K L
-1% KL,
0.2% KL,
-4: 1 DPPC:POPG(d-31)


-30 -20 -10 0


I'I
20 30


Chemical Shift (ppm)
Figure 3-8. Static 31P NMR spectra of 4:1 DPPC:POPG(d-3 1) MLVs with increasing amounts of
KL4.





























-60 -50 -40 -30 -20 -10 0 10 20 30
Chemical Shift (ppm)

Figure 3-9. DePaked 31P NMR spectra of 4: 1 DPPC:POPG(d-3 1) with increasing amounts of
KL4. The movement of the POPG peak upon addition of peptide is clearly visible


3% K L
1% KL4
0.2% KL,
~4:1 DPPC:POPG(d-31)


DPPC


POPG











10.00


e2 --o- DPPC
Q. 5.00-








0.00
0 0.5 1 1.5 2 2.5 3
m o1% KI..

Figure 3-10. The shift in dePaked frequency in ppm (Ao) for DPPC and POPG (in ppm) by KL4
in 4: 1 DPPC:POPG(d-31).












(A)


















(B)


-- DPPC(d-62)
-DPPC(d-62) 1.5mol%KL4


-60 -40 -20 0 20 40 60
Chemical Shift (ppm)


- POPC(d-31)


-POPC(d-31) with 1.5 mol%









-60 -40 -20 0 20 40 60

ppm

(C) -POPG(d-31)

-POPG(d-31) with 1.5 mol% KL4











-60 -40 -20 0 20 40 60
ppm

Figure 3-11. Static 31P NMR spectra of single lipid and lipid with 1.5 mol% KL4 A:
DPPC(d-62), B: POPC(d-31) and C: POPG(d-31).














DPPC(d-62)

DPPC(d-62) with 1.5 mol%
KL4


-30 -20

Chemical shift (ppm)


-POPC(d-31)

-POPC(d-31) with 1.5 mol%
KL4


-40 -30 -20 -10 0


I Shift (ppm)


POPG(d-31)

POPG(d-31) with 1.5 mol%
KL4


-40 -30 -20 -10
Chemical shift (ppm)


Figure 3-12. DePaked 31P spectra for (Top) DPPC(d-62), (Middle) POPC(d-31) and (Bottom)
POPG(d-31) with and without 1.5mol% KL4.




































2.5% K L


KL,
KL,


1.6%
S0.8%


0.4% KL,
0.2% K4
0.2% KL,

no KL,


-20000 -15000 -10000


0 5000 10000 15000 20000


-5000


Frequency (Hz)

Figure 3-13. 2H spectra of 3: 1 POPC(d-3 1):POPG MLVs with increasing amounts of KL4. The
2H spectra overlap indicating very little change in this lipid system as a function of
KL4.











-2.5% KL,
-1.6% KL,
-0.8% KL,
0.4% K4
0.4% KL,

-0.1% KL,
-no KL,


I 'I
-30000 -20000


10000 20000 30000


-10000


Frequency (Hz)

Figure 3-14. 2H NMR spectra of 4: 1 DPPC(d-62):POPG with increasing amounts of KL4.
















(A)


2 5% KL4
0 8% KL4
0 1% KL4
no KL4


4~ h

I :


-35.00 -25.00 -15.00 -5.00 5.00
Frequency (kHz)


15.00 25.00 35.00


(B)













-35.00 -25.00 -15.00 -5.00 5.00 15.00 25.00 35.00
Frequency (kHz)




Figure 3-15 DePaked 2H spectra for (A) 4: 1 DPPC(d-62):POPG MLVs and (B)
3:1 POPC(d-3 1):POPG MLVs with increasing amounts of KL4.


2.5% KL4
0.8% KL4
0.1% KL4
no KL4











3.0% KL4
1.0% KL4
0.2% KL4
no KL4


(A)


-20 -15 -10 -5 0 5 10 15 20
Frequency (Hz)


-3.0% KL4
-1.0% KL4
-0.2% KL4
-no KL4


B)


-40 -30 -20 -10 0 10 20 30 40
Frequency (kHz)


Figure 3-16 Deuterium NMR spectra for (A) 3:1 POPC:POPG(d-31) MLVs and (B) DePaked
spectra with increasing amounts of KL4.













(A)


-3.0% KL4
-1.0% KL4
-0.2% KL4
-no KL4


-20 -15 -10 -5 0 5
Frequency (kHz)


10 15 20

-3.0% KL4
-1.0% KL4

-, 0.2% KLn L4


(B)


-30 -25 -20 -15 -10 -5 0 5

Frequency (kHz)


10 15 20 25 30


Figure 3-17. Static 2H spectra for (A) 4: 1 DPPC:POPG(d-31) MLVs and (B) DePaked spectra
with increasing amounts of KL4.

















-- DPPC(d-62)
S1.5 mol% KL4





-35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35

Frequency (kHz)






POPC(d-31)
S1.5 mol% KL4




-35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35
Frequency (kHz)
(C)


POPG(d-31)
1.5 mol% KL4




-35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35

Frequency (kHz)

Figure 3-18. Static 2H NMR spectra of single lipid MLVs with and without 1.5mol% KL4. Blue
spectrum is single lipid, pink spectrum is lipid with 1.5mol% KL4. (A) DPPC(d-62),
(B) POPC(d-31) and (C) POPG (d-31).














0.25


A 0.2


v)
S0.15

E

ca 0.1



0 0.os


-*- o KL4
--*-.8% KL4
--e2.3% KL4


0 2 4 6 8 10 12 14 16 18
carbon number


0.25


^0.2



e 0.15






O 0.05


-*- no KL4
S0.8% KL4
-+- 2.3% KL4


0 2 4 6 8 10 12 14 16 18
carbon number


Figure 3-19. Order parameter profiles for (4:1i) DPPC(d-62):POPG MLVs with and without
KL4. Top:sn-1 chain Bottom: sn-2 chain












0.25


0.15-
d -e no KL4
~9a ~t0.8% KL4

0.. 0.12.3%KL4


0.05




2 4 6 8 10 12 14 16
carbon number
Figure 3-20. Order parameter profile for the sn-1 chain of 3:1 POPC(d-31):POPG MLVs with
and without KL4














(A)


5 carbon 3
H carbon 8
carbon 10
H carbon 12
H carbon 15


carbon 15
Carbon 10
carbon 3


0 0.1 0.2 0.

rnol% KL4


DaO '


2, 2, 2, D D, D2 Ol


(B)


1 2%

10%


6%

4%
2%
0%
0 0.1 0.2 0.4 0. 1.5 2.3

rnol% KL4


H carbon 3
H carbon 8
carbon 10
H carbon 12
H carbon 15


carbon 15
carbon 10
carbon 3


Figure 3-21. Three dimensional plot of change in order parameter for DPPC(d-62):POPG MLVs
as a function of mole percentage of KL4. The change in order parameter is color-
coded for individual carbons. The top graph is for the sn-1 chain and the bottom
graph is for the sn-2 chain.


v 8%
a, 6%

4%
~e2%
0%

















O%~

0%--
Scarbon 3
-1%
Fl I \\ \\ carbon 8
-2% carbon 10
v 15
Carbon 12
S-3% 12
carbon 15
m -4% -110

S-5%
carbon 15
carbon 12
-6% carbon 10
0 carbon 8
0.1 0.2 04carbon 3
0.8 1.
2.3
mol% KL4


Figure 3-22. Three dimensional plot of change in time averaged order parameter for 3:1
POPC(d-3 1):POPG MLVs as a function of mole percentage of KL4. The change in
order parameter is color-coded for individual carbons.



















(A)


^ 0.20i


S0.15


S0.10


o 0.05


-* no KL4
-C1% KL4
-A 3% KL4


0.00 L~-
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
carbon number


0.20



A 0.15-



E 0.10



o 0.05-


-eno KL4
- 1% KL4
3% KL4


2 34 56 7 8


9 10 11 12 13 14 15 16


carbon number


Figure 3-23. Order parameter profile for A) 4: 1 DPPC:POPG(d-31) MLVs and
B) 3:1 POPC:POPG(d-3 1) MLVs with varying amounts of KL4.














o~~uvo~ ___carbon 3

11 carbon 8
0.004~C 11 carbon 10
^ 11 carbon 12
cX -5.094 11 I carbon 15
K> 11 carbon 16

-10.094
carbon 15
carbon 10
-15.086
carbon 3

% KL4





carbon 3

(E3) HI carbon 8
11carbon 10

di -470 1 carbon 12

v 11carbon 15


carbon 10


0 1 carbon 3
% KL4

Figure 3-24. Change in order parameter values on (A) POPC:POPG(d-31) and (B)
DPPC:POPG(d-3 1) upon addition of KL4.




























Figure 3-25. Model of KL4 penetration in two lipid environments. Based on 2H NMR data, KL4
appears to "snorkel" in 4:1 DPPC:POPG lipid vesicles. This snorkeling may have
consequences in the lung, where these lipids are the most prevalent, since it may help
facilitate lipid shuttling and/or promote formation of different lipid phases. In
contrast, in 3:1 POPC:POPG MLVs lipids, a more peripheral interaction of KL4 with
the headgroup region is seen









CHAPTER 4
STRUCTURAL STUDIES OF KL4

This chapter describes structural measurements of KL4 in a heterogeneous lipid

environment using CD and ssNMR. This work was done in collaboration with Dr. Douglas

Elliot and Professor Joanna Long.

Characterization of KL4 Secondary Structure

In vitro and clinical studies show that KL4 minimizes surface tension and provides for

relief from respiratory distress. The linear charge distribution of KL4 is based on charge

distribution in the C-terminus of SP-B with the presumption that both SP-B59-80 and KL4 form

amphipathic helices. In Chapter 3, the effects of KL4 On lipid dynamics and order were

measured using 31P and 2H NMR. 31P NMR spectroscopy indicated the peptide predominantly

interacting with the PG headgroup and having small negative effects on acyl chain order

parameters in POPC:POPG. The opposite effects were found in DPPC:POPG LUVs where

deuterium-derived order parameters indicate KL4 Ordering the fatty acyl chains. We postulated

this increase in deuterium order parameters as being due to KL4 penetrating deeper into

DPPC:POPG bilayers as opposed to POPC:POPG bilayers. While the different effects on one

lipid system compared to the other is interesting, the question remains as to the secondary

structure of the peptide is when bound to DPPC:POPG and POPC:POPG and how its structure

allows shallower or deeper penetration. In Figure 3-25, it is assumed in the molecular models

that KL4 is helical and that it is superficial in one lipid system while embedded in the other. The

assumption of KL4 being helical in both membrane milieus is an assumption based on findings

that SP-B and SP-C are helical; particularly, SP-B59-80, which has been purported to be an

amphipathic alpha helix (11). However, even though SP-B59-80 iS presumed helical, it does not

necessarily implicate KL4 aS being an a-helix in a lipid environment. Furthermore, the structure









of KL4 in phospholipid bilayers has not been definitively resolved and may be contingent on

lipid composition. Infrared studies (148, 166) involving the orientation of the peptide in

different lipid environments give conflicting secondary structure adaptations. One IR study

shows KL4 being a transmembrane helix in 7:3 DPPC:PG bilayers (148); the other study,

combining IR measurements with surface pressure isotherms, postulated the peptide to be an

anti-parallel beta sheet in a 7:3 DPPC:DPPG membrane environment (166). Thus, even when

the lipid composition is constant, disagreements exists in the literature. The simple assumption

of KL4 being helical, based on sequence periodicity and charge distribution, cannot be readily

accepted and other biophysical methods need to validate the IR measurements and higher

resolution measurements could provide even more insight.

In this chapter, circular dichroism and ssNMR studies are described which were

undertaken to determine the structure of KL4 in the lipid environments of 3: 1 POPC:POPG and

4:1 DPPC:POPG LUVs as well as neat lipids. Circular dichroism represents the simplest and

most effective qualitative measure of global secondary structure in solution and in the presence

of LUVs. Under optimized CD conditions, one can rule out P-sheet or a-helical structures for a

peptide interacting with different lipid moieties. We undertook a systematic CD study of KL4 in

the presence of differing concentrations of POPC:POPG and DPPC:POPG LUVs. These are the

two lipid systems where different topologies of KL4 WeTO pOstulated in Chapter 3 based on NMR

studies examining phospholipid dynamics. As part of these experiments characterizing the

structure of the peptide, CD experiments were also performed to assess the influence of aqueous

buffer or helix-inducing solvents on the gross secondary structure of the peptide in the absence

of lipids.









In collaboration with Professor Joanna Long and Dr. Douglas Elliot, ssNMR studies were

performed on 13C-labeled KL4 peptides using the DQ-DRAWS pulse sequence (detailed in

Chapter 2). 13C labels were enriched in adj acent carbonyl leucines of KL4 to determine

backbone torsion angles phi (cp) and psi (uy). Each 13C-labeled peptide was completed to lipids

and the DQ-DRAWS sequence was used to first determine the torsion angle cp and then the

torsion angle uy. The technique provides for a more detailed structural characterization of KL4 18

the lipid environments. These structural studies can provide insight into the IR results and

possibly explain their discrepancies. They also demonstrate the utility of ssNMR in resolving

peptide secondary structures in complex environments; in this case, mixtures of phospholipids

are used to simulate eukaryotic membranes and membranes from the lung.

Materials and Methodology

Peptide synthesis: KL4 WAS synthesized via automated solid-phase peptide synthesis

(ABI 430, ICBR, UF) on a Wang resin. Peptide was cleaved from the resin with 90% TFA/5%

triisopropyl-silane/5% water and ether precipitated. The crude product was purified by RP-

HPLC using an acetonitrile/water gradient and purity was presence of KL4 WAS Verified by mass

spectrometry with a m/z ratio of 2471. Finally, the product was lyophilized and stored at -30 oC

until used in sample preparation.

Preparation of KL4 in buffer: Dried peptide was weighed and solublized in 1:1

methanol:water to an estimated stock concentration of ~200C1M. This ensured full dissociation

of the peptide and prevented any unwanted formation of aggregates. The solution of 1:1

methanol:water was placed under a stream of nitrogen to remove the methanol and then

concentrated HEPES buffer was added to achieve a final concentration of ~100C1M of KL4 111

5mM HEPES, pH 7.4.










Preparation of POPC:POPG and DPPC:POPG Vesicles For CD Measurements with

KL4: DPPC, POPC and POPG lipids dissolved in chloroform were purchased from Avanti Polar

Lipids (Alabaster, AL). The lipids were mixed at molar ratios of 3:1::POPC:POPG and

4:1:: DPPC:POPG and the CHCl3 WAS removed under a nitrogen stream. The dried lipid films

were dissolved in cyclohexane and lyophilized to remove residual chloroform. The lipids were

then reconstituted in 5mM HEPES, pH 7.4, and subj ected to 3 freeze-thaw cycles to facilitate the

formation of multilamellar vesicles (MLVs). Large unilamellar liposomes (LUVs) were prepared

by extrusion of the MLVs through 100 nm polycarbonate filters (Avanti Polar Lipids) at room

temperature. Extrusion of DPPC:POPG LUVs took place above the mixture' s phase transition

temperature.

Preparation of solution CD samples: CD spectra were collected on 150C1M KL4 in 1:1

methanol:water as well as hexafluroisopropanol. Since the helical content of KL4 is unknown in

solution, a spectrum of 40C1M KL4 in the helix-inducing solvent trifluoroethanol (TFE) (81) was

also collected to generate a baseline CD spectrum for the purely helical peptide. In this

preparation, stock KL4 (498C1M +/- 3 CM) in methanol solution was dried under compressed

nitrogen gas and the peptide film was solubilized in 1mL TFE to a final peptide concentration of

40C1M. The spectrum for KL4 in TFE was run at 45oC and subtracted from a spectrum of only

TFE.

Preparation of KL4/lipid samples in organic solvent for CD: A stock concentration of

KL4 in methanol (498C1M +/- 3 CM) was added to chloroform solution of lipids at 1, 2 and 3mol%

peptide relative to lipids. The peptide-lipid mixtures were allowed to dry overnight before being

dissolved with cyclohexane and lyophilized overnight. Samples were hydrated in 10mM HEPES










pH 7.4, 140mM NaCl and freeze-thawed 3-5 times over the course of 48 hours before extrusion.

Extrusion to generate LUVs were done as described above.

Preparation of CD samples with peptides and lipids mixed in buffer: 100C1M KL4 in

HEPES buffer was added to extruded DPPC, POPC, POPG, 4:1 DPPC:POPG, 3:1 POPC:POPG

vesicles in the same HEPES buffer. For samples containing DPPC, samples were extruded at

50oC, and 100C1M KL4 Stock, prepared as above, was added to freshly extruded vesicles to

achieve a final peptide:1ipid ratio of 1:33. The final concentration of peptide in these samples

was 40C1M and the final concentration of lipid was 1.33mM. CD spectra on these samples were

recorded at 45oC.

Collection of CD spectra: CD experiments were performed on an Aviv Model 215 at a

wavelength range 195-260nm with a step size of Inm for 10-20 scans. The averaging time for

each sample was 1 second. The settling time for each time was 0.3-0.6 seconds. All CD spectra

were buffer subtracted.

CD shows KL~4 to be helical

Circular dichroism provides a simple qualitative method for determining peptide secondary

structure in solution as well as for peptide bound to lipid. As an initial assessment of global

secondary structure, CD was performed on KL4 in 5mM HEPES pH 7.4. At three different

concentrations in 5mM HEPES buffer at pH 7.4, the peptide displayed spectra that are

characteristics of a helical peptide, although some other secondary structure elements can also be

seen (Figure 4-1). CD performed on KL4 at 150C1M, still show a minimum at 208nm, however

the appreciable loss of signal at 222nm may indicate the onset of aggregation of the peptide.

Given the highly hydrophobic nature of the peptide, aggregation or higher order peptide

structures is highly possible. In the presence of the helix-inducing organic solvent

hexafluoroi sopropanol (HFIP), the CD spectrum of 150CIM KL4 indicates helical structure









(Figure 4-2). These initial CD experiments indicate that KL4 is helical in buffer and HFIP

induces further helicity. At higher concentrations in buffer, peptide aggregation most likely

occurs. In methanol and water a reduced helical signature is seen. We then undertook CD

experiments to examine the secondary structure of KL4 when it is interacting with LUVs to

answer the question of whether lipid bilayers can serve as a substrate to induce secondary

structure formation.

Figure 4-3 shows CD measurements taken of 40C1M KL4 interacting with 1.33mM lipid

LUVs in which both peptide and LUVs were reconstituted separately in 10mM HEPES buffer

and then mixed. Helicity was assessed by examining the ellipticity at 208 and 222nm. KL4

added to neat POPG and DPPC LUVs yielded spectra with adsorption minima at 208nm and

222nm indicative of helical secondary structure. The mixture of 4: 1 DPPC:POPG LUVs with

3mol% peptide also showed a pronounced helical CD spectrum (Figure 4-3). CD recorded of

KL4 in the presence of neat POPC lipids resulted in a noisy spectrum with poor signal; however

when the same amount of peptide was added to 3:1 POPC:POPG LUVs, signal with clear helical

tendencies was recaptured (Figure 4-3). The following observations from CD supports 31P NMR

data that reflect an affinity of the peptide with the anionic phosphatidylglycerol lipid. Since KL4

shows helical propensity in solution and is clearly not unstructured (Figure 4-1), the role of

POPG may not necessarily involve folding of the peptide, yet the presence of the lipid clearly

results in changes in ellipticity at 222nm strongly implicating the modulation of helical content

by this phospholipid species. Solution CD of KL4 in 10mM HEPES buffer, pH 7.4 and 140mM

NaCl show a double minima that is characteristic of some helical qualities of the peptide, but the

CD spectrum indicates the peptide is not 100% helical, especially when compared to peptide in

TFE (Figure 4-4).









Ellipticity at 222nm (6222) has been found to increase linearly with the extent of helix

formation (169, 1 70). Shown in Table 4-1 are the 6222 ValUeS obtained by adding peptide to pre-

formed LUVs. Negative ellipticities at 222nm were found when KL4 WAS interacting with

DPPC, POPG and DPPC:POPG LUVs. When viewed as the dichroic ratio of 6222/ e208, the two

wavelengths that yield double minima in an alpha helical CD spectrum, it is clear that the helical

signatures of the peptide are changing relative to the peptide in solution. The CD data strongly

implicate PG headgroups and saturated PC lipids in enhancing the helical propensity of the

peptide.

Peptide and lipid mixed together in organic solvent and reconstituted also result in helical

CD spectra typified by the minima at 208nm and 222nm. Shown in Figure 4-4 are spectra for

40C1M KL4 in the presence of POPC:POPG and DPPC:POPG LUVs at a peptide molar ratio of

1%. To determine if we can discern structural changes in KL4 aS a function of concentration,

samples of 40C1M KL4 which was either at 2mol% and 3mol% with respect to the lipids were

also run. At 2mol% peptide, the double minima in both DPPC:POPG and POPC:POPG can be

seen, although the signal is degraded by light scattering from the lipids (Figure 4-5). 40C1M KL4

with POPC:POPG LUVs shows a CD spectrum that displays significant overlap in signal to

40C1M KL4 in HEPES/NaCl solution. In an environment of POPC:POPG LUVs, the peptide is in

a state that is helical but less so than in TFE. In comparison, the double minima at 208nm and

222nm is prominent at 2mol% peptide in DPPC:POPG LUVs. (Figure 4-5). At 3 mol% peptide,

the CD signal from KL4 is barely discernable when interacting with POPC:POPG LUVs,

suggesting it is disturbing the lipid phase properties leading to light scattering, but the double

minima is still prominent in DPPC:POPG LUVs (Figure 4-6). The data clearly indicate

concentration and lipid dependence in CD signal and can help explain the differing dynamics and









lipid ordering seen from 2H and 31P NMR based on KL4 Studied in POPC:POPG versus

DPPC:POPG vesicles. Figure 4-7 shows the CD signal generated as peptide levels are increased

in DPPC:POPG LUVs and POPC:POPG LUVs. The helical signature seen for KL4 in

DPPC:POPG bilayers, where we postulated the peptide is more embedded, is seen in Figure 4-7.

The signals at 208nm and 222nm seen in DPPC:POPG LUVs could also be a result of

contributions from other types of helices such as a xn-helix that do not make such contributions in

POPC:POPG LUVs. Table 4-2 shows the raw CD ellipticity (in millidegrees) of DPPC:POPG

and POPC:POPG at different molar percentages of KL4. COmparing the ellipticity values

amongst the lipid systems, we find that helical signatures in DPPC:POPG at higher peptide

levels than in POPC:POPG. However, it is not known if the changes seen in ellipticity are

representing changes at the residue level of KL4 Or entire sections of the peptide are assuming

different helical conformations as concentration increases relative to the lipids. If the spectrum

of KL4 in TFE assumes complete helicity, then the CD data indicate that both lipid systems

increase helical content of KL4 relative to that in solution. Comparing KL4 in buffer relative to

that in TFE solvent, it is clear that KL4 COntains a mixture of secondary structures rather than a

purely a-helical motif. These experiments were performed with 10-20 scans for signal

averaging; it is not known if an increase in the number of scans would allow better comparison

of KL4 Samples in an embedded bilayer relative to bound to the headgroup regions. It can be

seen however that differences can be seen when KL4 binds DPPC:POPG versus POPC:POPG

LUVs.

While the typical double minima characteristic of a-helices are seen in CD spectra of KL4

both with and without lipid, it is important to realize that such a method is only sensitive to

average properties of a molecule, and the distribution of helical and non-helical residues cannot









be determined (169). Additionally, CD does not allow clear distinction between different types

of helices. The peptide in combination with lipids may have signal due to formation of non-

standard helices. Our data do show, however, that KL4 has helical characteristics both in

solution and when interacting with DPPC, POPG, DPPC:POPG and POPC:POPG LUVs.

CD indicates that KL4 is helical in solution; however, the periodic placement of lysines

would make an a-helical conformation favorable in buffer, but not at a lipid interface where

amphipathic structures are favored. Placement of KL4 in helical wheel diagrams for different

types of helices show that in order for the peptide to bind to an amphipathic substrate, such as a

lipid interface, the peptide might form i, i+5 hydrogen bonds (Figure 4-8). Such a helical wheel

diagram suggests that a n helix has a favorable alignment of the hydrophobic leucine residues

and hydrophilic lysine residues for snorkeling into a lipid bilayer. Whereas modeling KL4 aS a

i4 i 4 helix shows hydrophilic lysines around the periphery of the helical wheel. This

secondary structure should be favored in the solution-state, where interactions occur

isotropically, but is disfavored in a lipid environment. This begs the question whether the

peptide adapts a canonical i j i+4 helix or another helix with a different hydrogen bonding

pattern in the context of a lipid environment. Torsion angle measurements using ssNMR were

used to answer this question.

Different Types of Helices KL4 May Adapt in a Lipid Bilayer

A standard i ji +4 helix has 3.6 residues per turn providing for the most stable atomic

arrangement thermodynamically, accounting for roughly 30% of secondary structure found in

proteins (1 71). Torsion angles used to define the backbone conformation at C,-N bond (defined

as the angle psi: uy), at the C,-C' bond (defined as the angle phi: cp) are -57o (cp) and -47o (uy) for

a classic a-helix. However, variations do exist, and on average values of q=-65o and uy =-450 are

seen in x-ray structures of crystalline proteins (1 72). Given the nature of lysine side chains









spaced every Hyve residues in KL4, hydrogen bonds to residues in an i j i 5 arrangement cannot

be disqualified as a resulting helical fold in the lipid environment. This can give the helix a

tighter coil which may help the peptide interact with heterogeneous environments and facilitate

the "snorkeling" model envisaged in Chapter 3. An i~ i 5 hydrogen bond pattern results in a

helix with 4.4 residues per turn and is termed a x helix (1 71) with 16 atoms between the

hydrogen bonds. This type of helix is considered to be thermodynamically unfavorable in free

solution (173).

Solid-State NMR Studies of KL4 in POPC:POPG and DPPC:POPG

Work in determining the helical nature of KL4 WAS undertaken by Professor Joanna Long

and Dr. Douglas Elliott. In these studies, KL4 WAS synthesized with 13C enrichment at specific

leucine C' positions in the peptide and the peptide was completed with 3:1 POPC:POPG MLVs

for CPMAS experiments. Using the DRAWS pulse sequence, the double quantum (DQ) state

between the 13 CI Spins was excited during mixing times in the pulse sequence. The mixing time

it takes to generate the DQ coherence is dependant on the distance between the spins.

Distances between adj acent 13C Spins were determined by a least squares fit of DQ buildup

curves to numerical simulations (Figure 4-9). As can be seen in this figure, the build-up curve is

consistent with a cp angle of -1000 to -105o. Using a 2D-DRAWS experiment, the relative

orientations of the amide planes were measured and compared to simulations. The X2-ftting of

the uy torsion angle based on comparison of experimental data to numerical simulations is shown

and the best fit torsion angles from these simulations were cp = -1050 uy = -26o and cp = -105o,

uy = 132o (Figure 4-10).

Using torsion angles of -105o and -26o in the molecular graphics program PYMOL renders

a helix where all the charged lysines lie on one side of the helix (Figure 4-1 1). Such a

configuration would indeed have biological implications; particularly in terms of the peptide









being able to embed to the membrane. However, the splaying of the lysines suggest it would not

be deeply embedded. From the deuterium NMR data performed in Chapter 3, POPC and POPG

acyl chain order parameters decreased slightly in the presence of KL4 COrroborating this model.

Similar experiments performed by Professor Joanna Long on KL4 in DPPC:POPG LUVs reveal

backbone torsion angles of cp =-65 and uy =-78. Placement of these torsion angles into the

molecular graphics program PYMOL also yields a helix (Figure 4-12). However, unlike what

was seen for the peptide bound to POPC:POPG vesicles, it is found that the lysine residues are

more aligned along one side of the helix. Comparing Figure 4-11 and Figure 4-12 one sees that

in POPC:POPG MLVs, the lysine residues have a more radial distribution around the axis of the

helix as compared to in DPPC:POPG MLVs. In POPC:POPG MLVs, the side chain lysines face

preferentially one side of the helix, but the first and last lysine residue are particularly out of

phase. In DPPC:POPG MLVs, the first and fifth lysine side chains are more parallel and in

phase with each other. The argument presented for lysine snorkeling in Chapter 3 from

deuterium N\MR data corroborates nicely with the structure Professor Long found for the 13C

labeled peptide in DPPC:POPG. The helical structure obtained from DQ-DRAWS aids in

explaining the increase in deuterium NMR order parameters found in the middle to the end of the

acyl chain. If the lysine residues of KL4 are mOre aligned in DPPC:POPG, the peptide can bury

itself deep into the bilayer, while the long side chains of lysine can extend out into the interface

and form a favorable electrostatic interaction with the phosphate headgroups. In POPC:POPG,

where the acyl chains become less ordered in the presence of peptide, the lack of lysine

alignment seen in the structure found from DQ-DRAWS measurements strongly implicate a

peripheral interaction of the peptide with the headgroup only. This is seen from our CD data

where in POPC:POPG the helical signature was lost at 3mol% peptide but can still be discerned









in DPPC:POPG. The CD data also rules out cp=-105 and uy =132 as one of the torsion angles

obtained from Professor Long's 2D-DRAWS simulations since such backbone torsion angles

would predict a P-sheet for the peptide. Hence, the findings from Professor Long and Dr. Elliott

show that headgroup conformation and acyl chain dynamics clearly affect the type of helix KL4

adapts in a lipid bilayer setting. Our deuterium NMR and CD data with the ssNMR data from

Professor Long show that in DPPC:POPG, KL4 adapts a structure that has more qualities of a x

helix. Based on CD and ssNMR torsion angle measurements, in POPC:POPG, KL4 adapts into a

structure that is helical but intermediate between an a-helix and a P-sheet.

Fodj e and Karadaghi recently re-evaluated the proteins in the PDB (1 71), and according to

their criterion, have found an underestimation in the amount of 2n helices accounted for. Based

on their algorithms, the authors have come up with mean dihedral angles (cp,u) of -76o and -41o,

which are in stark contrast to previous measurements of -57o and -700 (1 71, 1 72) but in good

agreement with the DPPC:POPG structure. Furthermore, Fodje and Karadaghi found significant

differences in dihedral angles from the i-4 to the i+4, residues within the helix. Hence it seems

probable that KL4 has characteristics (particularly in the middle of the sequence) similar to a x

helix, offering the advantages of partitioning the charged lysine side chains to adapt to a lipid

environment and reducing the surface area and volume occupied by the peptide (1 71) which

would be entropically favorable. xn helix formation may be modulated by amphilicity and may

also help to stabilize lipid-water or air-water interfaces (1 74). Thus, the types of helix resulting

from ssNMR data of KL4 TepreSent non-canonical structures due to residues changing alignment

in the presence of amphiphilic lipid substrates such as 3:1 POPC:POPG and 4: 1 DPPC:POPG

and could indicate how KL4 mOdulates lipid dynamics in the lung. The CD data show different

secondary structure signals contingent on the different lipid system which agree nicely with 31P









and 2H NMR data. Professor Long' s structural measurements of the peptide in DPPC:POPG and

POPC:POPG clearly indicate the peptide has a different structure in each environment

underscoring the observation that the peptide's structure, orientation, and depth penetration is

dependant on the saturation level of the acyl chains.

















-15uM KL4
-30uMKL4
40 -1 60uM KL4



S20-




105 35~Z5~ 245 255

-20-



-40-

Wavelength (nm)





300-


200-

-150uM KL4
Tii 100-




1535 \205 215 225 245 255

[i -100-


-200-


-300-

Wavelength (nm)




Figure 4-1. CD Spectra of KL4 in 5mM HEPES at pH 7.4. Below is a spectrum of 150yM KL4
indicating potential aggregation.
















15000


10000-


5000-





-soo -50


S-10000-


-15000-


-20000
190 200 210 220 230 240 250 260 270

Wavelength (nm)


Figure 4-2 CD spectra of KL4 in Organic solvents. Red is a spectrum of peptide in 150pM
hexafluroisopropanol and black is a spectrum in 50:50 MeOH:dH20.





3:1 POPC:POPG
DPPC
-4:1 DPPC:POPG
100pM KL4


















190 200 210 220 230 240 250 260 270


120 -

100 -

80 -

60 -
40-

40 -
0-




-20 -

-40 -

-60 -


Wavelength (nm)


Figure 4-3. CD Spectra of 40C1M KL4 added to 1.33mM LUVs. DPPC containing samples were
run at 45oC. Remaining samples were run at room temperature. Samples were in
10mM HEPES pH 7.4 and 140mM NaC1.



























-20-


-30-

190 200 210 220 230 240 250 260 270
Wavelength (nm)
Figure 4-4. CD spectra of 40C1M KL4 TOCOnstituted in 4mM 4: 1 DPPC:POPG and 3:1
POPC:POPG LUVs. Shown in comparison is 40C1M KL4 in 10mM HEPES, 140mM
NaCl and in TFE.


-DPPC:POPG LUVs with 1% KL,
-POPC:POPG LUVs with 1% KL,
-40ciM KL4 in HEPES/NaCI
40cLM KL4 in TFE












-DPPC:POPG LUVs with 2% KL,
- POPC:POPG LUVs with 2% KL,
-40pMM KL4 in HEPES/NaCI
40pLM KL4 in TFE


10
0-



-10


-20 -


-30 -


190 200 210 220 230 240

Wavelength (nm)


250 260 270


Figure 4-5. CD spectra of 40C1M KL4 TOCOnstituted in 2mM 4:1 DPPC:POPG and 3:1
POPC:POPG LUVs. Shown in comparison is 40C1M KL4 in 10mM HEPES, 140mM
NaCl and in TFE.












-DPPC:POPG LUVs at 3% KL,
-POPC:POPG LUVs at 3% KL,
-40tLM KL4 in HEPES/NaCI
40tLM KL4 in TFE


10
0-



-10


-20 -


-30 -


190 200 210 220 230 240 250 260 270

Wavelength (nm)

Figure 4-6. CD spectra of 40C1M KL4 reconstituted in 1.33mM 4: 1 DPPC:POPG and 3:1
POPC:POPG LUVs. Shown in comparison is 40C1M KL4 in 10mM HEPES, 140mM
NaCl and in TFE.























143







































___


-POPC:POPG with 1%
KL4
-POPC:POPG with 2%
KL4
-POPC:POPG with 3%
KL4


30

20
E
10


= 1

-20


240 250 260


Wavelength (nm)


-4:1 DPPC:POPG with
1% KL4
-4:1 DPPC:POPG with
2% KL4
-4:1 DPPC:POPG with
3% KL4


S20
*0
4 0

=11


-20


20210 220 2e/40 250 260


Wavelength (nm)


Figure 4-7. CD Spectra of (Top) POPC:POPG LUVs and (Bottom) DPPC:POPG LUVs with
increasing mol% of KL4.













Table 4-1. Ellipticity (in mdeg) of KL4 at 222nm and 208nm and ratio of helical
signatures 222nm and 208nm
208nm 222nm 222nm/ 208nm
40C1M KL4 in
POPC -5.4 -4.8 0.9
POPG -6.4 -14.1 2.2
3:1 POPC:POPG -5.2 -6.7 1.3
DPPC -17.9 -17.9 1.0
4:1 DPPC:POPG -16.3 -17.3 1.1
100C1M KL4 in
solution -45.7 -9.5 0.2












Table 4-2. Ellipticity (in mdeg) of 40C1M KL4 TOCOnstituted in LUVs from organic
solvent.
208nm 222nm 222nm/208nm
4:1 DPPC:POPG
1% KL4 -22.5 -16.1 0.7
2% KL4 -15.4 -13.5 0.9
3% KL4 -17.4 -15.2 0.9

3:1 POPC:POPG
1% KL4 -20.6 -16.2 0.8
2% KL4 -11.2 -9.4 0.8
3% KL4 -10.9 -9.6 0.9
40C1M KL4 -8.9 -9.0 1.0
(in 10mM HEPES, 140mM NaC1)
40C1M KL4 -24.9 -16.5 0.7
(in TFE)




















L7 L20 LB L12 l




KS L1

L10
L7 LI
L3a LD~ ~ l

512 p pa S L1 L17L18 LS
L21 K2


Figure 4-8. Heia he rjcin f L S lf)30hlx mide tnadahlxn
(righ) 2 elix












1.0 '



0.8 -Z



0.6 -/



2 0.4-. KL4-L2,L3
2 KL4-L4,L5

KL4-L7,L8
0.2 eKL4-L9,L10


---- --- phi = -120

0.0
0 2 4 6 8

M ixing tim e (m sec)

Figure 4-9. DQ-DRAWS buildup curves generated from spectra on 13C labeled KL4 with
POPC:POPG (3:1). Data taken with permission from Professor Joanna Long.


















100 -







50 ---













-100 -




-150 -



-110 -108 -106 -1 4 -102 -100 -98 -96 -94 -92 -90


(-1050, -260) 9


Figure 4-10. Ramachandran plot showing a X minimum at (p=-105 and y=-26 for KL4 in 3:1
POPC:POPG. Contours shown in blue, green and brown are 1,2, and 3 standard
deviations away from the minima. Data taken with permission from Professor Joanna
R. Long.











































Figure 4-11i. Model of KL4 based on torsion angles obtained from the 2D-DRAWS experiments.
The charged lysines (shown in blue) line up along one side of the helix.
























Figure 4-12. KL4 with torsion angles of cp = -65, uy= -78 obtained from ssNMR studies of KL4 in
a DPPC:POPG lipid environment. Different views of the peptide indicate the lysine
side chains line up along one side of the helix. In comparison, in POPC:POPG the
lysine side chains splay radially along the helical axis. Data taken with permission
from Joanna R. Long.









CHAPTER 5
COMPARATIVE BIOPHYSICAL STUDIES OF SP-B59-80

This chapter describeS 2H and 31P NMR studies of 4: 1 DPPC(d-62):POPG and 3:1 POPC(d-

31):POPG lipid systems on incorporation of SP-B59-80, the C-terminus of SP-B.

Fragments of SP-B have biophysical activity

While the entire 80 amino acid SP-B protein is essential for lung surfactant organization,

lung dynamics and respiration, subfragments of the native sequence have also shown significant

biophysical function, particularly peptides corresponding to the N-terminal and C-terminal 20-25

amino acids. While both ends of protein have interesting biophysical properties, to date, the N-

terminal region of the peptide has been more extensively studied in terms of functional properties

and possible structural adaptation in different lipid environments.

The N-terminal 25 residues of SP-B (SP-B1-25) have been shown to interact with specific

anionic lipids to facilitate squeeze-out of lipids based on surface-film studies (52). SP-B 1-25 has

also been shown to mediate mixing of lipid vesicles. Fourier-transform infrared measurements

and CD studies of the N-terminal peptide in methanol, SDS micelles, and egg yolk lecithin

(phosphatidylcholine) indicate the peptide has a high helical content. Spin-labeling of the first

phenylalanine residue for electron spin resonance studies show that the N-terminus of the peptide

retains mobility when bound to lipid (1 75). Recent molecular dynamics simulations of the

peptide in DPPC monolayers indicate that the most likely equilibrium conformation is an a-helix

parallel to the interface (1 76). However, the N-terminal fragment of SP-B has not been used in

any formulations of artificial lung surfactants in clinical use indicating that while the role of the

N-terminus may be of importance, it does not convey all the functionality needed for the protein

component of lung surfactant.









The C-terminal fragment of SP-B, specifically residues 59-80 (SP-B59-80), has shown

potential in in vitro assays measuring surface tension such as pulsating bubble surfactometry.

Isotherms relating surface pressure to surface area show that this peptide imparts lipid monolayer

stability under compression, preventing collapse at pressures where monolayers of lipids alone

collapse (133). Of particular clinical interest, the last 22 amino acids of SP-B served as the

template for designing KL4 aS discussed in Chapters 3 and 4 (67, 133). The basis of the design

for KL4 WAS modeling the charge distribution and hydrophilic/hydrophobic ratio of the primary

sequence of SP-B59-80. As with the N-terminus, the C-terminal peptide is believed to form an

amphipathic helix involved in headgroup ordering, but direct structural measurements in varying

lipid contexts have to date not been documented. Nonetheless, a solution NMR study of the

C-terminus of SP-B (residues 63-78) reconstituted in either SDS micelles or the organic solvent

HFIP was recently published. This study was unable to see structure in the first five residues,

but established that the rest of the sequence formed a helix in both SDS micelles and organic

solvent (177). Unlike SP-B1-25, FTIR or CD studies of SP-B59-80 interacting with lipids are not

documented and molecular dynamic simulations of SP-B59-80 do not exist. A previous CD study

(1 78) using TFE and SDS micelles and the solution NMR study are the only current structural

assessment of the C-terminal region of SP-B. Despite the scarcity of literature pertaining to

residues 59-80 of SP-B, it is widely believed that many in vivo activities of SP-B are fulfilled by

the C-terminal end based on in vitro studies (134, 177).

Interestingly, both N and C-terminal ends of the peptide have been reported to be cationic,

amphipathic helices which could serve in a functional role by interacting with anionic lipids,

particularly PG headgroups (11). Placement of SP-B59-80 in helical wheels based on standard a-

helical geometries as well as a 3 l0 and 2n helix are shown in Figure 5-1. While solution NMR









measurements indicate, and the helical wheel diagrams depicted in Figure 5-1 assume regular

periodicity to the helix, it should be noted that this may not be its exact structural adaptation in a

lipid environment. Also, an a-helical conformation leads to unfavorable placement of the

charged residues in a lipid milieu

Of particular interest to us, the KL4 Sequence originates from the primary sequence

residues 59-80 of SP-B. However, ifKL4 prOVides an essential function to artificial lung

surfactant, as the clinical studies suggest, a significant question that is raised is whether the

peptide behaves like SP-B59-80 Or SP-C. From infrared studies, SP-C was found to be a

transmembrane helix that can perfectly span the width of a DPPC bilayer and an FTIR study of

KL4 in lipids yielded similar results (5, 11, 148). However, our data described in Chapter 3 show

that KL4 is not transmembrane in DPPC:POPG and POPC:POPG bilayers, and therefore would

not orient in the same manner as SP-C.

Nonetheless, a significant question remaining is whether KL4 and SP-B59-80 act similarly,

despite clear deviations at the primary amino acid level. One key to answering this is to

determine if both peptides orient similarly in model lipid membranes with similar effects on their

dynamic properties using 2H and 31P NMR experiments. Additionally, one can compare their

effects on lipid phase transitions with DSC. The following studies were undertaken to determine

whether the biophysical activity of the native sequence closely followed that of KL4.

Materials and Methodology

Synthesis of SP-B residues 59-80: SP-B59-80, (Sequence from the N to C-terminus:

DTLLGRMLPQLVCRLVLRC SMD) was synthesized via solid-phase peptide synthesi s by Dr.

Alfred Chung at the University of Florida and cleaved from the resin with 90% TFA/5%

triisopropyl-silane/5% water and ether precipitated. The cleaved product was purified via a

HPLC with C18 Vydac column using a water/acetonitrile gradient with 0.3% TFA









(trifluoroacetic acid). The fractions corresponding to SP-B59-80 WeTO COllected and purity of the

product was verified by mass spectrometry with a mass to charge ratio of (m/z) of 2533.

DSC on 4:1 DPPC(d-62):POPG LUVs with SP-Bs9-so: DSC measurements were taken

on DPPC(d-62):POPG LUVs with varying levels of SP-B59-80. Samples were prepared by drying

peptide:1ipid complex from chloroform:methanol mixtures at a specific molar ratio, drying and

dissolution in cyclohexane. After lyophilization overnight, samples were hydrated with 5mM

HEPES buffer pH 7.4 containing 1mM EDTA and 140mM NaCl in a water bath maintained at

50oC. The samples underwent multiple freeze-thaw cycles to facilitate MLV formation and then

were extruded through 100nm filters a minimum of 15 times to generate LUVs Each sample

was run in triplicate at a temperature range of 10-70oC and a scan rate of loC/min.

Solid-state NMR sample preparation: Samples for ssNMR were prepared in an identical

manner to KL4 detailed in the Materials section of Chapter 3. Briefly, SP-B59-80 peptide was

dissolved in MeOH to a stock concentration of 1mM. DPPC(d-62), POPC(d-31), and POPG

were purchased from Avanti Polar Lipids (Alabaster, AL) in chloroform solutions and mixed to

the desired molar ratios. 4: 1 DPPC(d-62):POPG and 3:1 POPC(d-31):POPG solutions were

mixed with the peptide in MeOH solution to create a range of samples containing 0-3mol% SP-

B59-80. After mixing corresponding amounts of peptide and lipids to the desired molar

percentage, the samples were reconstituted by drying peptide/lipid mixture to a film under

compressed nitrogen gas, solubilizing them in 2mL cyclohexane and freeze-drying overnight.

The next day, approximately 20-30 mg each of dried sample was packed into a standard 5mm

NMR tube (Wilmad, Buena NJ) and hydrated with 150-200C1L 5mM HEPES buffer pH 7.4,

containing 1mM EDTA, and 140mM NaC1. NMR samples were freeze-thawed 3-5 times to









facilitate MLV formation. For samples containing DPPC:POPG, the samples were thawed in a

water bath maintained at 50oC.

The 31P NMR data were collected on a 600 MHz Bruker Avance system (Billerica, MA)

using a standard 5 mm BBO probe, and 25 k
were acquired at 3 temperatures with 1024-2048 scans for each spectrum and a 5 second recycle

delay between scans to minimize RF heating of the samples. 2H NMR data were collected on a

600 MHz Bruker Avance System (Billerica, MA) using a standard 5mm BBO probe and quad

echo sequence with a B1 field of 40 k
2048 scans and 0.5 second recycle delay. Depaking of 31P and 2H NMR spectra was performed

using a Tikhonov regularization method that takes into account macroscopic lipid alignment.

This algorithm was provided by Professor Edward Sternin (103, 124).

DSC Studies on SP-Bs9-so with DPPC:POPG LUVs

DSC thermograms for DPPC(d-62):POPG LUVs with varying amounts of SP-B59-80 aef

shown in Figure 5-2. Overall, in a concentration dependant manner, SP-B59-80 inCreaSCS C max and

AHeal for the phase transition from the Lp to the L, state in DPPC:POPG LUVs. In comparison to

Figure 3-1, where DSC thermograms performed on similar 4: 1 DPPC:POPG LUVs with varying

levels of KL4 indicate domain formation, a clear formation of 2 lipid phases on addition of

SP-B59-80 iS not seen. However, it is evident that SP-B59-80 CauSes an ordering of the lipids by the

increased amplitude of the transition seen in the DSC curves. This becomes manifest when

comparing AHeal, the integration of each DSC peak, for no peptide and with 3% peptide. AHeal is

approximately 2-3 times greater with 3% SP-B59-80 than without any peptide (Table 5-1). The

phase transition temperature is not dramatically influenced by SP-B59-80, Staying relatively

constant at 32oC.









Table 5-1 displays a thermodynamic evaluation of the effects of SP-B59-80 On the

DPPC(d-62):POPG phase transition. The most interesting and unexpected finding stemming

from these DSC studies is that, unlike KL4, SP-B59-80 does not produce phase separation in 4:1

DPPC:POPG. In a concentration dependant manner we see an increase in the enthalpy of the

DSC curves upon more addition of peptide. DSC studies on KL4, at a similar concentration

range, show a drop in enthalpy at concentrations where phase separation or domain formation

became obvious, which were at 1.5 and 2.2mol%. At 1.5mol% KL4, the DSC curve bifurcated

and was characterized by two Tm values, with one Tm value shifted towards the phase transition

of DPPC (Figure 3-1) suggesting lipid sequestration or phase separation by the peptide. No such

bifurcation or change in the appearance of the DSC curve occurred with addition of SP-B59-80 in

similar concentration ranges. The increase in enthalpies (both AHeal and AHyH) Seen as more

peptide is added, suggest SP-B59-80 Orders the lipids and stabilizes the DPPC:POPG L, state.

This is corroborated by the finding that AH>>AS indicating that the phase transition is enthalpic,

not entropic in nature. While addition of peptide seems to order the lipids and stabilize a

particular state, it has no substantial effect on cooperativity other than at 0.5% of peptide (Table

5-1). We cannot explain the sudden increase in peak-width at half height seen only at 0.5mol%

of peptide. However, the broadened lineshapes seen in the DSC curves strongly indicate that the

phase transition of the lipids in the presence of SP-B59-80 iS not a highly cooperative process.

However, with the exception of 0.5mol% peptide, SP-B59-80 Seems to decrease the cooperativity

of lipid motions and no phase separation is seen when the peptide is added to 4: 1 DPPC:POPG

LUVs. This is also verified from DSC calculations that measure indirectly the extent of

cooperativity by comparison of the Van Hoft and calorimetric enthalpies where it is seen that

their ratio decreases slightly on addition of peptide.









It is known that the addition of monounsaturated lipids markedly reduces the Tm when

added to saturated phospholipid dispersions, such as DPPC, thus aiding such mixtures to be more

fluid. This is important in lung surfactant because without such molecules, the enriched amount

of DPPC in the lungs would cause the monolayer to exist as rigid gel. In our DSC results with

SP-B59-80, We See an increase in the energy required for 4: 1 DPPC:POPG to shift to any other

state or transition. By keeping the thermodynamic barrier high for any such temperature

dependant transitions of 4: 1 DPPC:POPG it is possible to hypothesize that SP-B59-80 iS

destabilizing the fluidizing properties of the monounsaturated lipids, particularly POPG. This

would be beneficial in situations where DPPC needs to be selectively enriched, such as would

occur when the alveolar lipid monolayer needs to be compressed. However, the exact

implication of ordering of lipids by residues 59-80 of SP-B, as seen by DSC, is complex

especially with regard to the complicated lipid environment in the lung as well as in

DPPC:POPG. Still unanswered is whether the ordering seen is due to SP-B59-80 interacting with

DPPC, with POPG, or by stabilizing the mixed state of both species. Since there was no

significant shift in phase transition temperature (Tm) of the binary lipid system with SP-B59-80,

the exact role of the C-terminus of the peptide in altering lipid biophysics is not easy to interpret

based on DSC data.

31P NMR of Lipid MLVs Containing SP-Bs9-so Show POPG Interacting with the Peptide

Natural abundance phosphorous NMR for 4: 1 DPPC:POPG and 3:1 POPC:POPG MLVs

with varying levels SP-B59-80 WeTO COllected to assess the effect of this peptide on lipid phases

and headgroup dynamics in comparison to KL4 (Chapter 3). Shown in Figure 5-3 are 31P NMR

spectra for the DPPC(d-62):POPG samples. Only lamellar phases are observed and the

resonance for the POPG lipids moves with addition of peptide. Like the experiments with KL4,

macroscopic lipid alignment is seen and the extent of alignment was accounted for by the









dePaking algorithm. As with KL4, SP-B59-80 reduces this macroscopic alignment and generates

spectra that are more powder-like in nature. This is apparent by the concentration dependent

change in the intensity of the double-singularity seen in the compiled spectra (Figure 5-3).

DePaking the series of spectra (Figure 5-4) yields the parallel component of the powder spectra

for DPPC and POPG and allows a quantitative measurement of the change in 31P CSAs. Plotting

these changes relative to lipids alone reveals that addition of peptide causes a decrease in the

POPG 31P CSA by up to 10% but does not affect the DPPC 31P CSA (Figure 5-5). This is similar

to behavior seen for DPPC:POPG MLVs on addition of KL4 and argues strongly for a similar

interaction of the two peptides with the anionic POPG headgroups.

The 31P spectra for 3:1 POPC(d-31):POPG MLVs show that a lamellar phase is also

observed (Figure 5-6). Although the spectrum at 3% peptide potentially shows the onset of other

phase behavior; however, it has not been fully characterized. Examination of the dePaked 31P

spectra show that changes in the POPG peak occur upon increasing levels of peptide (Figure 5-

7). The dePaked spectra stacked together show that the PC headgroup remains largely invariant

while significant ppm shifts are seen in the PG headgroup (Figure 5-8). Interpretation of the

dePaked spectrum for the sample with 3mol% SP-B59-80 iS not straightforward and the shape of

the spectrum indicates a drastic change in headgroup dynamics at this specific concentration. As

with KL4, we did not see an inverted HII phase upon addition of peptide. However, at a higher

percentage of SP-B59-80, this might be observed. Also, similar to the non-natural peptide analog,

there seems to be a preferential electrostatic interaction with the PG headgroup. However, we

also see SP-B59-80 affecting the dynamics of POPC at higher concentrations, though the

physiological amount of SP-B believed to be in the lung is much lower (~0.2mol% relative to the

lipids) than the levels where these dynamics are seen by our 31P NMR measurements.









2H NMR Indicates that the Properties of KL4 are Similar to SP-Bs9-so.

Deuterium NMR was performed on DPPC(d-62):POPG and POPC(d-31):POPG MLVs

with varying levels of SP-B59-80 (Figure 5-9) to assess how the peptide affects the acyl chain

dynamics in the two lipid systems. If increasing order is seen, then it can be assumed that SP-

B59-80 penetrates deeply into the lipid bilayer much like the findings reported with KL4. Time

averaged deuterium order parameters , calculated after dePaking (Figure 5-10) and

assigning each C-D bond in the acyl chain, show that, just as with KL4, SP-B59-80 inCreaSes order

in acyl chains of DPPC in 4: 1 DPPC(d-62):POPG MLVs (Figure 5-11). These large scale effects

in ordering are not equal over the entirety of the acyl chain, which can be seen from the change

in order parameter profiles depicted in Figure 5-11; the largest shifts in are Seen from the

middle to the end of the acyl chains. Regions corresponding to the plateau region (carbons 2-8)

(145) have order parameter values changing on the order of 4% even at SP-B59-80 leVOIS of 2

mol%. Positions 9-15 show ordering even at 0. 1 mol% peptide. In 4: 1 DPPC(d-62):POPG

MLVs, the largest change in time averaged order parameter <6SCD> Occurs in C-D bonds at

positions 10-13 which show increases up to 10% in C-D bonds 10-12, and up to a 15% increase

for the C-D bond at position 13. This is seen in both the sn-1 and sn-2 chains of DPPC(d-62) in

the context of 4: 1 DPPC:POPG MLVs.

Similar experiments were run on 3:1 POPC(d-31):POPG MLVs containing SP-B59-80

(Figure 5-12) and dePaked (Figure 5-13) for calculation of order parameters. Looking at the

same sn-1 C-D positions 10-13 for POPC(d-31) in 3:1 POPC:POPG, we find that changes in

order parameters are negative and the ordering goes down by as much as 13-16%, with a 16%

decrease in ordering seen for position 13 (Figure 5-14). This strongly correlates to SP-B59-80

having minimal to little interaction with the acyl chains of POPC in 3:1 POPC(d-3 1):POPG

MLVs. Conversely, the peptide displays profound ordering of DPPC(d-62) in 4: 1 DPPC(d-









62):POPG MLVs. Since only the middle carbons seem to be ordered on addition of peptide

(Figure 5-11), a transmembrane orientation of SP-B59-80 iS unlikely. If SP-B59-80 were indeed

transmembrane in any of our lipid systems, we would expect ordering of the plateau carbons as

well as the middle and tail ends of acyl chains. This is what is seen for helical transmembrane

peptides including the WALP and KALP synthetic peptides (79, 127). More intriguing is the

finding that, like KL4, We See that when acyl chains of the PC lipids are deuterated, SP-B59-80

causes changes in order of the acyl regions dependent on whether the lipid is saturated or

monounsaturated.

The derived order parameters for each acyl chain C-D bond from 2H NMR spectra seem to

contradict literature 2H data studying the effects of full-length SP-B on DPPG (d-62) and

DPPC (d-62) lipids. These studies concluded that there was little effect of SP-B on the

orientational order parameters for perdeuterated acyl chains examined in the liquid crystalline

state (1 79, 180). In these papers, it was argued that SP-B perturbation was not localized at a

particular depth along the bilayer at concentrations of up to 11% by weight. However, they used

concentrated, non-physiological levels of SP-B to determine its effects and orientation in various

lipid environments. More recent findings by the same group indicate that full length SP-B can

reduce chain order in DPPC(d-62) (181) which even further contradicts our 2H data which shows

an ordering of the lipid in the presence of POPG. However, there are several plausible

explanations for these discrepancies. It should be noted that in these published works, order

parameters are not reported for each carbon along the acyl chain, which correspond to a more

discrete, quantitative measure of order derived from the dePaked spectra. Instead, a first spectral

moment (M1) is reported as a function of temperature. While the spectral moment reported is

proportional to the overall average orientational order parameter, it does not yield a thorough










analysis of the change in the order at each carbon of the acyl chain, and thus changes at

particular positions would be averaged out, or not seen, by such an analysis. It also cannot be

discounted that the sample preparation choices may have given rise to these conflicting findings.

Although Morrow, et al, and our preparations both involve reconstitution from organic solvents,

we used substantially lower concentrations of peptide in our experiments, which more closely

reflect physiologic levels and prevent aggregation of the peptide. Their findings that SP-B has

no effect on acyl chain order in DPPC and DPPG may stem from the amount of SP-B used in

these experiments. In one study, concentrations of 6-17 weight percent of full length SP-B were

used (182). At these levels, protein aggregation is highly possible, especially given the

hydrophobicity inherent within the full length protein. Higher order intermolecular complexes

such as dimers, tetramers and multimers cannot be ruled out at these concentration ranges used

for their NMR experiments. This may explain the little to no perturbation caused by the peptide

on DPPC acyl chains. The potential of full-length SP-B at these high, non-physiologic

concentrations to form higher order aggregates has not been fully addressed by the authors and

any minimal interaction seen with the acyl chain may thus reflect this. In our studies, we are

seeing dramatic effects on acyl chain order in DPPC with as little as 0.5mol% of the C-terminal

end of the peptide. Aggregation of SP-B59-80 at the levels of peptide we use is unlikely given the

concentration dependent effects on lipid ordering and disordering in this range; if aggregation

were indeed a concern we would expect minimal perturbation of these chains in a concentration

dependant manner. However, we cannot completely reconcile their findings with ours since our

studies used SP-B59-80 in binary lipid systems mimicking the lung and Morrow, et al used full

length protein SP-B in neat lipid systems. Also, we cannot rule out the regulation of activity by

the C-terminal end of SP-B by the full length protein; it also provides evidence that SP-B is









sensitive in its function to the lipids it is in complex with. Thus, our studies as well as those of

Morrow et al, and numerous others clearly underscore the vital notion that membrane protein

form and function is highly contingent on the lipid environment used to study it.

Evidence of Alternative Dynamics at High Concentrations of SP-Bs9-so?

In 3:1 POPC(d-31):POPG vesicles, the deuterium NMR order parameters decrease in a

concentration dependent manner (Figure 5-14). The downward sloping <6SCD> ValUeS SOCH in

Figure 5-14 are similar to the trends found for KL4. However, the changes seen with SP-B59-80

are larger. In conjunction with 31P NMR, these findings indicate that the SP-B59-80 iS clearly

stationed at the headgroup region of these lipids. However, at the highest concentration used in

these studies, we found a clear deviation in the typical powder spectrum in 3:1

POPC(d-31):POPG. With 3mol% SP-B59-80 COmplexed to 3:1 POPC(d-31):POPG, the 2H NMR

spectrum shows a large coalesced peak in the center of the spectrum (Figure 5-12). The

dePaked spectra for 3:1 POPC(d-31):POPG MLVs are shown in Figure 5-13, and while all

spectra were readily dePaked, the spectrum taken with 3% SP-B59-80 has less definition. The 31P

NMR spectrum for the sample with this concentration of peptide also resulted in a poor quality

dePaked spectrum that was uninterpretable (Figure 5-7). One possibility emerging from the 2H

NMR and 31P NMR data is that at 3mol% SP-B59-80, the formation of a second, non-bilayer lipid

phase is occurring. This averaging seen at this peptide concentration is due to additional fast

motions of the lipid acyl chains which could occur in a non-lamellar phase, such as an inverted

HII phase, addressed briefly in Chapter 2 or due the formation of small vesicles which tumble

quickly on the NMR timescale.

These results indicate that higher concentrations of SP-B59-80, the dynamics and motion of

the acyl chain are significantly affected. Not only is the finding shown in Figure 5-12 a possible

indicator of an inverted HII phase, but it may also implicate SP-B59-80 in lipid shuttling, lysis or









degradation. Lipid lysis and degradation has been reported to be one of the putative functions

attributed to SP-B that could be necessary for effective surfactant recycling and remediation.

Some reports implicate SP-B in anti-microbial function which could help explain and

corroborate the striking dynamics seen here, which should be of no surprise since SP-B is

classified into the saposin family of proteins (23, 30, 33, 71). However, further studies are

needed to differentiate between a role for SP-B59-80 in lipid degradation or alternative phase

formation. Surprisingly, we only saw this effect in 3:1 POPC(d-31):POPG, and not in

DPPC(d-62):POPG at 3 mol% SP-B59-80. If the above finding is indeed reproducible, it would

represent an exciting discovery of an activity for SP-B59-80 that can discriminate based on

saturation level of the acyl chain.

Comparisons of SP-Bs9-so and KL4

Even though the sequence of KL4 is based on SP-B59-80, there are some differences seen

from our data. First, the most striking difference is seen in the DSC data. In the same

concentration ranges, SP-B59-80 does not seem to influence the phase properties of

DPPC(d-62):POPG as markedly as KL4. While SP-B59-80 clearly affects the thermodynamic

properties of these LUVs, we saw clear affects of KL4 in the lipid miscibility and overall mixing

properties in DPPC(d-62):POPG LUVs and MLVs. Our data showed that during the initial

stages of lipid sequestration, KL4 lowered the Cpinax and AHeal of the peptide-lipid system, seen at

1.5 and 2.2mol% peptide in MLVs. Clear phase separation was also seen with KL4 added to

4:1 DPPC(d-62):POPG LUVs (Figure 3-1). SP-B59-80 does not cause similar behavior.

31P and 2H static NMR data show that KL4 and SP-B59-80 behave similarly in 4:1

DPPC(d-62):POPG lipid environments. 31P NMR data show that, after dePaking, very little

change occurs in DPPC parallel edge frequency, while some modest changes occur in POPG

parallel edge frequency. While an interaction of SP-B59-80 with anionic PG headgroup occurs is









seen, the placement of lysines in KL4 COuld allow for a more enhanced interaction with PG than

with SP-B59-80, which does not possess the distinct charge periodicity found in KL4 and whose

peptide sequence contains negative charges as well. The most striking changes seen on addition

of either peptide is in deuterium order parameters which indicate significant changes in the

middle to the tail end of the acyl chain corresponding to carbons 8-15. Increases in ordering of

these regions while a lack of ordering in the carbons 2-8 were seen on addition of both peptides

to DPPC:POPG MLVs. These findings discount a transmembrane orientation of either peptide

and predispose them to adapting a more peripheral orientation. In this regard, our observations

implicate KL4 and SP-B59-80 to orient similarly in DPPC(d-62):POPG lipid environments.

The 2H NMR data also show the KL4 and SP-B59-80 behave similarly in 3:1

POPC(d-31):POPG MLVs up to a concentration range of 3mol%. The substantial decrease in

time averaged order parameters indicate very little effect on the motional freedom of the POPC

sn-1 acyl chains mediated by both KL4 and SP-B59-80. In 3 mol% peptide, SP-B59-80 Seems to

facilitate additional lipid dynamics which needs to be addressed for reproducibility by 31P and 2H

NMR. If indeed the observations at 3 mol% are valid, SP-B59-80 changes the dynamics of lipid

systems based on the saturation of the PC acyl chains. This was seen with KL4 aS well, however,

the possibility of peptide-mediated change in lipid properties at high molar percentages of

SP-B59-80 iS ai novel distinguishing feature relative to KL4.

Static 31P NMR data reveal that in 4: 1 DPPC:POPG MLVs, both SP-B59-80 and KL4

interact with the PG headgroup. Both peptides displayed no predilection for an inverted HII

phase though as-of-yet uncharacterized dynamics were seen at 3 mol% SP-B59-80. In KL4, we

reconciled interaction with DPPC acyl chains and the POPG headgroup with domain formation

that stemmed from our DSC studies with the peptide. However, since we did not see any phase









separation from our DSC studies with SP-B59-80, we have no data correlating SP-B59-80 interacting

with DPPC acyl chains at the same time interacting with the PG headgroup. One way to

reconcile these findings is to conduct experiments with deuterated PG acyl chains, i.e 4:1

DPPC:POPG(d-3 1) and 3:1 POPC:POPG(d-3 1). If the deuterium order parameters for each C-D

bond increase, clearly the peptide is buried deep within the bilayer of the two lipids. If the

ValUeS decrease, then a more complicated molecular picture must be occurring whereby

the peptide somehow is buried with DPPC without facilitating de-mixing of the lipids. While

KL4 has proven to be a clinically functional and potent peptide for replacing SP-B in treating

respiratory distress and conventional lung surfactant formulations contain SP-B in full form, no

formulation currently used contains either the N or C-terminal peptide as a substitute for SP-B.

This raises the possibility that while SP-B59-80 and KL4 may have similar structural topologies in

lipid environments, KL4 may have additional functions that SP-B59-80 lacks due to the flexibility

imparted by using only leucines and lysines in the peptide.

Preliminary Molecular Model of SP-B59-80 with Lipids

With 31P NMR data looking at headgroup dynamics and 2H NMR data examining acyl

chain ordering, a low resolution molecular picture of SP-B59-80 interacting with lipid systems can

be hypothesized derived from NMR spectroscopy and DSC. It should be noted the following

model contains the important assumption that SP-B59-80 iS helical in a lipid environment. This

estimation is valid given the findings in the literature pertaining to the helical nature of SP-B

particularly at the C-terminal region; models describing this region as being helical are well

documented in the literature (87, 133, 134, 177). However, as seen with KL4, the type of helix

and the derived torsion angles may be non-canonical and deviate from those seen for an i, i 4

helix. To answer the question of the type of helix SP-B forms in a lipid environment, MAS

NMR experiments on 13C labeled peptide in complex with lipids need to be performed in









conjunction with the DQ-DRAWS pulse sequence. The laboratory of Professor Joanna Long is

currently testing various conditions for the expression of SP-B59-80 in a DNA vector for

production by E. coli using recombinant molecular biology techniques. Once optimal conditions

for expression are found, bacteria expressing the construct can be grown in isotopically enriched

13C medium for incorporation into the growing polypeptide chain for NMR experiments

With the assumption that SP-B59-80 iS helical in lipid systems, Figure 5-15 displays a

preliminary orientation of the peptide in lipid environment. It is important to realize that the

following model is theoretical and is only used to display the presumed orientation of the peptide

based on 2H and 31P NMR data. Since we saw an increase in ordering in 4: 1 DPPC:POPG, we

assume the peptide buries in DPPC acyl chains. In 3:1 POPC:POPG, the disordering in the acyl

chains and increased ordering of the headgroup region indicates that the peptide is peripheral in

this lipid system. Unlike KL4, which had its leucine content buried in a hydrophobic

environment, we predict that the acidic aspartic acid residues in SP-B59-80 pOint toward the

aqueous interface away from the phosphate headgroups. With the valid assumption of helicity

for SP-B59-80, we predict that the peptide is more peripheral in POPC:POPG than KL4.

The results of SP-B59-80 in DPPC:POPG may indicate snorkeling, which we postulated

occurs for KL4. The snorkeling hypothesis as predicted for KL4 appeafS Valid given it' s

periodicity in primary amino acid sequence. However, despite the periodic charge distribution

inherent in SP-B59-80, We CannOt account for snorkeling for the peptide unless we assume that the

long chain of aspartic acid, glutamine, and arginine extend to the interface to interact with the

phosphate headgroup. As of this moment, more experiments are needed utilizing NMR and EPR

on 13C labeled and spin labeled peptide in complex with lipid. This will yield more accurate

information in terms of orientation and depth penetration of the peptide.





















D1 RC M:l
L11
~ vrs


GSR1 T L17


thl


MMt M21



3, a rr







Figure 5-1 Putative helical wheel for each type of helix rendered for the C-terminal residues 59-
80 of SP-B.













3% SP-B
(59-80)
2% SP-B
(59-80)
1200 -( 1% SP-B
(59-80)
0.5% SP-B
(59-80)
1000 0.2% SP-B
o (59-80)
o 0. 1% SP-B
E soo (59-80)
no SP-B
ca (59-80)

> 600-


(1 400-


S 200-
"I





200
10 20 30 40 50 60

Temperature oC

Figure 5-2. DSC thermograms of 4:1 DPPC:POPG LUVs with varying molar percentages of
residues SP-B59-80












Table 5-1 Thermodynamic parameters derived from DSC on residues on 4: 1 DPPC (d-62):POPG LUVs with SP-B59-80.

Tn, A~a HVH as Cpnmax


%SP-B
(59-80)

no SP-
B(59-80)

0.1%

0.2%


(cal/mol/K) AT1 2 CU* kcal/mol/oC


(oC) (kcal/mol) (energy/mol)


32.1 &
0.4
32.6 &
0.3
32.8 &
0.3


32.44
+ 0.03
32.0 &
0.2
32.32
+ 0.06
32.44
+ 0.03


2.50 + 0.04

5.5 A 1.2

5.9 11.3



7.1 10.3

6.71 0.2

7.5 & 0.6


7.01 & 0.07


102 & 6

98 & 13

106 & 6



84 & 2.6

117 & 3

123 & 26


137 & 29


8.11 0.1

17.9 & 3.9

19.2 & 4.4



23.2 & 1.1

21.6 & 0.7

24.4 & 2.1


6.7 & 0.4

6.6 & 1.4

6.3 & 0.7



8.6 & 0.3

5.8 & 0.1

6.1 & 0.1


39 &2

18 &6

19 &4



12 & 1

18 &1

17 & 5


0.34 & 0.02

0.70 + 0.06

0.8 & 0.2



0.80 + 0.02

1.042 & 0.008

1.2 & 0.2


1.3 & 0.3


0.5%

1%

2%


3%


23.0 + 0.2 6.0 + 0.3 20 & 4












-4:1 DPPC:POPG
-0.1% SP-B(59-80)
- 0.2% SP-B(59-80)
-0.5% SP-B(59-80)
-1% SP-B(59-80)
-2% SP-B(59-80)
- 3% SP-B(59-80)


-50 -40 -30 -20 -10 0 10 20 30 40

Frequency (ppm)

Figure 5-3. 3 1P NMR data of 4: 1 DPPC:POPG MLVs with varying molar percentages of
SP-B59-80












-4: 1 DPPC: POPG
-0. 1% SP-B(59-80)
-0.2% SP-B(59-80)
-0.5% SP-B(59-80)
-1% SP-B(59-80)
-2% SP-B(59-80)
-3% SP-B(59-80)


-40 -30 -20 -10 0


Frequency (ppm)


Figure 5-4. DePaked 31P NMR data of 4: 1 DPPC:POPG MLVs with varying molar percentages
of SP-B59-80













12.0%

10.0%


S8.0%

c + DPPC
*6.0%
=POPG

ca 4.0%


2.0%

0.0% iI ,,*
00.5 1 1.5 2 2.5 3 3.5
-2.0%

mol% SP-Bse-so



Figure 5-5. Change in 31P CSAs for 4:1 DPPC:POPG MLVs as a result of increasing molar
percentages of SP-B59-80











-3: 1 POPC:POPG
-0. 1%o SP-B(59-80)
-0.5% SP-B(59-80)
-1% SP-B(59-80)
-2% SP-B(59-80)
-3% SP-B(59-80)


-50 -40 -30 -20 -10 0 10 20 30 40

Frequency (ppmn)


Figure 5-6. Phosphorous NMR data of 3:1 POPC:POPG MLVs with varying molar percentages
of SP-B59-80













-3:1 POPC:POPG
-0.1%~ SP-B(59-80)
-0.5% SP-B(59-80)
-1%~ SP-B(59-80)
-2% SP-B(59-80)
-3% SP-B(59-80)


-50 -40 -30 -20 -10 0 10 20

Frequency (ppm)


Figure 5-7. DePaked 31PNMR data of 3:1 POPC:POPG MLVs with varying molar percentages
of SP-B59-80













12.0%-

10.0%-

E 8.0%-

.c 6.% ** POPC

4.0% mPOPG
o + +
S 2.0%-

0.0%
0 0.5 1 1.5 2 2.5
-2.0%-

mol% SP-Bas-so


Figure 5-8. Change in 31P CSAs for 3:1 POPC:POPG MLVs as a result of increasing molar
percentages of SP-B59-80














-4:1 DPPC:POPG
-0.1%~ SP-B(59-80)
-0.2% SP-B(59-80)
-0.5% SP-B(59-80)
-1%o SP-B(59-80)
-2% SP-B(59-80)
-3% SP-B(59-80)


-30000 -20000 -10000 0 10000 20000 30000

Frequency (Hz)


Figure 5-9. Deuterium NMR spectra of 4:1 DPPC(d-62):POPG MLVs with SP-B59-80.















DP PC(d-62): POPG
0.1% SP-B(59-80)
0.2% SPB(59-80)
0o. 5% SPB(59-80)
-1% SPB(59-80)
-2% SPB(59-80)
3% SPB(59-80)









-40 -30 -20 -10 0 10 20 30 40

Frequency (Hz)

Figure 5-10. Stacked dePaked spectra of 4:1 DPPC(d-62):POPG MLVs with SP-B59-80.












12%


ao H0 carbon 3
S8%- H carbon 8
.5 / carbon 10
S6%
I /// ~ carbon 12
4%1 H carbon 15
2%
carbon 15
0% carbon 10
0 0.1 0.2 0.5 1 2 3 carbon 3

mol% SP-B59-80

12%

n_10%1 H carbon 3
8% carbon 8
I ~ H carbon 10
6%
c~ H carbon 12
S4%- H carbon 15
S2%
carbon 15
0% carbon 10
0 0.1 0.2 0.5 1 2 3 carbon 3


mol% SP-B59-80



Figure 5-11. Percent change in time averaged order parameter <6SCD> for DPPC (d-62) in 4: 1
DPPC(d-62):POPG MLVs on addition of SP-B59-80. Top: change shown in the sn-1 and Bottom:
the sn-2 chain.














-3:1 POPC:POPG
-0.1%~ SP-B(59-80)
-0.5% SP-B(59-80)
-1% SP-B(59-80)
-2% SP-B(59-80)
-3% SP-B(59-80)














-30000 -20000 -10000 0 10000 20000 30000
Frequency(Hz)




Figure 5-12. 2H NMR spectra of 3:1 POPC(d-3 1):POPG MLVs with SP-B59-80.














POPC(d-31): POPG
0.1% SP-B(59-80)
--- 0. 5% SP-B(59-80)
-1% SP-B(59-80)
--- 2% SP-B(59-80)
-3% SP-B(59-80)












-40 -30 -20 -10 0 10 20 30 40

Frequency (Hz)



Figure 5-13. Stacked dePaked spectra of 3:1 POPC(d-31):POPG MLVs with SP-B59-80












0%

Carbon 3
^n, a carbon 8
<4 aA carbon 10
\ carbon 12
S -5% cabn1

carbon 15
cabo 1

) carbon 10

-1 0% carbon 8
0 0.1 carbon 3
0.5

mol% SP-B59-80


Figure 5-14. Percent change in time averaged order parameter for POPC (d-31) in 3:1
POPC(d-31):POPG MLVs on addition of SP-B59-80. Shown is the change in the
sn-1 chain.
























DPPC:POPG POPC:POPG


Figure 5-15. Hypothesized orientational model of SP-B59-80 in two different lipid MLV systems.
SP-B59-80 iS assumed helical based on previous literature findings and is shown as a
yellow ribbon. The following models are preliminary and are based on 2H and 31P
solid state NMR spectroscopy results.









CHAPTER 6
CONCLUSIONS AND FUTURE EXPERIMENTS

Our data argue that KL4 has a different binding orientation in 3:1 POPC:POPG and

4:1 DPPC:POPG lipid systems. Deuterium NMR is a powerful tool to study the dynamics of the

lipid acyl chains based on the quadrupole splitting and the orientational order parameters

calculated therefrom. From these data, it is clear that KL4 has a perturbing effect on

DPPC(d-62):POPG. The addition of KL4 inCreaSes the ordering of the acyl chains near the

middle and the tail ends indicating that it is these regions of the fatty acyl chains undergoing the

most interaction with KL4. KL4 is modeled after the last 21 amino acids of SP-B, particularly

residues 59-80, in terms of hydrophilic and hydrophobic distribution of residues. Preliminary

measurements done in our lab on SP-B59-80 have shown that in DPPC(d-62):POPG, a similar

increase in order parameters is seen upon addition of peptide. It is believed that both KL4 and

SP-B59-80 l0dge similarly in DPPC(d-62):POPG lipids. When the POPG sn-1 acyl chain is

deuterated, we find that KL4 decreases the order parameters for both DPPC:POPG and

POPC:POPG lipid systems, arguing for very little interaction with the PG acyl chains. In

conjunction with the deuterated DPPC data, a possible scenario can be envisioned of phase

separation, with KL4 "Snorkeling" with the DPPC acyl chains, and interacting with the phosphate

headgroups of POPG. The DSC data strongly argue for the case of lipid phase separation at

concentration ranges of 1.5 mol%---such a concentration reported here is dependent on DSC

sample preparation.

While the orientation of these peptides may be the same, the question then becomes, what

is that orientation? For KL4, a transmembrane orientation has been proposed based on FT-IR

data, but the preparation of the sample warrants some doubt. In those experiments, KL4 WAS

dried with 7:3 DPPC:DPPG bilayers in non-physiological amounts. The transmembrane









orientation hypothesis contradicts many previous reports that postulated that the evenly spaced

charged lysine residues can ionically interact with the phosphate headgroup, thus imparting a

more peripheral orientation to the peptide. A second study using IR absorption spectroscopy

showed the peptide adapting an anti-parallel beta sheet structure in DPPC/DPPG bilayers while

being alpha-helical in DPPC. That work did predict KL4 to be neither transmembrane nor

helical. In light of these findings, our data show KL4 being helical by circular dichroism, at

concentration ranges that negate the effect of potential aggregation. Solid state NMR

experiments taken in conjunction with Professor Joanna Long and Dr. Doug Elliott on 13 ,

labeled KL4 WeTO perfOrmed in POPC:POPG and DPPC:POPG lipids. 13 g_13C' distances and

torsion angle measurements show KL4 to be helical albeit in a non-canonical form as mentioned

in Chapter 4. Using DQ-DRAWS, the peptide displayed torsion angles ((q=-105, uy=-26) that

were helical but had the lysines partially aligned along one side of the helix. Similar experiments

of 13C' labeled KL4 in DPPC:POPG show that the peptide follows a more xn-helical fold with the

lysines fully aligned along one face of the helix. In our final model depicted in Chapter 3 and in

the submitted manuscript, we model KL4 aS a helix in both DPPC:POPG and POPC:POPG lipids

based on the input of the torsion angles generated into the molecular graphics program PYMOL.

In light of the data borne out from DSC, and solid-state NMR experiments, it seems that the

preferred orientation of KL4 in 4: 1 DPPC:POPG bilayers is to be buried within the bilayer

beneath the headgroups. This represents a unique binding orientation of the peptide in lipid

compositions modeled after the lung. In model 3:1 POPC:POPG MLVs, we can state with some

degree of confidence that KL4 is bound peripherally to this lipid system at a shallower depth.

31P solid state NMR on static peptide-lipid samples show the peptide decreasing

spontaneous macroscopic orientation of the lipids, giving rise to more spherical shapes of the









MLVs. This was found in both lipid systems. As an unintended byproduct of samples run at

14. 1Tesla field strength, lipid samples exhibited spontaneous alignment, leading to a loss of the

parallel edge of the powder pattern. At a macroscopic level, this can be depicted as normal

spherical liposomes being deformed into ellipsoidal shaped vesicles. As part of Professor

Edward Sternin's dePaking algorithm, a parameter that reports the extent of orientation caused

by magnetic field alignment is reported. Adding KL4 primarily decreases this parameter

indicating the peptide affects the macroscopic ordering of lipids and converting ellipsoidal

deformed liposomes into spherical ones. Conversion of an ellipsoidal form of liposomes, as

mediated by KL4, to spherical ones may help in potentiating surface tension but this hypothesis

remains to be tested.

The interesting aspect of this work is what KL4 is doing in the lung. The snorkeling

hypothesis presented shows two seemingly conflicting points arising from the NMR data--

interaction with POPG headgroup and interaction with DPPC acyl chains at the interfacial region

of the tail. One way to reconcile these two results stemming from the 31P and 2H NMR data is

phase separation or lipid sequestration. This is seen in the DSC data on DPPC:POPG vesicles,

regardless of the method of sample preparation. With phase separation, we envisage a scenario

where one population of the peptide can sequester DPPC into a microdomain (of size which

cannot be determined from these experiments) and at the same time a second population of the

peptide can be interacting with the phosphates of POPG in a POPG-rich domain.

Similar 31P and 2H studies were also performed on SP-B59-80, the native sequence after

which KL4 is modeled, to see if the two peptides orient similarly. Our data show that SP-B59-80

presence increases acyl chain ordering in DPPC(d-62):POPG while decreasing it in POPC(d-

31):POPG. Similar to KL4, the ordering or disordering of the acyl chains is dependant on the









saturation status of the phospholipids. 31P NMR data also show SP-B59-80 interacting with PG

headgroups as was seen for KL4. Despite these similarities seen from ssNMR spectroscopy,

DSC measurements at increasing levels of SP-B59-80 Show no indication of phase separation.

This finding implies that the way the two peptides partition between the lipids is different.

Hence, SP-B59-80 Seems to orient similarly to KL4 but conveys a different biophysical function to

the lipid systems studied in this dissertation.

It was hoped that an inverted HII phase would be seen in our data when KL4 WAS added to

both our lipid systems. Such a phase can clearly be characterized by 31P NMR and a lipid

polymorphism of that arrangement was believed to be important for lung surfactant functioning.

Though we did not see such a lipid geometry in both our lipid systems, we believe that

snorkeling of KL4 in DPPC:POPG can induce a curvature strain on the lipids that can make these

lipids more prone to alternative geometries (like an inverted HII phase). However, because we

did not see an inverted HII phase, we can only speculate that this could be one advantage of

lysine snorkeling. Of interesting note, our findings show that at 3mol% SP-B59-80 a pOtential

alternative lipid phase was seen in 3:1 POPC(d-31):POPG lipids but not in 4: 1

DPPC(d-62):POPG lipids. This discovery needs to be further investigated to see if indeed the

peptide facilitates a concentration dependant change in the geometry that is sensitive to the level

of saturation of the lipid chain.

The spectroscopy and calorimetry experiments delineated above were performed on simple

binary lipid systems of defined molar ratio. Obviously, lung surfactant is much more

heterogeneous than just DPPC and POPG, and a whole host of other minor constituents exist

which even include di- and tri-unsaturated fatty acids. Future experiments should involve

addition of more layers of complexity to lung surfactant that typify what is found endogenously.









Such experiments have been performed on more native bovine lung surfactant extracts which

have been spiked with DPPC(d-62) to measure effect of albumin on phase separation and lipid

dynamics (183). The studies presented here can also be performed with the addition of palmitic

acid to our DPPC:POPG lipid mix to more closely simulate the clinical version marketed by

Discovery Labs and Tanaka' s original work (18). Finally, the studies presented here can be

extended to other peptides systems such as RL4 (lySines replaced with arginine residues),

SP-B1-25, and to Mini-B, a surface active fusion peptide of SP-B1-25 and SP-B59-80 (184). By such

work, it can be clarified whether peptide mimics based upon SP-B act via a common structural

intermediate and interact with lipids in a similar manner. It could also be possible that these

peptide mimics can have a substantially different effect of lipid thermodynamics and lipid NMR

time-scale dynamics. If so, it would imply more than one mechanism for simple peptides to

achieve surface tension lowering capabilities in the lung. Also, it would be interesting to note if

the helical secondary structure as reported here for KL4 tin Solution and by solid-state) is

essentially the overriding secondary structure needed for proper lung surfactant function. It

would be interesting to see whether even more simple peptides that follow the alpha-helical fold

can be rationally designed for the treatment of RDS. These short, small molecular weight

peptides may also be of aid in solving other problems involving interfacial biology or surface

tension mimimization.











APPENDIX A
CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS 4:1 DPPC(D-62):POPG WITH KL4 (MOLAR PERCENTAGES)

Table A-1 Order Parameters for sn-1 and sn-2 chain of DPPC(d-62)
sn-1 chain Order Parameter
no
Carbon number KL4 0.09% KL4 0.19% KL4 0.38%KL4 0.76% KL4 1.5% KL4 2.3% KL4
2 0.21 0.22 0.22 0.22 0.22 0.22 0.21
3 0.21 0.21 0.21 0.21 0.21 0.21 0.21
4 0.21 0.22 0.22 0.22 0.22 0.22 0.21
5 0.21 0.21 0.21 0.21 0.21 0.21 0.21
6 0.21 0.21 0.21 0.21 0.21 0.21 0.21
7 0.21 0.21 0.21 0.21 0.21 0.21 0.21
8 0.21 0.21 0.21 0.21 0.21 0.21 0.21
9 0.19 0.19 0.19 0.19 0.19 0.20 0.20
10 0.18 0.18 0.18 0.18 0.19 0.19 0.19
11 0.16 0.16 0.17 0.17 0.17 0.18 0.18
12 0.15 0.15 0.15 0.15 0.15 0.16 0.16
13 0.13 0.13 0.13 0.13 0.13 0.14 0.14
14 0.11 0.11 0.11 0.11 0.11 0.12 0.12
15 0.08 0.08 0.08 0.08 0.08 0.09 0.09
16 0.02 0.02 0.02 0.02 0.02 0.03 0.03
sn -2 chain
no 0.38%
Carbon number KL4 0.09% KL4 0.19% KL4 KL4 0.76% KL4 1.5% KL4 2.3% KL4
2 0.21 0.22 0.22 0.22 0.22 0.22 0.21
3 0.21 0.21 0.21 0.21 0.21 0.21 0.21
4 0.21 0.22 0.22 0.22 0.22 0.22 0.21
5 0.21 0.21 0.21 0.21 0.21 0.21 0.21
6 0.21 0.21 0.21 0.21 0.21 0.21 0.21
7 0.21 0.21 0.21 0.21 0.21 0.21 0.21
8 0.21 0.21 0.21 0.21 0.21 0.21 0.21
9 0.19 0.19 0.19 0.19 0.19 0.20 0.20
10 0.19 0.19 0.19 0.19 0.19 0.20 0.20
11 0.18 0.18 0.18 0.18 0.19 0.19 0.19
12 0.16 0.16 0.17 0.17 0.17 0.18 0.18
13 0.15 0.15 0.15 0.15 0.15 0.16 0.16
14 0.12 0.12 0.12 0.12 0.12 0.13 0.13
15 0.09 0.10 0.10 0.10 0.10 0.10 0.10
16 0.02 0.02 0.02 0.02 0.02 0.03 0.03











APPENDIX B
CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS 4:1 DPPC:POPG(D-31) WITH KL4 (MOLAR PERCENTAGES)

Table B-1 Order Parameters for deuterated sn-1 chain of POPG
palmitoyl chain Order Parameter
No 3.0%
carbon KL4 1.0% KL4 KL4
2 0.20 0.19 0.18
3 0.20 0.19 0.18
4 0.20 0.19 0.18
5 0.20 0.19 0.18
6 0.20 0.19 0.18
7 0.20 0.19 0.18
8 0.20 0.19 0.18
9 0.18 0.18 0.17
10 0.17 0.17 0.17
11 0.16 0.16 0.16
12 0.14 0.15 0.14
13 0.12 0.13 0.13
14 0.10 0.11 0.11
15 0.08 0.08 0.08
16 0.02 0.02 0.02
0.16 0.16 0.15











APPENDIX C
CALCULATED 2H ORDER PARAMETERS (TO TWO SIGNIFICANT FIGURES) FOR
DEUTERATED LIPID S 3:1 POPC(D-3 1):POPG WITH KL4 (MOLAR PERCENTAGES)

Table C-1 Order Parameters for deuterated sn-1 chain of POPC


palmitoyl chain
Carbon number
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16


Order Parameter
no KL4 0.09% KL4
0.20 0.19
0.20 0.19
0.20 0.19
0.20 0.19
0.20 0.19
0.20 0.19
0.20 0.19
0.17 0.17
0.16 0.16
0.14 0.14
0.13 0.12
0.11 0.11
0.09 0.09
0.07 0.07
0.02 0.02


0.19% KL4
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.17
0.16
0.14
0.12
0.11
0.09
0.06
0.02


0.38% KL4
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.17
0.16
0.14
0.12
0.11
0.09
0.06
0.02


0.76% KL4
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.17
0.16
0.14
0.12
0.10
0.09
0.06
0.02


1.5% KL4
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.17
0.16
0.14
0.12
0.11
0.09
0.07
0.02


2.3% KL4
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.17
0.16
0.14
0.12
0.11
0.09
0.07
0.02











APPENDIX D
CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS INT 3:1 POPC:POPG(D-31) WITH KL4 (MOLAR PERCENTAGES)



Table D-1 Order Parameters for deuterated sn-1 chain of POPG


palmitoyl chain

Carbon
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16


Order Parameter
no
KL4 0.2% KL4
0.19 0.18
0.19 0.18
0.19 0.18
0.19 0.18
0.19 0.18
0.19 0.18
0.18 0.18
0.17 0.16
0.15 0.15
0.14 0.13
0.12 0.12
0.10 0.10
0.08 0.08
0.06 0.06
0.02 0.02


1.0%
KL4
0.18
0.18
0.18
0.18
0.18
0.18
0.18
0.16
0.15
0.13
0.12
0.10
0.08
0.07
0.02


3.0%
KL4
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.15
0.14
0.12
0.11
0.09
0.08
0.06
0.01











APPENDIX E
CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS IN 4:1 DPPC(D-62):POPG WITH SP-B(59-80) (MOLAR
PERCENTAGES)

Table E-1 Order parameters for sn-1 and sn-2 chain of DPPC with SP-B(59-80)
Order Parameter
sn-1 chain
carbon no SP-B 0.1% SP-B 0.2% SP-B 0.5% SP-B 1.0 SP-B 2.0% SP-B 3.0% SP-B
2 0.22 0.22 0.22 0.22 0.23 0.23 0.23
3 0.21 0.21 0.21 0.21 0.22 0.22 0.22
4 0.22 0.22 0.22 0.22 0.23 0.23 0.23
5 0.21 0.21 0.21 0.21 0.22 0.22 0.22
6 0.21 0.21 0.21 0.21 0.22 0.22 0.22
7 0.21 0.21 0.21 0.21 0.22 0.22 0.22
8 0.21 0.21 0.21 0.21 0.22 0.22 0.22
9 0.19 0.19 0.19 0.20 0.20 0.20 0.20
10 0.18 0.18 0.18 0.19 0.19 0.20 0.19
11 0.16 0.16 0.16 0.17 0.18 0.18 0.18
12 0.15 0.15 0.15 0.16 0.16 0.16 0.16
13 0.13 0.13 0.13 0.14 0.14 0.14 0.14
14 0.11 0.11 0.11 0.12 0.12 0.12 0.12
15 0.08 0.08 0.08 0.09 0.09 0.09 0.09
16 0.02 0.02 0.02 0.02 0.03 0.03 0.03
sn-2 chain
carbon no SP-B 0.1% SP-B 0.2% SP-B 0.5% SP-B 1.0 SP-B 2.0% SP-B 3.0% SP-B
2 0.22 0.22 0.22 0.22 0.23 0.23 0.23
3 0.21 0.21 0.21 0.21 0.22 0.22 0.22
4 0.22 0.22 0.22 0.22 0.23 0.23 0.23
5 0.21 0.21 0.21 0.21 0.22 0.22 0.22
6 0.21 0.21 0.21 0.21 0.22 0.22 0.22
7 0.21 0.21 0.21 0.21 0.22 0.22 0.22
8 0.21 0.21 0.21 0.21 0.22 0.22 0.22
9 0.19 0.19 0.19 0.20 0.20 0.20 0.20
10 0.19 0.19 0.19 0.20 0.20 0.20 0.20
11 0.18 0.18 0.18 0.19 0.19 0.20 0.19
12 0.16 0.16 0.16 0.17 0.18 0.18 0.18
13 0.15 0.15 0.15 0.16 0.16 0.16 0.16
14 0.12 0.12 0.12 0.13 0.13 0.13 0.13
15 0.10 0.10 0.10 0.10 0.10 0.10 0.10
16 0.02 0.02 0.02 0.02 0.03 0.03 0.03













APPENDIX F
CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR
DEUTERATED LIPIDS INT 3:1 POPC(D-31):POPG WITH SP-B(59-80) (MOLAR
PERCENTAGES)


Table F-1 Order Parameters for deuterated sn-1 chain of POPC with SP-B(59-80)
Order Parameter


3.0% SP-B(59-
80)
0.18
0.18
0.18
0.18
0.18
0.18
0.18
0.15
0.13
0.13
0.11
0.10
0.08
0.06
0.02


Carbon
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16


no SP-B(59-80)
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.16
0.15
0.13
0.12
0.10
0.08
0.06
0.02


0.1%~ SP-B(59-80)
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.16
0.15
0.13
0.12
0.10
0.08
0.06
0.02


0.5% SP-B(59-80)
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.16
0.15
0.13
0.12
0.10
0.08
0.06
0.02


1.0%~ SP-B(59-80)
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.17
0.15
0.13
0.12
0.10
0.08
0.06
0.02


2.0% SP-B(59-80)
0.19
0.19
0.19
0.19
0.19
0.19
0.19
0.15
0.14
0.13
0.11
0.10
0.08
0.06
0.02










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204









BIOGRAPHICAL SKETCH

Vijay C. Antharam got his Bachelor of Science degree in microbiology and cell science at

the University of Florida, where he graduated in 3 years. He joined the PhD program in

biomedical sciences at the University of Florida in the Fall of 2002 and j oined the lab of Joanna

R.Long in 2003. His interests include biophysics, structural biology, medicine, and playing

chess.


205





PAGE 1

1 BIOPHYSICAL CHARACTERIZATION OF PEPTIDE MIMICS OF LUNG SURFACTANT PROTEIN-B By VIJAY C. ANTHARAM A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Vijay C. Antharam

PAGE 3

3 To my Mom.

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4 ACKNOWLEDGMENTS I thank my m entor Dr. Joanna Long for providing me the opportunity for working in her laboratory. I thank her for her support, patienc e, sense of humor, and kindness. I hope to acquire some of those qualities th at she has as I continue to ma ture and develop. I would also like to offer my gratitude to members of my committee: Dr. Art hur Edison, Dr. Robert McKenna, Dr. Susan Frost and Dr. Ron Castellano. I appreciate th eir support and their help. I am particularly grateful for Dr. Susan Frost for giving me some extra support with regards to my presentation and for her willingness to afford so me of her time to look over my dissertation. I want to extend my heartfelt gratitude and si ncere thanks to members of the laboratory throughout these years. These include Dr. Frank Mills, Dr. Mini-Samuel Landtiser, Dr. Doug Elliott, Seth McNeill, Suzanne Farver, David Fleishmann, Julie Vanni, Joanne Anderson, and Jonathan Lane. I also want to thank some ve ry special people I have met: Sonny Flores, Nelson Klahr, Saurav Chandra, Karen vonDeneen and Mariam Rahmani. I am grateful and fortunate to have known Sonny, Nelson, Jonathan, and especially Ma riam--all of them are very special to me. Lastly, I thank my family members, mainly my mother Urmila Antharam. I feel very blessed and grateful for all that they have done for me.

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5 TABLE OF CONTENTS page 0ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 LIST OF ABBREVIATIONS........................................................................................................ 14 ABSTRACT...................................................................................................................................17 CHAPTER 1 3DISCOVERY AND HISTORY OF LUNG SURFACTANT................................................ 19 1The Alveoli and Surface Tension........................................................................................... 20 1Chemical Composition and Origin of Lung Surfactant..........................................................22 5Cellular Biology of Lung Surfactant...............................................................................24 5Important Surface-Acting Proteins of Lung Surfactant...................................................25 1Lung Surfactant Protein B (SP-B) and RDS........................................................................... 27 2Mammalian Lung Surfactant Cycle........................................................................................ 31 2Exogenous and Artificial Lung Surfactants............................................................................31 2 4BIOPHYSICAL TECHNIQUES TO PROBE PEPTIDE STRUCTURE AND PEPTIDE-LIPID INTERACTIONS....................................................................................... 45 2Circular Dichroism.................................................................................................................45 2Differential Scanning Calorimetry (DSC).............................................................................. 47 2Solid state NMR Spectroscopy............................................................................................... 50 2Spin Interactions Commonly Seen in ssNMR........................................................................50 5Chemical shift..................................................................................................................50 5Dipole-dipole couplings..................................................................................................51 5Quadrupole couplings...................................................................................................... 52 2Applications and Methodologies in Solid-State NMR........................................................... 52 5Pake Powder Pattern........................................................................................................52 5Chemical Shift Anisotropy (CSA)...................................................................................53 5Magic Angle Spinning NMR........................................................................................... 54 5Range of NMR Time Scales............................................................................................55 2NMR-Active Nuclei and Importance to Biological Molecules.............................................. 57 631P (Phosphorus) NMR....................................................................................................57 62H (Deuterium) NMR...................................................................................................... 60 2DePaking.................................................................................................................................62 2Magnetic Field Orientation.....................................................................................................63

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6 3 5SURFACTANT PEPTIDE KL4 DIFFERENTIALLY MODULATES LIPID COOPERATIVITY AND ORDER IN DP PC: POPG AND POPC: POPG LIPID VESICLES....................................................................................................................... .......80 3Relevance of KL4 to Lung Surfactant Biology....................................................................... 81 3Methodology to Study KL4 with Lipids.................................................................................83 3KL4 Affects Lipid Phase Behavior......................................................................................... 85 331P NMR: Addition of KL4 Leads to Changes in Orient ation of the PG Headgroups............ 87 3KL4 Effects on Lipid Acyl Chain Ordering De pendent on the Saturation of the Acyl Chains..................................................................................................................................88 3KL4 in Relation to other Peptides of Similar Size, Composition and Length........................ 91 3KL4 Shares Many Properties with Cholestero l and Transmembrane Helices in DPPC......... 94 3Molecular Model of KL4 with POPC:POPG and DPPC:POPG............................................. 94 4 6STRUCTURAL STUDIES OF KL4.....................................................................................125 3Characterization of KL4 Secondary Structure....................................................................... 125 3Materials and Methodology..................................................................................................127 4CD shows KL4 to be helical..................................................................................................129 4Different Types of Helices KL4 May Adapt in a Lipid Bilayer........................................... 133 4Solid-State NMR Studies of KL4 in POPC:POPG and DPPC:POPG.................................. 134 5 7COMPARATIVE BIOPHYSICAL STUDIES OF SP-B59-80...............................................152 4Fragments of SP-B have biophysical activity....................................................................... 152 4Materials and Methodology..................................................................................................154 4DSC Studies on SP-B59-80 with DPPC:POPG LUVs............................................................ 156 431P NMR of Lipid MLVs Containing SP-B59-80 Show POPG Interacti ng with the Peptide. 158 42H NMR Indicates that the Properties of KL4 are Similar to SP-B59-80................................160 4Evidence of Alternative Dynamics at High Concentrations of SP-B59-80?...........................163 4Comparisons of SP-B59-80 and KL4.......................................................................................164 5Preliminary Molecular Model of SP-B59-80 with Lipids.................................................... 166 6 8CONCLUSIONS AND FUTURE EXPERIMENTS............................................................ 184 APPENDIX A 9CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS 4:1 DPPC(D-62):POPG WITH KL4 (MOLAR PERCENTAGES)................................................................................................................. 189 B 1CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNFICANT FIGURES) FOR DEUTERATED LIPIDS 4:1 DPPC:POPG(D-31) WITH KL4 (MOLAR PERCENTAGES)................................................................................................................. 190

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7 C 1CALCULATED 2H ORDER PARAMETERS (TO TWO SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS 3:1 POPC(D-31):POPG WITH KL4 (MOLAR PERCENTAGES)................................................................................................................. 191 D 1CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS IN 3: 1 POPC:POPG(d-31) WITH KL4 (MOLAR PERCENTAGES)................................................................................................................. 192 E 1CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS IN 4:1 D PPC(d-62):POPG WITH SP-B(59-80) (MOLAR PERCENTAGES)................................................................................................................. 193 F 1CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS IN 3:1 POPC(D-31):POPG WITH SP-B(59-80) (MOLAR PERCENTAGES)................................................................................................................. 194 1LIST OF REFERENCES.............................................................................................................195 1BIOGRAPHICAL SKETCH.......................................................................................................205

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8 LIST OF TABLES Table page 1-1 Approximate weight percentages of lipids in ma mmalian lung surfactant........................ 39 1-2 Table of artificial lung su rfactants used clinically............................................................. 43 3-1 Thermodynamic parameters obt ained from DSC therm ograms........................................ 99 3-2 CSA span for phosphate headgroup in 3:1 POPC:POPG and 4:1 DPPC:POPG MLVs with and without KL4.......................................................................................................100 4-1 Ellipticity (in mdeg) of KL4 at 222nm and 208nm and ratio of helical signatures 222nm and 208nm............................................................................................................ 145 4-2 Ellipticity (in mdeg) of 40M KL4 reconstituted in LUVs from organic solvent........... 146 5-1 Thermodynamic parameters derived from DSC on residues on 4:1 DPPC (d62):POPG L UVs with SP-B59-80......................................................................................170 A-1 Order Parameters for sn-1 and sn-2 chain of DPPC(d-62).............................................. 189 B-1 Order Parameters for deut erated sn-1 chain of POPG..................................................... 190 C-1 Order Parameters for deut erated sn-1 chain of POPC..................................................... 191 D-1 Order Parameters for deut erated sn-1 chain of POPG..................................................... 192 E-1 Order parameters for sn-1 and sn-2 chain of DPPC with SP-B(59-80)...........................193 F-1 Order Parameters for deuterated sn-1 chain of POPC with SP-B(59-80)........................ 194

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9 LIST OF FIGURES Figure page 1-1 Cartoon illustration of surface tension............................................................................... 37 1-2 The alveolar environment.................................................................................................. 38 1-3 Hypothesized interactions of SP-B and SP-C with lipid lame llae..................................... 40 1-4 Histology of SP-B mutation............................................................................................... 41 1-5 Mammalian lung surfactant homeostasis........................................................................... 42 1-6 Primary amino acid sequence of SP-B and KL4................................................................44 2-1 CD spectra from various secondary structure elements..................................................... 64 2-2 Schematic of a differentia l scanning calorimeter (DSC). .................................................. 65 2-3 The gel to liquid-crystalline phase transition of phospholipids bilayers........................... 66 2-4 A) DSC thermogram of 2mM DPPC larg e unilame llar vesicles (LUVs) dispensed in 5mM HEPES pH 7.4.......................................................................................................... 67 2-5 Time scale of motional processes for nuclear spins in NMR............................................ 68 2-6 31P NMR static spectrum of DPPC hydrated vesicles (approximately 60mg). The spectrum was taken at 44 degrees on a 600M Hz Bruker instrument with 1024 scans...... 69 2-7 31P NMR of static hydrated DPPC vesicles (approximately 60mg) taken on a 600 MHz Bruker instrument (1024 scans) at 240C, well below its phase transition temperature,................................................................................................................... ....70 2-8 Graphical depiction of shielding tensor for a nucleus with an axis of symmetry. ............. 71 2-9 Phosphorous NMR lineshape patterns for lipid mesophases............................................. 72 2-10 Magic angle spinning (MAS) spectra of the 13C nucleus in glycine................................ 73 2-11 DRAWS pulse sequence of DRAWS employed during MAS for dipolar recoupling...... 74 2-12 Collective motions of each methylene posit ion along the acyl chain roughly averages out to a conicall shape........................................................................................................ 75 2-13 Deuterium spectra of (4:1) DPPC:P OPG(d-31) (mol/mol) with POPG ful ly deuterated on the sn-1 acyl chain...................................................................................... 76

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10 2-14 Example of order parameter profile generated from dePaking 2H NMR spectra, for perdeuterated acyl chains...................................................................................................77 2-15 Example of 31P NMR (blue) and the dePaked result (red).................................................78 2-16 Lipid vesicles when placed in a magnetic field can deform to for m an ellipsoidal shape..................................................................................................................................79 3-1 Differential scanning calorimetry on KL4 with 4:1 DPPC:POPG vesicles with KL4 at the indicated molar percentages......................................................................................... 98 3-2 Static 31P NMR spectra of 3:1 POPC(d-31) :POPG MLVs with the increasing addition of KL4................................................................................................................101 3-3 Static 31P dePaked NMR spectra of 3:1 POPC(d-31):POPG with increasing amounts of KL4...............................................................................................................................102 3-4 Static 31P NMR spectra of 4:1 DPPC(d-62):POP G MLVs with increasing addition of KL4...................................................................................................................................103 3-5 Static 31P dePaked NMR spectra of 4:1 DPPC(d-62):POPG with increasing amounts of KL4...............................................................................................................................104 3-6 Static 31P NMR spectra of 3:1 POPC:POPG(d-31) MLVs with increasing amounts of KL4...................................................................................................................................105 3-7 DePaked 31P NMR spectra of 3:1 POPC:POPG( d-31) with incr easing amounts of KL4...................................................................................................................................106 3-8 Static 31P NMR spectra of 4:1 DPPC:POPG(d-31) MLVs with increasing amounts of KL4...................................................................................................................................107 3-9 DePaked 31P NMR spectra of 4:1 DPPC:POPG( d-31) with incr easing amounts of KL4...................................................................................................................................108 3-10 The shift in dePaked frequency in ppm ( ) for DPPC and POPG (in ppm) by KL4 in 4:1 DPPC:POPG(d-31)................................................................................................109 3-11 Static 31P NMR spectra of single lipi d and lipid with 1.5 mol% KL4 A: DPPC(d-62), B: POPC(d-31) and C: POPG(d-31)................................................................................110 3-12 DePaked 31P spectra for (Top) DPPC(d-62), (M iddle) POPC(d-31) and (Bottom) POPG(d-31) with and without 1.5mol% KL4..................................................................111 3-13 2H spectra of 3:1 POPC(d-31):POPG MLVs with increasing amounts of KL4...............112 3-14 2H NMR spectra of 4:1 DPPC(d-62):POPG with increasing amounts of KL4................113

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11 3-15 DePaked 2H spectra for (A) 4:1 DPPC (d-62):POP G MLVs and (B) 3:1 POPC(d31):POPG MLVs with increasing amounts of KL4.........................................................114 3-16 Deuterium NMR spectra for (A) 3:1 POPC:POPG(d-31) MLVs and (B) DePaked spectra with increasing amounts of KL4..........................................................................115 3-17 Static 2H spectra for (A) 4:1 DPPC:POPG(d-31) MLVs and (B) DePaked spectra with increasing amounts of KL4......................................................................................116 3-18 Static 2H NMR spectra of single lipid MLVs with and without 1.5mol% KL4. ............ 117 3-19 Order parameter profiles for (4:1) D PPC(d-62):POPG MLVs with and without KL4. Top:sn-1 chain Bottom: sn-2 chain..................................................................................118 3-20 Order parameter profile for the sn-1 ch ain of 3:1 POPC(d-31):POPG MLVs with and without KL4......................................................................................................................119 3-21 Three dimensional plot of change in order parameter for DPPC(d-62):PO PG MLVs as a function of mole percentage of KL4......................................................................... 120 3-22 Three dimensional plot of change in ti me averaged order parameter for 3:1 POPC(d31):POPG MLVs as a function of mole percentage of KL4............................................ 121 3-23 Order parameter profile for A) 4: 1 DPPC:POPG(d-31) MLVs and B) 3:1 POPC:POPG(d-31) MLVs ...............................................................................................122 3-24 Change in order parameter valu es on (A) POPC:POPG(d-31) and (B) DPPC:POPG(d-31) upon addition of KL4.......................................................................123 3-25 Model of KL4 penetration in two lipid environments......................................................124 4-1 CD Spectra of KL4 in 5mM HEPES at pH 7.4. Below is a spectrum of 150M KL4 indicating potential aggregation....................................................................................... 138 4-2 CD spectra of KL4 in organic solvents. Red is a spectrum of peptide in 150M hexafluroisopropanol and black is a spectrum in 50:50 MeOH:dH20.............................139 4-3 CD Spectra of 40M KL4 added to 1.33mM LUVs. DP PC containing samples were run at 45oC.......................................................................................................................140 4-4 CD spectra of 40M KL4 reconstituted in 4mM 4:1 DPPC:POPG and 3:1 POPC:POPG LUVs......................................................................................................... 141 4-5 CD spectra of 40M KL4 reconstituted in 2mM 4:1 DPPC:POPG and 3:1 POPC:POPG LUVs......................................................................................................... 142 4-6 CD spectra of 40M KL4 reconstitu ted in 1.33mM 4:1 DPPC:POPG and 3:1 POPC:POPG LUVs......................................................................................................... 143

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12 4-7 CD Spectra of (Top) POPC:POPG LUVs and (Bottom) DPPC:POPG LUVs with increasing mol% of KL4...................................................................................................144 4-8 Helical wheel projections of KL4 as: (left) 310 helix, (middle) standard -helix, and (right) helix...................................................................................................................147 4-9 DQ-DRAWS buildup curves generated from spectra on 13C labeled KL4 with POPC:POPG (3:1)...........................................................................................................148 4-10 Ramachandran plot showing a 2 minimum at =-105 and =-26 for KL4 in 3:1 POPC:POPG....................................................................................................................149 4-11 Model of KL4 based on torsion angles obtained from the 2D-DRAWS experiments.....150 4-12 KL4 with torsion angles of = -65, = -78 obtained from ssNMR studies of KL4 in a DPPC:POPG lipid environment.................................................................................... 151 5-1 Putative helical wheel for each type of he lix rendered for the C-term inal residues 5980 of SP-B..................................................................................................................... ...168 5-3 31P NMR data of 4:1 DPPC:POPG MLVs with varying molar percentages of SPB59-80......................................................................................................................... .....171 5-4 DePaked 31P NMR data of 4:1 DPPC:POPG MLVs with varying molar percentages of SP-B59-80.......................................................................................................................172 5-5 Change in 31P CSAs for 4:1 DPPC:POPG MLVs as a result of increasing molar percentages of SP-B59-80...................................................................................................173 5-6 Phosphorous NMR data of 3:1 POPC:POP G MLVs with varying molar percentages of SP-B59-80.......................................................................................................................174 5-7 DePaked 31PNMR data of 3:1 POPC:POP G MLVs with varying molar percentages of SP-B59-80 ................................................................................................................... 175 5-8 Change in 31P CSAs for 3:1 POPC:POPG MLVs as a result of increasing molar percentages of SP-B59-80...................................................................................................176 5-9 Deuterium NMR spectra of 4:1 DP PC(d-62):POPG MLVs with SP-B59-80. ................ 177 5-10 Stacked dePaked spectra of 4:1 DPPC(d-62):POPG MLVs with SP-B59-80....................178 5-12 2H NMR spectra of 3:1 POPC(d -31):POPG MLVs with SP-B59-80.................................180 5-13 Stacked dePaked spectra of 3:1 POPC(d-31):POPG MLVs with SP-B59-80....................181 5-14 Percent change in time averaged order parameter < SCD> for POPC (d-31) in 3:1 POPC(d-31):POPG MLVs on addition of SP-B59-80.................................................... 182

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13 5-15 Hypothesized orientational model of SP-B59-80 in two different lipid MLV systems...... 183

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14 LIST OF ABBREVIATIONS AAA am ino acid analysis AP alveolar proteinosis BAL bronchoalveolar lavage Bo external magnetic field C-D carbon-deuterium bond Cp max maximum heat capacity CD circular dichroism CPAP continuous positive airway pressure CPMAS cross polarization with magic angle spinning CSA chemical shift anisotropy CU cooperativity unit D or d deuterium atom (2H) DPPC 1,2-Dipalmitoylsn -Glycero-3-Phosphocholine DRAWS dipolar recoupling w ith a windowless sequence DSC differential scanning calorimetry Eo electric field vector FTIR Fourier-transfor m infrared spectroscopy P pressure across alveoli m gyromagnetic ratio of a NMR nucleus Go change in Gibbs free energy (standard state) Hcal change in calorimetric enthalpy HvH change in van Hoft enthalpy change in shift in ppm HFOV high frequency oscillatory ventilation

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15 HPLC high performance liquid chormatography ITC isothermal titration calorimetry KL4 KLLLLKLLLLKLLLLKLLLLK (sinapultide) LUVs large unilamellar vesicles L gel phase of phospholipid bilayer L liquid-crystalline phase of phospholipid bilayer MLVs multilamellar vesicles NMR nuclear magnetic resonance P ripple phase of a phospholipid bilayer PA palmitic acid PDB protein data bank POPC 1-Palmitoyl-2-Oleoylsn -Glycero-3-Phosphocholine POPG 1-Palmitoyl-2-Oleoylsn -Glycero-3-[Phosphorac -(1-glycerol)] R universal gas constant (1.987 cal K-1 mol-1) rad radians RDS respiratory distress syndrome time averaged order parameter of a C-D bond at position i ssNMR solid-state NMR SP-A Lung surfactant protein A SP-B Lung surfactant protein B SP-C Lung surfactant protein C SP-D Lung surfactant protein D SUVs small unilamellar vesicles S change in entropy p surface tension

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16 Tm phase transition temperature T peak-width at half height in a DSC trace

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17 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOPHYSICAL CHARACTERIZATION OF PEPTIDE MIMICS OF LUNG SURFACTANT PROTEIN-B By Vijay C. Antharam May 2008 Chair: Joanna R Long Major: Medical Sciences-Biochemistry and Molecular Biology Lung surfactant is a lipid rich fluid that co ats the inner surface of the alveoli, the primary units of respiration where oxygen diffuses into the bloodstream. Due to the small radii and high curvature of the alveoli, significant pressure is required to overcome surface tension and to inflate the lung. Lung surfactan t is a biological coating that lowers surface tension and minimizes the work required for breathing. It is primarily composed of two phospholipids, the zwitterionic 1,2-dipalmitoylsn -Glycero-3-Phosphocholine (DPPC) and the anionic 1-Palmitoyl2-Oleoylsn -Glycero-3-[Phosphorac -(1-glycerol)] (POPG). The 79-amino acid surfactant protein B (SP-B), however, is an absolutely essential component of lung surfactant. Although present at low levels (approxi mately 1% by weight), mutation or loss of SP-B results in respiratory distress syndrome (RDS). The design of simple peptides to mimic the charge distribu tion and the hydrophobic characteristics of SP-B was inve stigated in the early 1990s. A peptide composed of only the basic residue lysine and hydrophobic leucine systematically repeated (KLLLLKLLLLKLLLLKLLLLK) has demonstrated clin ical efficacy in the treatment of RDS when administered with DPPC, POPG and palmitic acid.

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18 In this dissertation, solid-state NMR spectrosc opy (ssNMR) was used to examine in detail the phase properties of these lipids and the effects of KL4 on lipid organization and dynamics. Structural measurements were also performed to determine the sec ondary structure of KL4. These studies were carried out using two model lipid systems: 1) 3:1 POPC: POPG, a bilayer system used in many studies probing peptide:lipi d interactions, including amphipathic antibiotics of similar size and hydrophobicity to KL4, and 2) 4:1 DPPC:POPG, which is similar to the lipid composition in the lung. Comple mentary studies using differential scanning calorimetry (DSC) and circular dichroism (CD) were also carried out. Comparative experiments were also performed on residues 59-80 of SP-B (SP-B59-80), the C-terminal region upon which the sequence of KL4 was derived. Based on these studies, a molecular m odel incorporating the structure of KL4 and its interactions with 3:1 POPC:POP G bilayers and 4:1 DPPC:POPG b ilayers was developed. This model can serve as a guide for understanding ho w proteins modulate surface tension and for developing more effective peptid e mimetics for clinical use.

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19 CHAPTER 1 DISCOVERY AND HISTORY OF LUNG SUR FACTANT In 1929, Swiss physiologist Kurt von Neergard di scovered that the pressure required to fill the lungs with air was much greate r than the pressure required to fill the lungs with water. He hypothesized that surface tension was the force responsible fo r inhibiting the expansion of the lung with air ( 1-6 ). It was not until two decades later th at an absolutely essential surface active material was found in the lungs for overcoming th is force and reducing the work of breathing. Research describing the difficulties the l ung undergoes during alveolar expansion and contraction can be traced back to 1854, including contributions made by Alexander Graham Bell before he shifted interests a nd help invent the telephone ( 1). The discovery of lung surfactant as a material that minimizes alveolar surface tensio n, and its pivotal role in respiratory distress syndrome (RDS) has a history th at contains contributions from physicists, physiologists, biochemists, and medical doctors. The knowledge generated by this diverse community has led to an understanding of the mechanics involved in lung function as well as better diagnoses and treatments of RDS. RDS is a disease prevalent amongst premature in fants, but it also o ccurs in adults and children affected by significant inju ry or inflammation to their l ungs. General treatment of the disease involves mechanical ventilation, a proced ure that forces air into the lungs by positive pressure. Epidemiologic estimates indicate that ons et of adult respiratory distress occurs in 75 of 100,000 individuals per year ( 7 ); and the incidence of respiratory distress after delivery is 1% of all newborns. The advent of preventative surf actant instillation therapies has greatly improved survival outcomes, especially in premature in fants where mortality decreased by 6% from 1989 to 1990 ( 8). Recent estimates indicate less than a thousand infa nts annually are diagnosed with RDS as compared to 10,000-15,000 in the 1950s and 1960s ( 9). However, improvements need

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20 to be made in terms of therapy, and basic clinical and scientific questions remain to be answered which can aid in the developm ent of therapeutic agents. Originally, RDS in infants was incorrectly di agnosed as hyaline membrane disease, based on the mistaken assumption that the strain and grunting noises that occurred when premature infants took their first breath was caused by the inspirati on of amniotic fluid or milk, leading to the appearance of glassy membranes found duri ng lung autopsy. In the mid-1950s, however, physicians Dr. Mary Ellen Avery and Dr. Jere Mead found that ba bies who died from RDS had no residual air in their lungs implying that premat ure lungs were unable to retain air. At the same time, Dr. John Clements, a physiologist wo rking at the United States Army Chemical Center, designed a surface balan ce where a movable barrier was a llowed to cyclically compress and expand a shallow trough containing minced lung tissue while forces were measured. Such an apparatus, akin to a conven tional Langmuir-Wilhelmy balance ( 6), allowed Clements to monitor surface tension as surface area was varied. He found that surface tension rose as the surface area containing lung extracts was expande d, but dropped from 45 to 10mN/m when the layer was compressed wi thout any collapse (6, 10 ). The evidence pointed to a fluid that modulated surface tension in the lu ng that Clements first referre d to as an anti-atelectasis factor but was then later renamed ( by Clements) as pulmonary surfactant. Countless research by innumerable investigators contributed to the surmounting evidence of the importance of this surface active material This has lead to the current and almost indisputable claim that an absence or delayed app earance of pulmonary surfactant material is the prevailing cause of RDS in premature infants. The Alveoli and Surface Tension The alveoli are the respirator y units of the lung and contai n an air/water interface for oxygen diffusion into the bloodstream. It is es timated that hum an lungs contain approximately

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21 350 million alveoli with diameters ranging from 75 to 300 microns ( 11 ). Unique molecular forces arise at interfaces compared to molecules in solution or in a bulk phase. Surfactants are substances that have an energetic preference for interfacial regions and affect intermolecular forces there. A critical force that exists at the alveolar interfacial re gion is surface tension. Surface tension arises due to unbalanced forces th at exist for molecules residing at the surface, particularly since these molecules have diminished interaction with the bulk fluid phase (Figure 1-1). As a result of this imbalance, the surface tends to minimize in area. Surface tension is the work needed to expand the surface area of a sy stem, in respiration this is the work from expanding the alveolar air sacs. Surface tens ion is defined as the change in free energy per unit surface area, or equivalently it can be interpreted as work done on a surface, with units of force per distance (mN/m) ( 11). In the lung, amphipathic phospholipid molecules serve as the surface f ilm that separates the air volume of the alveoli from the fluid laye r (Figure 1-2). These lipid surface films have been found to lower surface tension values during in vitro dynamic compression experiments. The most abundant lipid in lung surfactant, 1,2-Dipalmitoyl-sn -Glycero-3-Phosphocholine (common name and abbreviated DPPC) contains two saturated 16-carbon fatty acid chains esterfied to a zwitterionic phosphatidylcholine he adgroup. The rigid nature of the disaturated fatty acyl chains allows DPPC to form tightly packed surface film s that can lower surface tension to values of less than 1mN/m ( 11). Hence, the addition of a phos pholipid film found in the lung greatly alleviates the surface tension problem faced by the lung. The essential need of the alveolus to have an inner coating of surfactant becomes clearer when considering the law of Young and Laplace This law states that the pressure difference normal to surface must be greater than twice (o r equal to at equilibrium) the surface tension

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22 divided by the radius of the object: p=2 /r (where represents surface tension) for inflation to occur ( 4 ). In terms of expansion and contrac tion of pulmonary alveoli, the Young-Laplace equation suggests considerable force is required to overcome the surface tension in the highlycurved alveoli. Theoretically, values fall to values near 0 upon expiration in the lung, and is estimated to be roughly 30mN/m in the trachea, and about 15mN/m in the airways; consequently, a surface tension gradient exists during exhalation ( 12). Methods to study surface tension invitro include the Langmuir-Wilhelmy balance, ca ptive bubble surfactometer, and pulsating bubble surfactometer ( 13 ). Such methods for measuring the properties of surfactant are highly dependant on lung surfactant composition and the buffer salts present in the hypophase chosen to simulate an air/water interface. Chemical Composition and Or igin of Lung Surfactant Knowing the exact chemical constitution of lu ng surfactant is im por tant to understanding how it lowers surface tension. Initial analys es using material lavaged from bovine lung indicated the dominant fraction was lecithin ( 14 ), commonly known as phosphatidylcholine. Of phosphatidylcholine, the primary fraction was the dipalmitoylated form, or DPPC. However, the chemical composition of lung surfactant can best be described as hete rogeneous mixture of proteins and lipids with the prot ein fraction constituting ~8% of th e total dry weight and the lipid constituting ~92% of the total dry weight. In the lipid fr action, DPPC is predominant, comprising up to 70%, followed by phosphatidylglyc erols, comprising about 8% of the lipid mass ( 5, 9). However, variations amongst species do exist ( 15 ). In humans, the second most prominent lipid in lung surfactant is palmitoyl oleoylphosphatidylglycerol (POPG). With the advent of mass spectrometry and other analytical techniques, quantification of surfactant material after lavage treatment has revealed a far more assorted mixture of lipids ( 16 ). Table 1 lists the most common lipid and protein species found in lung surfactant. Othe r surfactant components

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23 include phosphatidylinosito ls, phosphatidylethanolamines, sphingomyelins, lysophosphatidylcholines cholesterol, Ca+2, and free fatty acids ( 5, 15, 17 ). Despite the heterogeneity present in lung surfactant, most ar tificial formulations used as candidates for clinical treatments contain only a few component s, particularly DPPC and a monounsaturated phospholipid (usually POPG) ( 18). The lipid and protein component s of lung surfactant are also evolutionary conserved. Surfactant has been discovered in the Australian lungfish Neoceratodus forsteri which dates back more than 300 million years, raising the ques tion of whether the anti-adhesive properties of surfactant may have served in the primitive respiratory system and led to the evolution of gascontaining organs ( 19 ). Surfactant still remains one of the most highly conserved systems in vertebrate biology ( 17). Lung surfactant contains four proteins named in chronology of their discovery: (surfactant protein A) SP-A, SP-B, SP-C and SP-D. Both SP-A and SP-D are large multi-component proteins with an extensive collagenous stru cture and carbohydrate recognition domains. These two proteins have been implicated in immune surveillance of the alveolar structure of the lung, although SP-A has been found to also have roles in surface activity as well as intracellular surfactant processing ( 20 ). The presence of SP-A and SP-D and their close structural resemblance to components of the complement system (a glycoprotein cascade found in the blood for clearance of viral and bacterial pat hogens) raises interesti ng questions regarding divergent evolution of the immune system and lung surfactant (20). While SP-A is critical to surfactant homeostasis, and SP-D is important to immunological recogniti on, the focus of this thesis is on factors modulati ng surface tension, particularly the molecular basis for SP-B changing the phase behavior of lipids in the lung.

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24 Cellular Biology of Lung Surfactant Pulmonary lung surfactant is synthesized by the highly differentiated type II alveolar epithe lial cell derived from the alveolar epithelial lining ( 11, 15, 21-23 ). These cells are distinguished from type I cells by their flat appe arance, apical microvilli, and the presence of lamellar bodies in the cytoplasm. Type II epithelial cells comprise 5% of total alveolar cell surface area, account for 15% of peripheral lung cells, and have a surface area of 250m2 per cell ( 21). In addition to producing the four major surfactant proteins, type II alveolar epithelial cells have recently been demonstrated to be an importa nt line of defense in innate immunity. Various anti-microbial and anti-inflammatory substances ha ve been found to be secreted by type II cells, including lysozyme, defensins, cathelicidi n, lipocalin 2, as well as SP-A and SP-D which agglutinate fungi, bacteria, and viruses ( 21). Also, since lung surfactant consists of primarily lipids, a high level of lipogenesis occurs in these cells. The upre gulation of fatty acid biosynthetic enzymes such as fatty acid synt hase, acetyl-CoA carboxylase, ATP citrate lyase, and stearoyl CoA desaturase is stimulated by transcription factors SREBP-1c and C/EBP alpha delta. These transcription factors are in turn up regulated in response to various growth factors, most prominently keratinocyte growth factor (KGF) ( 21). Due to the critical role of the type II cell, it has often been dubbed as the great pneumocyte and the defender of the alveolus ( 22 ), since it not only produces the neces sary lung surfactant lin ing the alveoli, but also functions in maintaining sterility within the lung. Lung surfactant is synthesized in utero beginning at 28-32 weeks of gestation and babies born before 35 weeks of gestationa l age typically suffer from RDS ( 11, 24 ) due to inadequate levels of lung surfactant. The phos pholipid profile in amniotic fluid has historically been used as a diagnostic marker for the onset of lung surfact ant synthesis. Tests for predicting the likelihood of RDS typically analyze amniotic fluid for the presence of phosphatidylglycerol, the second

PAGE 25

25 most abundant phospholipids in lu ng surfactant; its absence is a highly predictive marker for RDS. Historically, preventive measures incl ude stimulation of lung surfactant production by type II cells through treatment with corticoste roids or glucocorticoids, which increase the enzymes necessary for lipogenesis ( 15). Lung surfactant production has been found to be regulated by hormonal agents ( 25 ) and metabolism of the lipid/protein species are under the control of granulocyte-macrophage co lony stimulating factor (GM-CSF) ( 26). Prior to the development of lung surfactant repl acement therapy, the clin ical course for the management of newborns with RDS was mechanical ventilation. Variations in ventilatory management give rise to differing outcomes of the disease, and treatments included continuouspositive airway pressure (CPAP) and high-frequency oscillatory ventilation (HFOV). Both methods involve heavy oxygenation of the lung bu t high frequency-ventilation has become the preferred course due to its adva ntages of reducing lung distensi on and minimizing shear forces. In this procedure small volumes airway of gas are pumped into the airways of the lung at rates of 15 Hz ( 27). Important Surface-Acting Prot eins of Lung Surfactant A funda mental problem associated with air-b reathing stems from the dynamic nature of the lung: the need to reduce surface tension in sma ll, gas-containing, aqueous lined structures which are constantly changing in volume. For most mammals, the task of reducing this force is achieved by the proteins SP-B and SPC, which directly interact wi th lipids specific to the airwater interface. Initial sequenc ing of isolated protein show ed SP-B to be extraordinarily hydrophobic (~41% to ~52%) with a high relative fr action of the amino acids valine, leucine, isoleucine, alanine, phenylalanine, and tryptophan ( 12, 23, 28). Later studies found the protein to also have a net cationic ch arge > +6 at physiologic pH ( 29, 30 ). Based on electrophoretic mobility in sodium-dodecyl polyacrylamide gels, SP-B has a molecular weight of approximately

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26 5-8 kDa ( 31 ). The native form contains 6 cysteines that form intramolecu lar disulfide bonds and a seventh cysteine residue that forms an intermolecular disulfide bond ( 32). In the lung, SP-B exists primarily as a 15-18 kDa homodimer with the monomers connected via a disulfide bond at cysteine residue 48 (23, 30, 31). The conservation of the 6 cysteines forming intramolecular disulfide bridges, the pattern of disulf ide formation, and the high number of hydrophobic residues place SP-B as the most hydrophobic member in the saposin family of proteins. Other proteins in the saposin fam ily include amoebapores from Entamoeba histolytica acid sphingomyelinase, acyloxyacyl hydrolase, and sphingolipid activator proteins A-D (saposins A-D). Saposin family members are important to both lipid homeostasis and lipid physical properties as they can f acilitate lipid fusion or bind lipids to increase the activity of lipid modifying enzymes. Saposins have been characterized as being heat-s table, with a helical secondary structure (33 ). What differentiates SP-B from ot her saposin family members is its hydrophobicity, rendering the protein insoluble in aqueous solutions, and the seventh cysteine residue, used in forming the homodimer, is unique to SP-B ( 33). The second surface-acting protein in lung surf actant is SP-C. SP-C is a 35 amino acid peptide of molecular weight 4.2 kDa. Its amino acid sequence lacks any known homologs and possesses a high valine, leucine, and isoleucine content. An NMR structure of porcine SP-C determined in organic solvent reve als a helical secondary structure ( 29). Two cysteine residues near the N-terminus are palmitoylated in human SP-C. The protein has an overall hydrophobicity of ~66% and is c onsidered the most hydrophobic pr otein ever to be found in a biological system (11, 28, 29). While SP-C has been deemed essential in respiratory dynamics, mutations in, or of knock-out of, the gene have not been proven to be lethal ( 34). SP-C null mice show normal respiratory kinetics Nonetheless, mutations in SP-C have been linked to lung

PAGE 27

27 disease and susceptibi lity to infection (34). It has also been shown that genetic knockout of SPB causes aberrant processing and misfolding of SP-C ( 35) and can lead to structures reminiscent of amyloid fibrils causing pulmonary alveolar proteinosis, a condition in which pathologically high protein levels are found in the airspaces ( 36). Models of the struct ure and interaction of SPB and SP-C with lipids are shown in Figure 1-3. Lung Surfactant Protein B (SP-B) and RDS The critical role of SP-B has been highlighted by targeted disruption of the gene in mi ce, as well as the identification of genetic mutati ons and polymorphisms in humans correlated with respiratory distress ( 23, 37, 38 ). The difficulty in breathing asso ciated with respiratory distress stems from an altered histopathology seen in the l ungs of patients who have inadequate levels of mutated SP-B. Altered morphology of the lung air spaces as well as pert urbations in the normal surfactant processing, secreti on and storage are seen ( 23, 38). SP-B mutations or deletions lead to disorganization of lamellar bodies and alveol ar type II cells, and alterations of the lung surfactant cycle leading to severe di sruption of respiratory mechanics. ( 30, 39 ). Besides the general abnormal morphology of type II cells la cking SP-B, an accumulation of distorted, or irregularly shaped multivesicular bodies in these cells suggests an impairment in lipid packaging and secretion as seen in SP-B(-) mice (Figure 1-4) ( 40). Depletion of SP-B also causes distortion or disorganization of tubular myelin ( 23), resulting in lower lung co mpliance resulting from the pathological organization of lame llar bodies in the Type II cell. The most prevalent nucleotide mutation of SP-B linked to RDS is an insertion of 2 bases at position 121 leading to a frameshift in the open reading frame ( 23, 41). The above findings clearly indicate the centr al importance of SP-B in lipid packaging, cycling and the lung surfact ant cycle. Knockout studies and gene tic analysis clearly indicate that SP-B is essential in the reorgani zation and restructuri ng of lipids at the air/water interface to

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28 allow for respiratory function. While inadequa te levels of functional SP-B is linked to respiratory distress, an excess of lung surfactan t proteins also leads to the clinical condition known as alveolar proteinosis (AP) Patients afflicted with AP often have 100-fold higher ratios of SP-B to disaturated lipids compared to normal subjects ( 42 ) and tend to suffer from dyspnea. Typically 75% of individuals with AP are smokers. To date, only saposin-B of the saposin fam ily has had its structure solved via x-ray crystallography. Recombinant expression of saposin B in Escherichia coli gave rise to crystals diffracting to 2.2 ( 43, 44). The structure of saposin B revealed an -helical rich dimer enclosing a hydrophobic cavity ( 45). Structures of saposin-A saposin-B, and saposin-D have been characterized by 15N solution NMR and also show an -helical rich fold ( 46). Modeling of one subunit of SP-B based on the sequence of NK-lysin has also been reported ( 47 ). While these structures provide insight for modeling the conformation of SP-B, its hydrophobicity and tight association with lipids make it an unlikely candidate for eith er x-ray crystallography or solution (high resolution) NMR, which can yield a more definitive structure as compared to modeling. Efforts to recombinantly express SP-B have been met with limited success (48). SP-B was first isolated in 1986 (49). The initially discovere d 6 kDa protein fragment, based on Edman sequencing of protein isolated from pig lung lavage, was found to lower surface tension when reconstituted in lipids (50). Studies directly linking SPB to RDS were done in the mid-1990s ( 37, 39, 51). A structural model of how protei n reduces surface tension was first predicted in 1993 by Longo ( 52). Isolation and purification procedures for SP-B are rather extensive and time-consuming with low yields ; new methods were developed to simplify purification, but many different protocols for SP-B isolation exist in the literature (53-56 ). Isolation of SP-B generally incl udes a low speed centrifugation of lung lavage fluid to remove

PAGE 29

29 cells, followed by a high speed centrifugation to pellet large surfactan t aggregates. The hydrophobic constituents are extracted into chloroform, the lipid s are removed using a Sephadex LH-20 column, and a final reverse phase C18 column chromatography step can be used to ensure highest purity (23, 56). Human SP-B is encoded on the short arm of chromosome 2 and the gene has 9500 base pairs and 11 exons. Translation of the protein re sults in the expression of a precursor protein of 381 amino acids containing both N-terminal and Cterminal signal sequences. The N-terminal signal sequence directs the precurs or to the endoplasmic reticulum (ER) while the C-terminal signal, containing a glycosylated asparagine, appears to have no ro le in the trafficking of the protein ( 23). Cleavage of the precursor protein by prot eases in the ER results in mature protein which transits from the ER to the Golgi appara tus where it associates with lipids that selfassemble to form a multi-lamellar concentric structure, called a lamellar body, containing SP-B, SP-C and SP-A ( 3), which act as a functional storage uni t of lung surfactant containing both the essential lipids and proteins. Exocytosis from th e type II pneumocyte results in unraveling of the lamellar bodies into a meshwork film known as tubular myelin. Electron microscopy images of tubular myelin show an inte rlocking lattice of lipids ( 17). Tubular myelin is the lipid reservoir from which adsorption and resorption occur at the air/water interface ( 23 ). Mutations in or deletions of SP-B have been demonstrated to lead to breakdown of the mu lti-lattice organization of tubular myelin which is essential for the maintenance of this interface ( 23, 39). Functions ascribed to SP-B include the promotion of rapid phospholipid insertion at the air-liquid interface during lung expression, promotion of tubular myelin formation, and an impact on the overall ordering and organization of phospholipids ( 5).

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30 Lipids cannot account for the near zero surface tension seen in lung tissue during exhalation or its surface compressi on characteristics; the presence at low molar ratios of SP-B and SP-C is required. Direct atomic level meas urements on how surfactant proteins associate with lipids to achieve surface tension minimization has previously not been accomplished. Preliminary models of SP-B and SP-C structure and orientation in phosph olipid environments are based on examining charge distributions a nd presumed secondary structures based on FTIR and Raman measurements ( 6, 23, 57). An important considerati on in determining structure and interactions of SP-B and SP-C with lipids is the cy cle that occurs at the air-water interface during compression and expansion of the alveoli. The interface separating the ai r space of the alveoli from the adjacent liquid lining is a lipid film that is enrich ed in DPPC, but its physical composition must be under constant flux between a liquid-expanded phase and a condensed film and there may also be cyclic changes in lipid composition ( 58 ). The saturated acyl chains of DPPC provide a surface film that can withstand compression of the surface, as would be the case during exhalation, but due to the molecules poor re spreading qualities, requi res the assistance of unsaturated lipids to aid in surface expansion that is required during the subsequent inhalation. Western blots probing for hydrophobic surfactant proteins often show no presence of SP-B from BAL (bronchoalveolar lavage ) samples from premature infant s with RDS. Some infants with chronic cases of RDS have been found positi ve for antibody to an aberrant 40-42kDa proform of SP-B indicating inadequate proteolytic processing, not just genetic mutation, as a contributor to disease ( 41 ). A highly sensitive technique em ploying HPLC in conjunction with Western blotting and light scatte ring detection on human BAL fluid showed SP-B from normal individuals to be on the orde r of 740 ng/mL. Premature in fants with respiratory tract infections or chronic bronchitis have SP-B levels 10-15% of normal ( 59).

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31 Mammalian Lung Surfactant Cycle A cycle exists that allows for a chan ge of film composition when the surface film in the alveoli expands (inhalation) and contracts (exhalation). The cla ssical hypothesis of the lung surfactant cycle has been described in a review by Weaver and Conkright (1991) as well as Veldhuizen and Hagsman (2000) and will be briefly described here: (1) afte r transit from the ER to the Golgi, lung surfactant proteins SP-A, SP-B and SP-C are pack aged with associated lipids to form a concentric organization of lipids known as lamellar bodies, (2) once secreted from type II pneumocytes, lamellar bodies unravel to form an extended mesh structure known as tubular myelin, (3) tubular myelin inter acts with the lipid monolayer film at the air/water interface by aiding in the adsorption of DPPC enriched lipids during film expansion, and promoting selective squeeze out of non-DPPC lipids occu rs during film compression, thus allowing for DPPC enrichment leading to a more stable compre ssed film, (4) the residual surfactant particle that is squeezed out can then (5) undergo ca tabolism by nearby macrophages, or (6) be taken up by a type II pneumocyte and recycled (Figure 15). Data disagreeing with this classical hypothesis exist, most notable is the appearance of lip ids undergoing a buckled structure at the air/water interface, rather than a complete selective squeeze out( 58). Furthermore, recent fluorescence and Brewster angle microscopy data al so seem to contradict the classical hypothesis and support the notion that the interface remains compositionally heterogeneous during film compression and not DPPC enriched as previously believed ( 58, 60). Exogenous and Artificial Lung Surfactants As the chemical composition of lung surfactan t was elucidated, replacem ent therapy, or instillation of the most critical components of lu ng surfactant become anot her mode of treatment for RDS. Administration of micr o-aerosolized DPPC resulted in negative results, while slight changes in breathing profiles were seen by blowi ng DPPC and PG powders into the trachea of

PAGE 32

32 premature infants ( 61). Better outcomes were observed for in stillation of lung su rfactant isolated from BAL of animals or animal lung homogenate s. The first clinical success using exogenous lung surfactant was reported by Fujiwara, et al who administered bovine lung homogenate via an endotracheal tube. The result was improved oxyge nation in 9 of the 10 infants in the trial ( 61, 62). Despite such procedural successes, concerns about viral and other pathogenic contamination from these animal sources remain ed significant. Furthermore, development of synthetic surfactants for clinical treatment overc omes the expense of isolation and permits better levels of quality control ( 32). Initial success obtained from administration of exogenous lung surfact ant heralded a new avenue of exploration using lung surfactant isolated either from lung lavage or tissue homogenates for the treatment of premature new borns afflicted with respiratory distress. Application of lung tissue homogenates from bovine, porcine sources, or la vage material are now standard treatments for RDS, and formulations are currently marketed by several pharmaceutical companies. A comprehensive list of availa ble lung surfactant formul ations used for the treatment of respiratory distre ss syndrome is compiled in Tabl e 1-2 and includes formulations using derivatized compounds as well as peptide mimetics not found biologically. The first concept of testing the bare mini mum components for surface activity came in 1981. Tanaka, et al, tested the surface properties of 25 lipid mixtures in Langmuir trough assays as well as in rabbits to examine the mini mum composition necessary to minimize surface tension. His findings concluded that the best an d most minimal requirements for a successful administration of artificial l ung surfactant (in rabbits and in vitro) were DPPC, a saturated fatty acid (either palmitic or stearic), an ani onic phospholipid (phosphatidylglycerol or phosphatidylserine), and a yet to be discovered lipid bound protein ( 18 ). The protein referred

PAGE 33

33 to by Tanaka was a not yet fully characterized form of SP-B. Subsequent work has shown that in the presence of DPPC, palmitic ac id acts as a spreading agent facilitating monolayer expansion as the surface area increases (63, 64). The need to assist in monolayer expansion is critical since DPPC itself has poor respreadability and adsorptive characteristics ( 60, 63, 65 ). Artificial lung surfactants greatly simplify the problem of studying the underlying biophysical aspects of lung su rfactant function ag ainst the complicated, heterogeneous background existing in vivo and allow the possibility of designing simpler replacements of SP-B with peptide mimetics (66 ). Non-natural peptide analogs of SP-B that exploit the molecules helical and amphipathic quality, such as KL4, can circumvent the need to isolate SP-B to sufficient purity. Oligo N-substituted glyc ines (phenylethyl, 2-butyl, or 4-aminobutyl substituents attached to the nitrogen of glycine) or peptoids, have also been employed to mimic the helicity of SP-B and show potential ( 67). Most lung surfactant formulations have in common DPPC and a chemical agent that facilitates in terfacial spreading such as palmitic acid or tyloxepol. The role of palmitic acid in spreading has been investigat ed by grazing incidence xray diffraction and by fluorescence microscopy on D PPC:PA monolayers. It has been found PA enhances membrane fluidity and causes the DPPC chains to be more rigid and ordered. Higher surface tensions of greater than 40mN/m cause pha se segregation of PA into condensed domains ( 64). KL4 is a 21 amino acid peptide designed to mi mic the activities of SP-B. To date, KL4 has enjoyed the most research focus and clin ical success as a replacement for SP-B ( 61, 68, 69). The primary amino acid sequence of KL4 is KLLLLKLLLLKLLLLKLLLLK with a pattern of hydrophobic leucines punctuated by the basic, hydrophilic lysines. KL4-based surfactant therapy relies on a formulation of the peptide with DPPC POPG, and a spreading agent (palmitic acid).

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34 Indicators used to measure improvements in oxyge nation after administrati on of lung surfactant include assaying the ratio of arterial/alveolar partial pressure of oxygen (a/AO2) (which must not fall below 0.22), the fraction of inspired oxygen (FiO2), and mean airway pressure (MAP). Declines in MAP, and FiO2, and an increase in a/AO2 are markers for improvement in the respiratory status of a premature infant ( 69). KL4 improves respiratory status and has been found to prevent the appearance of di ffuse, granular radiopacity seen in chest radiogra phs typical of RDS and meconium aspiration syndrome ( 69, 70). A recent antimicrobial function of KL4 has also been suggested, in line with some othe r recent reports concer ning the anti-bacterial properties of SP-B ( 71, 72). However, the biophysical properties of KL4 and their relation to alleviating RDS remains the focal point of current investigations, particularly in regard to its structure and organization in lipid environments. Sections of SP-B have also been assayed fo r their surface active properties including the C-terminal 21 amino acids. These peptides are am enable to solid-phase peptide synthesis and can circumvent some of the problems associated with isolation of the full-length mature protein; the C-terminal segment has demonstrated surface activ ity and an effect on surface tension values in phospholipids films ( 67, 73 ). Figure 1-6 shows the primar y amino acid sequence of SP-B and KL4 which was designed based on th e charge distribution in SP-B59-80. In this thesis, the effects of KL4 and SP-B59-80 on the biophysical properties of two lipid systems, DPPC:POPG MLVs and POPC:POPG ML Vs, are described based on an array of techniques for biophysical characterization of bot h the peptides and the lipids. DPPC:POPG in a 4:1 molar ratio was chosen as a model lipid syst em because it represents the native lipid profile in lung surfactant and artificial lung surfactants. POPC:POPG in a 3:1 molar ratio was chosen as a second model system because it has conveni ent physical properties a nd has been used in

PAGE 35

35 characterizing other amphipathic peptides. Th e POPC:POPG phase transition temperature is below 0oC allowing for easy manipulation of samples at room temperature; furthermore, the composition of PC and PG headgroups in this ratio is typically f ound in eukaryotic and prokaryotic membranes. Differe ntial scanning calorimetry (DSC), circular dichroism (CD), and a variety of solid-state NMR (ssN MR) techniques were employed to assess the structure of these peptides in solution and bound to lipids, and thei r impact on lipid self-assembly. A model of these peptides interacting with both these lipi d systems is presented and described in the following chapters. The integrati on of the structure of these peptid es with their orientations in the lipid bilayer is a critical first step toward s obtaining an atomic leve l understanding of how lung surfactant functions and may lead to develo pment of more potent pe ptide mimetics. The power of ssNMR in examining bi omolecules in heterogeneous envi ronments was critical in the development of this model. Such measurements were previously unattainable using x-ray crystallography, where peptide:lipid complexes are typically not amenable to the crystallization process. Furthermore, solution state NMR, where purity and size of the complex place limitations on samples, was unsuitable. Chapter 2 discusses background and theory be hind the biophysical techniques used in these experiments. Chapter 3 describes 2H NMR, 31P NMR and DSC studies of the lipid systems and the effects of KL4 on their properties. Chapter 4 di scusses structural studies of KL4 interacting with phospholip ids by applying ssNMR techniques to isotopically 13C labeled KL4 peptides, as well as circular dichroism. Th e results discussed in Chapter 4 were done in collaboration with Professor Joanna Long and Dr Douglas Elliott. Chapter 5 introduces DSC, 2H and 31P NMR data taken on SP-B59-80, the C-terminal 21 amino acids of SP-B from which KL4 was derived, and compares these results to results for KL4. Finally, Chapter 6 provides

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36 conclusions regarding this work, thoughts on future experiments, and ques tions that stem from these studies.

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37 Gas Liquid 2 1 Gas Liquid 2 1 Figure 1-1. Cartoon illustration of surface tension. Molecules at the surface experience an enhanced attraction (shown as the darkened arrows near molecule 2) The result is net attractive inward force causing the surface to curve seeking a minimal area, thus generating surface tension.

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38 Figure 1-2. The alveolar environment. In the alve oli, fluid coats the inner surface of the alveoli. The air/fluid interface is demarcated by a m onolayer enriched in DPPC as shown in the inset. The periphery is composed of 95% Type I pneumocytes and 5% Type II pneumocytes. The type II pneumocytes se crete lung surfactant. Reproduced with permission from Current Opinion in Structural Biology 2002 Aug,12(4):487-94.

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39 Table 1-1. Approximate weight percentages of lipids in mammalian lung surfactant (Adapted from Lung Surfactants: Basic Science and Clin ical Applications by Robert H. Notter) Lipid^ Lavaged lung surfactant Phosphatidylcholine Phosphatidylglycerol Phosphatidylinosit ol & Phosphatidylserine Phosphatidylethanolamine Sphingomyelin Other 80.0 .9 6.8.4 5.4.3 3.7.4 2.0.3 2.0.3 Protein* SP-A SP-B SP-C 5% 1-2% 1-2% ^ Percent weight of total lipid *Percent weight of total lung surfactant. Lung surfactant proteins constitute approximately 10% by weight of lung surfactant

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40 Figure 1-3. Hypothesized interactio ns of SP-B and SP-C with lipid lamellae. Left: SP-B is a homodimer with the two monomers lining ad jacent lamellae. Right: SP-C is believed to exist as a transmembrane helix within DPPC bilayers. SP-C is palmitoylated at cysteine residues near the N-terminus. S S N C

PAGE 41

41 SP-B (+/-) (+/-) (-/-) (-/-) SP-B (+/-) (+/-) (-/-) (-/-) SP-B (+/-) (+/-) (-/-) (-/-) Figure 1-4 Histology of SP-B mutati on. Figure taken with permission from JC Clark, originally appearing in PNAS 92(17):7794-8. Lung surf actant from mice heterozygous (+/-) for SP-B is shown in the left column while lung surfactant from SP-B knockout mice (-/-) is shown to the right. Clearly seen is th e loss of lamellar bodies and tubular myelin in homozygous (-/-) knock-out mice.

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42 Type II cell macrophageAlveolar Air-water interface2. Unravelling off lamellar body to form Tubular Myelin 1.Lamellar Body 4. Recycling of Lung Surfactant Particle to Macrophage S S N C 3. Interfacial adsorption/ resorptionaided by SP-B and SP-C Expansion Compression5. Recycled Lung surfactant Particle into Type II cell ~200M Type II cell macrophageAlveolar Air-water interface2. Unravelling off lamellar body to form Tubular Myelin 1.Lamellar Body 4. Recycling of Lung Surfactant Particle to Macrophage S S N C S S N C N C 3. Interfacial adsorption/ resorptionaided by SP-B and SP-C Expansion Compression5. Recycled Lung surfactant Particle into Type II cell ~200M Figure 1-5. Mammalian lung surfactant homeostasis: (1 ) Lung surfactant is p ackaged into its the functional storage unit, lamellar bodies, in Type II pneumocytes, (2) lamellar bodies are excreted and unravel to form a networ k of lipids known as tubular myelin, (3) lipids adsorb to the air-fluid interface, (4) non-DPPC lipids resorb into the hypophase to be degraded by macrophages or (5) type II cells recycle lung su rfactant material to reinitiate lamellar body formation.

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43 Table 1-2..Table of artificial lung surfactants used clini cally. With permission from TA Merritt. Reproduced from Acta Paediatr. 2006 Sep;95(9):1036-48. Brand Name Generic Name Source/constituents Company Protein containing Alveofact SF-RI 1 Bovine-lung lavage Boehringer (Germany) BLES Bovine lipid extract surfactant Bovine-lung lavage BLES Biochem (Canada) Curosurf Poractant alfa Porcine-lung lavage Chiesi Pharmaceuticals (Italy) Dey, LP (USA) HL-10 Porcine-lung tissue Leo Pharmaceutical (Denmark) Human amniotic fluid surfactant NA Human amniotic fluid at term University of California, San Diego, USA, Univesity of Helsinki, Finland Infasurf Calfactant CLSE Bovine-lung (calf) lavage Forest Pharmaceuticals (USA) Newfacten Bovine-lung Yuhan (Korea) Surfacten Surfactant-TA Bovine-lung homogenate Mitsubishi Pharma (Japan, Korea) Surfaxin Lucinactant Synthetic (DPPC, POPG, PA, KL4 peptide) Discovery Laboratories (USA) Survanta Beractant Bovine-lung tissue Abbot Laboratories (USA) Venticute rSP-C surfactant Synthetic (DPPC, POPG, PA, rSP-C) Altana Pharmaceuticals (UK) Non-protein containing Adsurf Pumactant (ALEC: artificial lungexpanding compound) Synthetic (DPPC, PG) Britannia Pharmaceuticals (UK) Exosurf Colfosceril palmitate Synthetic (DPPC, hexadecanol, tyloxepol) GlaxoSmithKline (UK)

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44 Figure 1-6. Primary amino acid sequence of SP-B and KL4. KL4 is modeled after the charge distribution in residue s 59-80 of SP-B. KL4, SP-B(1-25), and SP-B(59-80) affect the surface activity in lipid monol ayers at air/water interfaces. Basic residues are highlighted in blue and acidic re sidues are highlight ed in red. FPIPLPYCWLC R ALI KR I QAMIP K G S P B (1-25) DTLLGRMLPQLVC RLVLRCSMD SP-B (59-80) KLLLLKLLLLKLLLLKLLLLK KL4 (sinapultide) SP-B FPIPLPYCWLC RALIKRIQAMIP KG ALAVAVAQVC RVVPLVAGGI CQCLAERYSVILL DTLLGRMLPQLVC RLVLRCSMD

PAGE 45

45 CHAPTER 2 BIOPHYSICAL TECHNIQUES TO PROBE PE PTIDE STRUCT URE AND PEPTIDE-LIPID INTERACTIONS This chapter details biophysical techniques us ed in this thesis to study peptide-lipid interactions. These techniques have also been applied in the study of membrane proteins, aggregated proteins (amyloid fibrils), and other complex biom olecular systems. The following descriptions are by no means comprehensive treatments on the rich and detailed history, intricacies, and science behind each technique, but are intended as primers on the information obtainable from each technique in its applicati on to understanding molecu lar interactions and structure in lung surfactant; references are incl uded for the reader wishing to obtain a more thorough understanding. Circular Dichroism Circular dichroism (CD) represents a strai ghtforward method to assess global secondary structure in proteins via detecti ng the interaction of polarized electromagnetic waves (light) with the chiral protein backbone. T ypically, the light source is circul arly polarized light generated by a xenon or a helium lamp and filtered to select a pa rticular circular polarization. Light waves are composed of electric and magnetic field waves propagating perpendicularly to each other. Circularly polarized light requ ires two of these waves (of equal magnitude and frequency) traveling together but 90o out of plane, allowing the magnitude of the electric field to remain constant but its direction to trace the path of a circle. This polarized light differentially excites electronic transitions in proteins leading to CD spectra that ar e reflective of these excitation energies with both positive and negative abso rption. When light passe s through an optically active chiral solution, such as amino acids in a protein, th e right handed and left handed circularly polarized light are absorbed to different levels at each wavelength.

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46 The differential absorption of left handed and right handed circular polarized light is characterized by RL (2-1) and gives rise to the CD signal ( 74-77 ) The raw CD signal is denoted as ellipticity (in units of millidegrees). The CD signal normalized to the protein concentration is denoted as molar ellipticity me and is given by the following equation: lMAA m m ][# deg (2-2) where #AA is the number of amino acids in the protein, [M] is the molar concentration of the protein, m deg is the raw CD signal in millidegrees, and l is the path length of the sample cell cuvette (in centimeters). Protein secondary structures such as -helices, -sheets, and turns have spectra of distinctive shape and magnitude in the far-UV regi on. The overall shape of the spectra is due to the periodicity of the amide bonds inherent in each type of secondary structure. For instance, the CD spectrum for an alpha helix has a minimum absorption at ~190nm representing the excitation of (nb) to as well as double minima at ~205 to ~220nm reflecting the excitation of a lone pair oxygen electron to the state ( 76 ). Typical signature CD spectra found for helix, -sheet and random coil conformations are shown in Figure 2-1. CD spectra have been used extensively to mon itor secondary structure in proteins free in solution as well as for proteins bound to lipid ve sicles at lipid concentrations of 2-10mM ( 7880). Factors such as the concentration of the protein, path lengt h, buffer composition, and dielectric constant of the buffer, temperature and pH play a role in determining the resultant CD spectra obtained. Helical secondary structure can often be induced in unfolded or random coil

PAGE 47

47 protein by addition of helix-enha ncing solvents such as fluo ro or alkyl alcohols including trifluoroethanol (TFE) and he xafluoroisopropanol (HFIP) ( 81 ). Differential Scanning Calorimetry (DSC) Differential scanning calorime try (DSC) is a technique used to study the temperature dependent phase behavior of lipids, due to their tendency to self-assemble in both model and biological systems ( 82). The technique has been used to examine the thermally induced transition of lipids from an ordered crystalline state (L) to the disordered liquid-crystalline state (L), which occurs at a characteristic temperature Tm for a particular lipid or lipid mixture ( 82, 83). Such a transition is char acterized by an increase in the trans-gauche isomerization rates along the fatty acyl chains, a decrease in bilayer thickness, the onset of axial diffusion, and an increase in the lateral cross section occupied by the phospholipid molecules ( 82, 84). Pure phospholipids have sharp symmetric phase transitions indicating the gel-liquid crystalline transition is a first order process; the Tm at which this transition occurs depends on the headgroup composition, fatty acyl chain lengths, and the de gree of saturation in th e fatty acyl chains. Broad, asymmetric DSC peaks are commonly seen in biological membranes where appreciable headgroup and acyl chain heterogeneity is present. While the L to L transition is the most common transition measured by DSC, other phase changes such as pre-melting transitions, liquid-liquid phase separati ons, and domain formations have b een seen and characterized by this technique ( 15, 82, 85, 86 ). The principle of DSC is relatively straightforw ard (Figure 2-2). A sample cell (containing sample) and a reference cell (containing solvent) are heated at very controlled rates. As the temperature is increased linearly, the temperatur e difference between the reference and sample cell is kept at zero via a feedback loop. This is done by heaters connected to the sample and reference cell. When the sample undergoes a thermally-induced even t, such as a phase

PAGE 48

48 transition, a temperature differential is sensed between the reference and sample cell and corrected by varying the power input to the individual cells. The power required to maintain both cells at the same temperature scan rate is measured. This raw diffe rential power signal is converted to heat capacity at constant pressure (Cp) and graphed versus the sample temperature ( 82, 87, 88). More accurately denoted, th e DSC instrument measures the excess specific heat, the amount by which the apparent specific heat of a particular solute transition exceeds the baseline specific heat required for heating the reference cell. Figure 2-3 illustrates the output generated after the excess specifi c heat is converted to heat capacity by the DSC instrument. DSC thermograms can be analyzed to extract the thermodynamic parameters for a lipid phase transition. The calorimetric enthalpy Hcal of a transition is obtained by integration of the peak related to the transition peak: ( 84 ) dCHT T calp2 1 (2-3) The vant Hoff enthalpy is the enth alpy calculated as a function of Tm; assuming a twostate process, the formula for HvH is: ( 89) cal p m vHH C RTH max 24 (2-4) where R is the universal gas constant and Cp max is the peak heat capacity measured for the transition. Another parameter which can be extracted is the cooperativity of the transition between phases. In some cases the phase transition can be instantaneous and the DSC thermogram will be highly dependant on the scan rate. In other sc enarios, the process might first be initiated by domains or islands of lipids a nd gradually spread throughout the sample as the temperature is changed. The cooperativity can be assayed by co mparing the values of the derived enthalpies

PAGE 49

49 Hcal and HvH ( 89) and evaluating their ratio HvH / Hcal. When this ratio is less than unity, intermolecular cooperation domina tes the transition mechanism; however, when the ratio is greater than unity, intermediate states are significantly populated. The effects that exogenous agents such as chol esterol, proteins and antimicrobial peptides have on the Tm of model lipid systems have been extensively investigated using DSC ( 79, 9092). DSC has also been used to delineate pep tide induction of alternat ive lipid polymorphisms, such as the HII phase, which has been shown to occur when transmembrane -helices enriched in the amino acid leucine are added to lipids (93). (This is of particular importance since the peptide under study (KL4) has a high percentage of leucines). Thus, DSC is a powerful tool to study the effects peptides have on the thermo tropic phase behavior of lipid systems. It should be noted that the p re-transition peak often seen in DSC on pure lipids (such as that seen in Figure 2-4) has been fully evaluated in terms of th e structural changes within the bilayers. For phosphatidylcholines, the pr etransition occurs in the range of ~5o below the main phase transition temperature and has been denoted the P or ripple phase. Since it is due to a transformation from a pure lamellar phase to a tw o-dimensional monoclinic lattice, in which the bilayer contains undulations (86, 88, 94 ). Addition of peptides or other exogenous agents have been shown to abolish or modulat e the appearance of this pre-tr ansition phase. Also shown in Figure 2-4a is the information content yielded from a DSC thermogram. The Tm corresponds to the temperature where the maximal heat capacity Cp max of the system is observed for each transition; the integration of any peak reflects Hcal, and the PWHH or T1/2 measures how broad the phase transition is. A broad peak (which implies a large PWHH and hence a greater temperature span or T1/2) indicates less lipid cooperativity du ring the phase tran sition. Adding

PAGE 50

50 monounsaturated lipids to pure DPPC has conse quences in terms of thermodynamics, lipid cooperativity and phase transition temperature (Figure 2-4b). Solid state NMR Spectroscopy Solid state NMR spectroscopy (ssNMR) has advan ced significantly as a tool to study the structure and dynam ics of solid systems ranging from glasses to polymers to catalysts. Advances and progress in the field have now been incr easingly focused on developing methodologies for complicated biomolecules such as membrane proteins ( 95, 96 ). Unlike solution NMR, where narrow resonances result due to rapid reorientation of molecule s and averaging of anisotropic interactions, ssNMR must deal with unaveraged interactions ( 97, 98). On the NMR time scale (Figure 2-5), interacti ons such as chemical shielding, dipole-dipole coupling, and quadrupole coupling are time aver aged in the solution state by the rapid reorientation of the molecule. Slower motions of molecules in the solid state give rise to a superposition of many overlapping resonances representing the many possible orientations of the molecules with respect to the magnetic field; the resulting broad spectrum can be referred to as a powder pattern ( 96, 98 ). These powder patterns observed in ssNMR spectra suffer from low resolution, low sensitivity, and (for the most part) ve ry little extractable st ructural information. However, magic angle spinning (MAS), describe d briefly below, can yield liquid-like spectra from solid-state experiments; and clever pu lse sequences during MAS can be used to reintroduce and to measure an isotropic information ( 99). A discussion of time scales used to measure molecular processes by NMR is detailed later in this chapter. Spin Interactions Commonly Seen in ssNMR Chemical shift When placed in a strong ma gnetic field, nuclei in different parts of a molecule experience varying fields due to their elect ronic environment. This phenomenon is referred to as chemical

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51 shielding and is caused by the external magnetic field ( Bo) generating currents in the molecules electron cloud, which in turn gene rates induced fields. The magnit ude of this induced field at a given nuclear site depends on the orientat ion of the molecule with respect to Bo and on the location of the nuclear spin within the molecule. This variation in chemical shielding leads to different nuclei of the same is otope resonating at di fferent frequencies or chemical shifts. In liquids the chemical shielding is averaged and is referred to as the isotropic chemical shift. This chemical shift depends upon the gyromagnetic ratio of the nuclear spin, the shielding by local electron currents, and the influence of local low-lying electronic ground states ( 100 ). For a single crystal sample, the NMR spectrum l ooks similar to a solution NMR spectrum in resolution (neglecting di pole-dipole couplings) but chemical sh ifts depend on the orientation of the crystal with respect to Bo. Therefore, by rotating the crysta l relative to the magnetic field, the resonance positions change. In powder samples, where all such orientations are present, the chemical shift spectrum is broad reflecting th at it is a superposition of spectra from each crystallite. This is described in more detail below. Dipole-dipole couplings The interaction of nuclear spins with one anot her also affects NMR sp ectra. Since each NMR active nucleus has a m agnetic moment, each spin experiences the field generated by other nuclear spins nearby. This interaction is called the (direct) dipolar coupling. Dipolar couplings (d) are through-space intera ctions and provide important structural information. The dipolar coupling is directly proportional to the gyromagnetic ratio of each participating spins (1 and 2) and inversely proportional to the cube of the distance between the spins (r) (100 ). The spatial dependence of the dipola r coupling with respect to the external magnetic field is accounted for in the angular term 2 1cos32 :

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52 3 2 21)1cos3( 2 1 r d (2-13) The gyromagnetic ratio describes th e size of the Zeeman interaction for a spin with respect to the magnetic field and determines its Larmor frequency. For example, the value for a 1H spin is 2.68x108 rad/sec/Tesla (101 ), corresponding to 600MHz in a 14.1T field. In a powdered NMR samples, dipolar, quadrupolar, and (i n part) chemical shift interactions scale by a factor of -0.5 to 1 based on the angular dependence of these interactions on the term (3cos2 -1)/2 ( 102, 103 ). Quadrupole couplings Quadrupole couplings represent the interactions of spin > 2 1 nuclei with the electric fields from their non-spherical nuclear charge distribution. The measure of a nucleuss effective ellipsoidal shape is called its quadrupole moment (Q, often reported as eQ). The strength of the quadrupole coupling is a good measure of a nucleuss mobility. For example, in this study, spin 1 deuterium (2H) has a quadrupolar coupling constant: h qQe2 used to assess the mobility of lipid acyl chains where e is the charge of an electron, q is the principle component of the electric field gradient tensor, and h is Planks constant. Applications and Methodologies in Solid-State NMR Pake Powder Pattern Large biomolecular systems such as membrane proteins, polymers, or inorganic complexes give rise to difficulties in structure determina tion because the orientation dependencies of the NMR interactions described above are not time averaged. Resultant NMR spectra yield broad lineshapes known as Pake powder patterns or powder spectra (104), after George Pake who derived the equations for these patt erns in the late 1940s. A typical 31P chemical shift spectrum for the phospholipid DPPC above its Tm is shown in Figure 2-6 and shows a powder pattern

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53 over a range of frequencies covered due to multiple orientations of the lipids with respect to the external magnetic field (98, 105). Below a phospholipids Tm, the motion of the phospholipid is non-axial and results in a spectrum shown in Figur e 2-7. In lipid dispersions taken above the phase transition temperature the ons et of motion about an axis of symmetry leads to a lineshape that has distinct parallel edge and perpendicular edges relative to the static magnetic field. All other intermediate orientations of the symmetry ax is relative to magnetic field are also enveloped in the powder pattern. The relative intensity at each point in the pattern is based on the probability distribution of that orientation. For a phospholipid dispersion, the powder pattern can take different lineshapes, not only dependant on Tm, but also dependent on the dynamics present for various lipid polymorphisms, such as hexagonal or inverted hexagona l phases (Figure 2-9). Chemical Shift Anisotropy (CSA) In an unoriented sample, many orientations of a particular molecule to the magnetic field are present. The chemical shifts seen are dependent on the distribution of orient ations in the sample. It is the chemical shift an isotropy that determines the 31P spectra in this work. The chemical shift anisotropy for a spin nucleus (such as 31P) can be described in its principle axis system by th ree values, designated as 11, 22, and 33 (Figure 2-7). By convention, 33 has the largest difference from the is otropic value and lower intensity and corresponds to the parallel e dge in the powder pattern, 22 corresponds to the hi ghest intensity of the powder pattern, and 11 has an intermediate intensity a nd corresponds to th e perpendicular edge in a powder pattern. The isotropic peak, iso, seen in MAS spectra and solution NMR, is equal to the average of the principal values. Th ese values are dependent on the electronic field surrounding the nucleus, which shield s the nucleus from the external magnetic field. Since the shielding is three dimensional, it is represen ted by a second rank tens or where the principal values correspond to the diagonal elements in a principal axis system (PAS), where this 3x3

PAGE 54

54 matrix is diagonalized. This interaction tensor ca n be visualized as an ellipsoid whose center is the active NMR nucleus of interest and the axes of the ellipsoid coincide with the principal values of the CSA tensor in Cartesian space. If the molecular orientation changes with respect to the magnetic field, then so does the nuc leus and its corresponding CSA tensor (98) (Figure 2-8). For phospholipid bilayer assemblies, a symmetry axis due to rotationa l averaging around the director axis perpendicular to the plane of the bilayer, (or the bilayer normal) avereages the 31P CSA leading to axial symmetry where 11 = 22 as shown in Figure 2-5 (94). This onset of motion causes rotational averaging of the 31P CSA tensor around the direct or axis and as a result, the z-axis of the time averaged CSA, or 33 PAS, coincides with the bilayer normal and remains unchanged. In such a scenario, =< PAS 22> . The isotropic chemical shift iso is defined as the average of the th ree main components of the tensor ) ( 3 133 22 11iso (94, 98) (2-14) and corresponds to the frequency if the samp le were tumbling isotropically in solution. The lower than usual signal at seen in Figure 2-6 is due to the fact that DPPC lamellae orient in high magnetic fields which is addressed below. Magic Angle Spinning NMR If the NMR sample is spun at a magic angle (54.74o) relative to the magnetic field, the orientation dependent term, (3cos2-1), is zero. At fast e nough spinning speeds, dipolar couplings and chemical shift anisotropies (CSA ) can be completely averaged, thus removing their anisotropic effects in resulting NMR spectra (95). MAS thus improves resolution and subsequently the applicability of ssNMR to complicated biomolecular spectra (99). Figure 2-10 illustrates the phenomenon of MAS and its effects on a chemical shift spectra (106). However, with the averaging of dipolar couplings, important structural information is lost. But

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55 dipolar couplings that are av eraged under MAS conditions can still be observed by the introduction of radiofrequency pulses synchroni zed with the spinning of the sample (dipolar recoupling). Pulse sequences such as DRAMA (dipolar recovery at the magic angle) use /2 RF pulses applied twice per rotation period to interrupt the averaging of th e dipolar coupling caused by MAS (95). A variation of the DRAMA sequence called DRAWS (dipolar recoupling with a windowless sequence) uses additional pulses to retain more of the di polar interactions, while still averaging CSA interactions. DRAWS is therefor e more effective for nuclei with large CSAs such as carbonyls (Figure 2-11). Structural aspects of the molecule in questi on can also be determin ed independent of the application of external RF pulse s, simply by modulating the speed of the spinning rotor while the sample is under MAS conditions. The presence of spinning side bands at spin rates of 30004000 Hz allows the determination of CSAs. Thus observation of any molecular motions of peptides in lipid MLVs as a functi on of sample composition is possible. Range of NMR Time Scales While solid state NMR is used to look at molecules that are slowly tumbling on the NMR time scale, it is important to realize that NMR can probe molecular motion over a wide array of time scales ranging from picoseconds to several s econds. The following sect ion serves as a brief primer on the vast ranges of frequencies that an NMR experiment can detect. The internal dynamics of molecule s allow nuclei to oscillate to a net average position. These molecular vibrations are on the or der of picoseconds or shorter. Internal dynamics within the molecule allow for large rotation of substituents within the molecule as well, such as a methyl group or an amine. If the substituent has an axis of symmetry such as around a local three-fold axis existing in a CH3 group, then such motion can be on the order of internal molecular vibrations (picoseconds), but if the same CH3 group is hindered by another nearby substituent,

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56 as can be expected during the folding of a protein or in a lipid bilayer environment, then such rotational motion can occur on the timescale of milliseconds to seconds. Hence, the boon of NMR stems from its ability to detect molecular motions as well as the nu mber of molecules in different states (100). Time scales which can be probed using NMR experiments are vastfrom hundreds of picoseconds (molecular rotations ), to many seconds (macroscopic diffusion and chemical exchange processes) (Figure 2-5). Dependi ng on the sampling time during signal acquisition, one can discriminate between types of motion being measured. Very fast motions in the picosecond to nanosecond timescale are on the orde r of the Larmor frequency representing the shortest time available to specifically encode informationanything faster is encoded as an average. However, NMR is not generally sample d this quickly and the totality of different actions must also be considered. For example, vibrational motion and molecular rotation can average out direct dipole-dipole couplings (o ften in the millisecond domain) in liquids (100). Motions on this timescale often form the basis for T1 relaxation measurements. Motional time ranges on the order of microsecond to milliseconds affect the typical spectral timescale strongly. As a result, lineshape pertur bations such as a broa dening, are seen. An example of the changes in NMR spectra within this regime is hindered rotation. In this type of experiment, a molecule has two (or more) non-eq uivalent conformations with a significant energy barrier between them. The first NM R experiment is run cold and shows each conformation as a distinct reso nanceindicating that their intraconversion is slow on the timescale. As the temperature is raised, the spect ra first broaden and then coalesce into a single peakindicating that their intraconv ersion is fast on the timescale.

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57 Lastly, molecular motion that ranges from millis econds to seconds can also be probed from NMR. Macroscopic diffusion or fl ow occurs via the transport of molecules from one region of space to another occurs on this timescale. Such a phenomenon forms the basis of diffusion NMR, whereby an inhomogenous magnetic field is generated and used to quantitate molecular diffusion and flow. Depending on what frequency of mo tion one is interested in ex amining, the NMR experiment can be correspondingly devised. For natural abundance 31P NMR experiments, the timescale of motion being probed are on the order of 10-9 to 10-11 seconds. The frequency range of a 31P CSA span is 50 ppm or approximately 30,000 Hz for an NMR experiment run at 600MHz. Headgroup vibrations, lateral diffu sion, and rotational motion along th e lipid axis are faster than this, so their effects are seen as primarily narrowing the spectra to an average value. Likewise, the frequency range of quadrupolar interaction are on the order of approximately 167,000 Hz. The 2H NMR experiments, looking at the dynamics of the acyl chain also involve motions that are faster than the spectral frequency so again, averaging of the spectra components are seen. Finally, in solids there is incomplete motional averaging of the internal spin interactions, so both intramolecular and intermolecular spin interactions retain their dependence on the orientation of the sample with respect to the magnetic field (100). NMR-Active Nuclei and Import ance to Biological Molecules 31P (Phosphorus) NMR Phosphorous is the spin NMR-active nucleus that gives rise to the powder lineshape mentioned above. 31P NMR is one of the tools that can be used as a non-perturbing probe to measure the orientation and conformation of th e phosphate headgroup in a lipid molecule (94). Another advantage of 31P NMR is that no isotopic incorporation is necessary since the target nucleus is 100% naturally a bundant. Furthermore, dipole-di pole interactions between 31P and 1H

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58 in phosphates are very small, and can easily be decoupled. As detailed above, lipid geometries important in membrane-membrane mediated even ts such as the lamellar and hexagonal phase, are sensitive to the onset of axial diffusion and molecular motion. These can be discriminated by 31P NMR; also, the technique is sensitive to head group orientation and charge interaction effects. 31P NMR spectra of small, sonicated vesicles have a single resonance at their isotropic chemical shift. This is due to the ve sicles being small enough to tumble rapidly on the NMR timescale. The tumbling rate of 4000 lipid molecules in a 30-50nm small unilamellar vesicle of radius 250 has been estimated to be ~1MHz. This is in stark contrast to lamell ae which have a tumbling frequency estimated at <1Hz (107). Sharp peaks at the isotropi c chemical shift seen in NMR spectra of vesicles are also due to a sma ll radius of curvature and lateral diffusion (107). The types of lipid polymorphisms described above have had their spectra distinguished and characterized by 31P NMR (108). Lipid phases seen by 31P NMR are essentially field independent; however, orientati on of lipid assemblies can caus e variations in the relative intensities of and edges with higher Bo fields (94). While 31P NMR of phospholipids ensembles yield specific lineshapes due to CSA interactions, it is also critical to mention that sharp, nearly Lorentzian lineshapes at a particular resonance frequency can occur on hydrated phospholipids if th e assemblies are highly ordered with respect to the magnetic field. Similarly, single crys tals of phospholipids produ ce single Lorenztian lines, which when the crystal is rotated with respect to the magnetic field, change their resonance position. In fact, this has been exploited by many research labs th at create oriented bilayers on glass plates, giving rise to a single orientation of the bilaye rs. The glass plates can then be placed in the magnet with the director axis perpendicular to the Bo yielding a single resonance at the perpendicular edge of the oriented lineshape. This approach has also been pivotal in using

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59 15N spectroscopy to determine the orientation of 15N backbone labeled peptides in bilayers (106, 109-111). The potential detergent and lyti c properties of antibiotics a nd anti-microbial peptides have also been examined by 31P NMR. Addition of molar percenta ges of down to 4 mol% of these molecules have been found to lead to an appearance of an isotropic peak in 31P spectra indicating significant membrane degrad ation or perturbation (112, 113). In addition to assessing the geometry and polymorphisms of phospholipids phases, 31P NMR has found use in a multitude of applications including muscle and tissue research diagnosing epileps y, and in specif ic sectors of the food industry (114-116). An unoriented static 31P NMR spectrum of a phospholipid is due to the CSA of the 31P nucleus in the phosphate headgroup. CSA values have been measured for various phospholipid headgroups in crystallized lipids (94). Furthermore, the size and sign of the phosphate CSAs can differentiate between different lipid geometries. As discussed above, lamellar phase lipids have asymmetric spectra below their Tm due to restricted motion, and above their Tm, have axially symmetric lineshapes which are approximately 50 ppm broad. Another lipid mesophase, the hexagonal phase, has an additional axis about which the averaging of the CSA occurs. The hexagonal phase describes lipids packed as elongated cylindrical structures. It is divided into two types: the HI and HII phase. The HI phase contains a cylindrical array of lipid molecules with the polar head group facing an aqueous exterior and the acyl chains facing inward. In the HII phase, or inverted hexagonal phase, the headgroup face an inner aqueous cavity of water and the acyl chains face outward stacked in a tubular form (15, 94). Figure 2-9 shows lipids in lamellar and HII phases and their resulting 31P NMR spectra. Lipids arranged in a hexagonal phase have the same motions as lamellar phases, and an additional degree of motional averaging

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60 occurs due to lateral diffusion of th e lipids about the cylindrical axes (94). The spectral lineshape for a hexagonal phase is half the width and opposite in si gn to the corresponding lamellar phase since this motion is perpendicu lar to the axial diffusion of the individual molecules. 2H (Deuterium) NMR Deuterium (2H or D) is a spin 1 nucleus with a natural abundance of 0.016%. Hence, any NMR signal obtained from sample unambiguously be longs to deuterons speci fically incorporated into the molecule of interest provide d deuterium free solvents were used (107, 117). Due to the spin 1 nature of the deuterium nucleus, 2H NMR powder spectra are characterized by two overlapping and opposite in sign lineshapes, due to the two possible transitions -1 0 and 0 1. These lead to characteristically symmetri c perpendicular peaks, often referred to as the Pake doublet (102). Deuterium spectra of acyl chain deuterated lipids provide structural information since averaging of the Pake powder patterns depends on motions of the individual methylene segments (118). The time averaged order parameter can be assigned for each C-D bond along the length of the acyl chain. This order parameter is a discrete value assigned for the collective ensemble motions that occur for each C-D bond and average the powder spectrum for that position. Such a value provides information on the internal motions of the fatty acyl chain. Motions of phospholipids pertinent to the NMR time scale (10-5 seconds) which average the quadrupolar interaction include oscillatory motions of bond lengths, bond angles, and torsion angles (10-12 seconds), gauche/trans isomerizations (10-10 seconds), axial diffusion (10-8 to 10-9 seconds), fluctuations of the director axis known as wobble (10-8 seconds), and translational diffusion along bilayer surfaces (10-7 seconds) (84, 107). The motion of a phospholipid molecule can be approximated as a cy linder that axially rotates (119) with each C-D bond wobbling as a

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61 cone along the length of the acyl chain (Figure 2-12) and determ ines overall averaging of the 2H patterns at all positions along the acyl chain. Additional gauche/trans isomerization motions affect the averaging at each C-D along the acyl chain differently since there is less steric hindrance encountered by methyl ene segments going toward the methyl terminus. The order parameter for a specific methylene position labeled i is defined with a second order Legendre polynomial 1cos3 2 12 i iS (2-15) where i is the angle between the symmetry axis of the molecule and the C-D chemical bond. The brackets indicate a time-average ensemble of all molecular motions of the C-D bond that are fast on the NMR time scale (107, 119-122). The bilayer normal is coincident with the symmetry axis for lipids in a fluid lipid bilayer due to the fast axial rotation of the individual molecules (84). The deuterium NMR spectra of lipids with pe rdeuterated acyl chains contain powder patterns from all the positions in the acyl ch ains. Full deuteration of both the sn-1 and sn-2 acyl chains of DPPC results in 30 2H NMR lineshapes correlating to each methylene position in each of the acyl chains. Each lineshape is also symmetric, with a Pake doublet or quadrupolar splitting (vq, measured in kHz) that can be used to calculate the order para meter for that particular acyl position (Figure 2-13). The frequency splitting vq for each C-D position is used to calculate the time averaged order parameter for the position according to the following simple relation: CD qS kHz 2 1673 (2-16)

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62 The quadrupolar coupling constant expected for a deuterium atom in a saturated C-D bond in the static limit is 167kHz (84). This value was determined by measuring deuterium quadrupolar coupling constants on deuterated paraffins such as ethane and butane at low temperatures (123). A numerical procedure, termed dePaking, is us ed to deconvolute the spectra. The methylene positions can then be easily assigned since they give rise to single resonances rather than lineshapes. These assignments can then be used to create an order parameter profile. Resonance assignments for each position were initially done by selectively deuterating individual positions along the acyl chain and m easuring the NMR spectra (117, 118). From the dePaked 2H NMR spectra, a characteristic order parameter prof ile is generated by gra phing the calculated values against the methylene positions in the acy l chain (Figure 2-14). The order parameters decrease as the carbon number of the acyl chain is increased reflecting the increased motional freedom of the acyl chains near the center of the bilayer (121, 124). DePaking As stated above, dePaking is a numerical dec onvolution procedure that takes the lineshape arising from randomly oriented mo lecules and converts it to an oriented spectrum. The procedure can be applied to any lineshape resulting from the spatial dependence of the spectrum having the form of a second order Legendre polynomial, i.e. ( 2 1cos32). Through a mathematical transform, the spatial component is removed resulting in an oriented spectrum in which the signal is refocused at the frequency corresponding to the parallel edge of the powder lineshape (125). For our work, the dePaking procedure employs an algorithm provided by Edward Sternin and colleagues at Brock University, which has been used to generate both profiles and determined the extent of spontaneous magnetic field orientation of lipids (102, 103). The result of the transform is a frequency spectrum

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63 corresponding to when the lipid bilayer normal is parallel to Bo (126, 127). This is the equivalent to the parallel edge for each C-D powder pattern in 2H NMR and likewise the parallel edge for each lipid phosphorous moiety in 31P NMR. An example of dePaked 2H and 31P spectra are shown for 4:1 DPPC(d-62):POPG in Figur e 2-13b and Figure 2-15 respectively. By measuring the dePaked frequency as a function of added agent such as a peptide, the extent and level of interaction of the lipids with the additive can be probed. Magnetic Field Orientation An additional complexity arising when interpreti ng NMR spectra for lipids being placed in large magnetic fields is macroscopic lipid alignment, particularly for lamellar phase lipids. The macroscopic orientation is due to the negative diamagnetic susceptibility ( <1) of phospholipid molecule assemblies (128). The phenomenon of a preferred orientational alignment has been well documented in the literature for many phospholipids classes, ranging from synthetic to biological extracts from E.coli and has been observed at field strengths as low as 7T (300 MHz for 1H) (103, 129-132). This complicates analysis of NMR spectra, particularly dePaking algorithms, since the typical powde r distribution of angles no l onger holds. The formulae and mathematical procedures involved when the powde r pattern is distorted by lipid alignment are discussed in Chapter 3. When multilamellar vesicles orient, the spherical shape becomes distorted to a geometry that is ellipsoidal (Figure 2-16). The de Paking algorithms used take into account the fact that the probability distributi on of the bilayer normal vector is no longer proportional to sin( ), as it would be for a totally random distribution of MLVs. To account for this discrepancy, the dePaking algorithm written by Professor Edward Sternin generates a orientation parameter, which accounts for the extent of spontaneous orientation of the lipid vesicles in the magnetic field Bo, assuming the MLVs deform to more ellipsoidal geometries.

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64 Figure 2-1 CD spectra from various secondary structure elements. Reproduced with permission from Omjoy K. Ganesh.

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65 Figure 2-2. Schematic of a differential scanning calorimeter (DSC). Information pertaining to its operation and output are described in Chapter 2. Temperature Sensor Reference Cell Sample Cell Heater Heater Adiabatic shield Electronics to regulate heat to sample and reference cells Data Output Computer Interface Temperature Sensor Reference Cell Sample Cell Heater Heater Adiabatic shield Electronics to regulate heat to sample and reference cells Data Output Computer Interface

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66 Gel (L) Liquid-crystalline (L) Tm Gel (L) Liquid-crystalline (L) Tm Figure 2-3. The gel to liquid-cr ystalline phase transition of phospholipids bilayers. When the temperature of a phospholipid dispersion is above its characteristic main phase transition temperature, Tm, the acyl chains acquire additional degrees of freedom causing the individual molecules to freely ro tate and an expans ion of the bilayer volume occurs.

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67 25303540455055 0 2 4 Cp (kcal/mole/oC)Temperature (oC)41.6oCCp max T1/2 HcalTmP |(A) (B)20 30 40 50 60 0 1000 2000 3000 4000 5000 Heat Capacity ( cal/mole/oC)Temperature (oC) DPPC 4:1 DPPC:POPG 25303540455055 0 2 4 Cp (kcal/mole/oC)Temperature (oC)41.6oCCp max T1/2 HcalTmP |(A) (B)20 30 40 50 60 0 1000 2000 3000 4000 5000 Heat Capacity ( cal/mole/oC)Temperature (oC) DPPC 4:1 DPPC:POPG20 30 40 50 60 0 1000 2000 3000 4000 5000 Heat Capacity ( cal/mole/oC)Temperature (oC) DPPC 4:1 DPPC:POPG Figure 2-4 A) DSC thermogram of 2mM DPPC large unilamellar vesicles (LUVs) dispensed in 5mM HEPES pH 7.4. Scan rate was at 1 degree per minute and temperature scans were from 10 to 70 degrees Cels ius. The phase transition at 41oC indicating the L to L is clearly shown. Th e pre-transition or the P phase is also shown. B) The effect on the DSC thermogram by the addition of monounsaturated POPG to DPPC LUVs.

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68 SLOW FAST very slowslowfastvery fast ultra-fast fs ps ns s ms s MACROSCOPIC DIFFUSION FLOW CHEMICAL EXCHANGE MOLECULAR ROTATIONS MOLECULAR VIBRATIONSLarmor Spectral RelaxationTimescale: SLOW FAST very slowslowfastvery fast ultra-fast very slowslowfastvery fast ultra-fast fs ps ns s ms s MACROSCOPIC DIFFUSION FLOW CHEMICAL EXCHANGE MOLECULAR ROTATIONS MOLECULAR VIBRATIONSLarmor Spectral RelaxationTimescale: Figure 2-5. Time scale of motional processes for nuclear spins in NMR.

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69 -40-30-20-10010203040 Chemical Shift (pp m ) 33= 22= 11= Perpendicular edge of powder pattern Parallel edge of powder pattern Bo -40-30-20-10010203040 Chemical Shift (pp m ) 33= 22= 11= Perpendicular edge of powder pattern Parallel edge of powder pattern Bo Bo Figure 2-6. 31P NMR static spectrum of DPPC hydrated vesicles (approximately 60mg). The spectrum was taken at 44 degrees on a 600M Hz Bruker instrument with 1024 scans. Above its phase transition, Tm, the powder pattern takes on a shape with axial symmetry. A clear perpendicular and parallel edge are seen.

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70 -75-50-250255075 Chemical Shift (ppm)332211 ) ( 3 133 22 11iso-75-50-250255075 Chemical Shift (ppm)332211 ) ( 3 133 22 11iso Figure 2-7. 31P NMR of static hydrated DPPC vesicles (approximately 60mg) taken on a 600 MHz Bruker instrument (1024 scans) at 240C, well below its phase transition temperature, Tm. Below Tm, the powder pattern reflects in complete averaging of the asymmetric 31P CSA tensor. Shown are the prin cipal values of the CSA.

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71 Bo Bo Nucleus at site of symmetry Phospholipid molecule Bo Bo Nucleus at site of symmetry Phospholipid molecule Figure 2-8. Graphical depi ction of shielding tensor for a nucleus with an axis of symmetry. The CSA tensor is illustrated as an ellipsoid with the center bein g at the nucleus of interest, in this case the phosphate headgroup of a phospholipid. When the molecular orientation changes, so does the orientation of the interaction tensor with respect to the magnetic field. For liquid-crystallin e phospholipids in a lamellar phase, the symmetry axis is the bilayer normal. Shown in orange and green are principal axes of the ellipsoid, while the dark black line is the axis of rotation. The phosphate headgroup is shown as a red sphere and wa vy lines represent the acyl chains.

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72 5 005 0 Chemical Shift (ppm) Bilayer HIIphase -50 0 50 Chemical Shift (ppm) 5 005 0 Chemical Shift (ppm) Bilayer HIIphase -50 0 50 Chemical Shift (ppm) Figure 2-9. Phosphorous NMR lines hape patterns for lipid mesophases. Shown above is the lineshape for an inverted HII hexagonal phase common in many lipid-lipid mediated events. The isotropic peak at 0 frequenc y represents partial degradation of the sample. Below is the standard powder patt ern seen in the bilayer lamellar phase.

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73 3 kHz 1 kHz 100 150 200 250ppm 6 kHz 8 kHz 3 kHz 1 kHz 100 150 200 250ppm 6 kHz 8 kHz Figure 2-10. Magic angle sp inning (MAS) spectra of the 13C nucleus in glycine. Sample was packed in a rotor and spun at the magic angle of 54.74 degrees at spin speeds between 1 and 8kHz. Clearly seen are the spinning side bands which gra dually disappear as the spin speed becomes comparable with the CSA; the isotropic peak is seen in the center. Data provided with permi ssion from Dr. Manish Mehta.

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74 2 CP CP 2 2 2 R DRAWS 2 n 2 DRAWS n t12 R CW Decoupling R RRR 2 x2 -x 2Y 2 x2 x2 -x2 -x2 -x2 x 2Y 1H13C R t2 DRAWS Pulse Sequence 2 CP CP 2 2 2 R DRAWS 2 n 2 DRAWS n t12 R CW Decoupling R RRR 2 x2 -x 2Y 2 x2 x2 -x2 -x2 -x2 x 2Y 1H13C R t2 DRAWS Pulse Sequence Figure 2-11. DRAWS pulse sequence of DRAWS em ployed during MAS for dipolar recoupling. Each rectangle represents a RF pulse that rotates the magnetiza tion either 90 or 360 degrees. CP is cross-polariza tion, the transfer of magneti zation from protons to lower gamma 13C nuclei. R and R corresponds to a MAS rotor period.

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75 Headgroup Tail C DD C D D Headgroup Tail C DD C D D C DD C DD C D D C D D Figure 2-12. Collective motions of each methylene position along the acyl chain roughly averages out to a conicall shape. The met hylenes near the hea dgroup trace out a cone of smaller radius while due to greate r motional freedom and lack of steric interference, the acyl chains at the terminal end trace out a cone of larger radius. Shown on the right are the diffe rence orientations of one C-D bond. With respect to other carbons in the chain the C-D bonds can be all trans or gauche-trans-gauche.

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76 -20-15-10-50 5101520Frequency (kHz) -30-25-20-15-10-5051015202530 Frequency (kHz)(A) (B) -20-15-10-50 5101520Frequency (kHz) -30-25-20-15-10-5051015202530 Frequency (kHz)(A) (B) Figure 2-13. Deuterium spectra of (4:1) D PPC:POPG(d-31) (mol/mol) with POPG fully deuterated on the sn-1 acyl chain. Spectrum was taken on a 600 MHz Bruker instrument with 1024 scans. Each pair of peaks corresponds to the perpendicular edges of a powder pattern for a methylen e position along the acyl chain. The most intense peaks near 0 frequency corres pond to the terminal methyl group while overlapping peaks with the largest quadrupolar splitti ng corresponds to C-D bonds near the headgroup region. B) The corre sponding dePaked spectrum allowing clear determination of the order parame ter at each methylene position.

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77 0 0.05 0.1 0.15 0.2 0.25 1234567891011121314151617 carbon numberorder parameter 0 0.05 0.1 0.15 0.2 0.25 1234567891011121314151617 carbon numberOrder parameter 4:1 DPPC(d-62):POPG 3:1 POPC(d-31):POPG 0 0.05 0.1 0.15 0.2 0.25 1234567891011121314151617 carbon numberorder parameter 0 0.05 0.1 0.15 0.2 0.25 1234567891011121314151617 carbon numberOrder parameter 4:1 DPPC(d-62):POPG 3:1 POPC(d-31):POPG Figure 2-14. Example of order parame ter profile generated from dePaking 2H NMR spectra, for perdeuterated acyl chains. The time averag ed order parameter for each methylene along the fatty acyl chains were calculated from spectra of 4:1 DPPC(d-62):POPG (above) and 3:1 POPC(d-31):POPG) (below). The order parameter profile for the sn-2 chain of DPPC is shown although both chains were deuterated. Only the sn-1 chain of POPC shown was deuterated.

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78 -60-50-40-30-20-100102030405060 Chemical Shift (ppm) DPPC(d-62) DePaked Figure 2-15. Example of 31P NMR (blue) and the dePaked result (red). From the dePaking, a single frequency, corresponding to the para llel edge of the pow der pattern, is generated. This spectrum was collected on 25mg of DPPC(d-62) on a 500MHz spectrometer with 3072 scans at 44 degrees.

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79 )sin()( p 22 E]cos)1(1)[sin()( pa bBo Bo 2 b a )sin()( p )sin()( p 22 E]cos)1(1)[sin()( pa bBo Bo 2 b a Figure 2-16. Lipid vesicles when placed in a ma gnetic field can deform to form an ellipsoidal shape. An orientational order parameter designated is the square of the ratio of the minor to major axis of the el lipsoid and yields information pertaining to the extent of magnetic field alignment by phospholipid molecules. A random distribution, as seen in a classic powder patter n, gives rise to intens ities proportional to sin( ), where is the angle between the bilayer normal and Bo. When lipids align, the probability of finding a particular bilayer nor mal orientation relative to Bo changes and is proportional to the orientational order para meter, according to the equation shown at the bottom. Equations taken from Ster nin E in J Magn Res on. 2001 Mar;149(1):1103.

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80 CHAPTER 3 SURFACTANT PEPTIDE KL4 DIFFERENTIALLY MODULA TES LIPID COOPERATIVITY AND ORDER IN DPPC: POPG AND POPC: POPG LIPID VESICLES The following is a manuscript in preparation to be submitted to the journal Biochemistry. The final version submitted may differ from what is presented in this dissertation due to revisions and corrections during th e peer review process and jour nal format specifications. KL4 is a 21-residue peptide employed as a func tional mimic of lung surfactant protein SP-B, an essential protein which lowers surf ace tension in the alveol i. In this study, 31P and 2H NMR were utilized to study the effects of KL4 on lipid organization in 3:1 POPC: POPG and 4:1 DPPC: POPG MLVs. NMR spectra recorded at 14.1T indicate a high degree of lipid alignment, particularly for DPPC: POPG MLVs. The addition of KL4 decreases this alignment in a concentration dependent manner. 31P NMR spectra of the phos pholipids clearly indicate KL4 affects the orientation of the anionic hea dgroups in a concentration dependant manner. 2H NMR spectra of the deuterated lipid acyl chains clearly show KL4 effects the ordering of the bilayer interior in a manner which is dependant on the degree of satura tion in the fatty acid tails. Substantial increases in the acyl chai n order parameters were observed in 2H NMR spectra of DPPC(d-62):POPG MLVs with increasing levels of KL4. The largest changes in order occur at carbon acyl position 9-15 suggesting the peptide deep ly penetrates into DPPC:POPG bilayers. Conversely, 2H spectra of POPC(d-31):POPG, DPPC:POPG(d-31), and POPC:POPG(d-31) MLVs showed smaller but measurable decreases in the acyl chain order parameter on addition of KL4. Thus, fatty acid saturation has a mark ed effect on the insertion depth of KL4 into phospholipid MLVs and lipid miscibility. The effects are seen to be approximately linear with KL4 concentrations up to 3mol% peptide, the high est percentage studied. The influence of KL4 on lipid phase transitions was also monitored fo r 4:1 DPPC:POPG MLVs via DSC. Addition of KL4 led to slightly higher lipid phase transiti on temperatures, supporting NMR data suggesting

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81 the peptide causes lipid domain separation in a concentration dependant manner. Based on these findings, a model of how KL4 interacts with these lipids is presented. Relevance of KL4 to Lung Surfactant Biology Lung surfactant is a lipid -rich substance containing key prot eins that lines the inner layer of the alveoli. The primary functions of lung surfactant are to minimize surface tension at the alveolar air-fluid interface and to provide a barrier against disease (2, 15, 60, 133-135). Inadequate protein levels are a leading cause of respiratory distress syndrome (RDS) in premature infants as well as in adults and children experiencing lung trauma or respiratory infections (39, 51, 66). Current therapies for RDS prim arily rely on administration of lung surfactant from exogenous sources (1, 136). This reliance on xenogeni c surfactant is due to the critical role of SP-B, a highl y hydrophobic, 79 residue protein wh ich functions as a homodimer containing 7 disulfide bridges (29). In mice, deletion of SP-B at the genetic level is lethal (16) and disruption via mutation causes respiratory failure (39, 137). Peptide-based lung surfactant replacements designed to replicate the propertie s of SP-B have received noticeable attention (67, 138) as the use of synthetic analogs would re move the immunologic risks associated with animal-derived surfactant and allow for greater therapeutic consistency (136). The lipid constitution in mammalian lung surfact ant is heterogeneous, but is dominated by zwitterionic DPPC (dipalmitoylphosphatid ylcholine) (~60%) and anionic POPG (palmitoyloleoylphosphatidylglycerol) (~10%). Leve ls of the lipids are re latively conserved in the lungs of vertebrates (4, 16, 29). SP-B is present at low levels 0.7-1.0% by weight or a molar percentage of <0.2% relative to the lipids (16). A multitude of roles for SP-B in surface tension minimization, intracellular and extracellular surf actant trafficking and respiratory dynamics in general have been proposed and experimentally established (23). Lung surfactant undergoes a cycle of lipid adsorption and resorption at the air-fluid interf ace which is postulated to be

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82 facilitated by SP-B (23). Lipid polymorphisms that change the geometry and arrangement of lipid headgroups have also been posited as critical for the functional properties of lung surfactant, as well as in other membrane-membrane mediated events (15, 108, 139). Molecular level information on how syntheti c peptides, unrelated at the pr imary amino acid level to SP-B, can modulate surface tension in alveolar compartments is lacking, yet KL4 in combination with POPG, DPPC, and PA (palmitic acid) has been appr oved as an agent for treatment of RDS due to its efficacy (136). In-vitro assays as well as animal stud ies have shown the ability of KL4 to lower surface tension in different lipid systems (133, 134). Clinically, administration of a KL4 surfactant preparation (lucinactant ) to very premature infants wa s markedly more effective in treating RDS than other commercially available formulations (140). Thus, understanding how KL4 affects the molecular and biophysi cal properties of the lipids is of particular relevance to the treatment of various forms of RDS. Peptide mimics which rely on a presumption of helicity and amphipathicity have been pursued both therapeutically a nd as model systems for understa nding the unique properties of SP-B. For example, KL4 was designed based on the charge distribution of the C-terminal residues 59-80 (133, 134). Of particular interest is that KL4 retains many of the macroscopic and biophysical properties of SP-B despite bearing litt le resemblance to the primary sequence of the protein other than a similarity in charge distribution at the C-terminus. The use of differential scanning calorimetry in combination with NMR can provide insight into how KL4 induces changes in lipid phase properties and/or geometric arrangement. In this study, 2H NMR, 31P NMR, and DSC were employed to investigate the properties of 4:1 DPPC:POPG and 3:1 POPC:POPG lipid vesicles on addition of KL4. The former composition was selected to mimic the composition of lung su rfactant while the latter composition is similar

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83 to formulations used in numerous NMR studies of membrane-active an timicrobial peptides, allowing us to compare the properties of KL4 to other amphipathic, helical peptides. Methodology to Study KL4 with Lipids Synthesis of KL4: KL4 (KLLLLKLLLLKLLLLKLLLLK) was synthesized via automated solid-phase peptide synthesis on a Wa ng resin (ABI 430, ICBR, UF). The peptide was cleaved from the resin with 90% TFA/ 5% triisopropyl-silane/5% water and ether precipitated. The crude product was purified by RP-HPLC using an acetonitrile/water gradient and purity was verified by mass spectrometry. Dried peptide was weighed and dissolved in methanol to a stock concentration of approximately 1mM. Aliquots were analyzed by amino acid analysis to allow for a more accurate determination of concentration (Molecular Structure Facility, UC Davis). Preparation of Peptide:Lipid Samples for DSC: DPPC and POPG were purchased (Avanti Polar Lipids, Alabaster, AL) as chloroform solutions and concentrations were verified by phosphate analysis. The lipids were mixed at a molar ratio of 4:1 DPPC:POPG in chloroform, aliquoted, and a methanol solution of KL4 was added as needed to make samples with peptide:lipid ratios ranging from <1:1000 to >1:50. The peptide-lipid samples were dried, dissolved in cyclohexane and freeze-dried overnight to remove residual solvent. Each resultant peptide-lipid powder was solubili zed in 5mM HEPES buffer at pH 7.4, with 140mM NaCl, 1mM EDTA, and 5mM CaCl2 to a final lipid concentration of approximately 3mM. Samples were placed in a 50oC water bath to facilitate solubilizati on accompanies by 3-5 freeze thaw cycles to facilitate solubilization and equilibration. Peptide:lipid MLVs were extruded through 100nm filters (AvantiPolar Lipids, Alabaster, AL) to form LUVs and degassed just prior to DSC. DSC experiments were conducted over a range of 10-70oC at a scan rate of 1oC/min and run in triplicate. Following DSC, aliquots of each sample were removed and assayed by phosphate

PAGE 84

84 analysis to determine final phospholipid concentra tion. All assayed concen trations were within 10% of initial estimated concentrations. Data analysis was performed with Origin v6.0. For heat capacity measurements, the molar lipid concentration in the DSC sample cel l was based on inorganic phosphate assays. Hcal was determined by integration of the phase transiti on peaks in the DSC thermograms after baseline correction. The vant Hoff derived enthalpy (HvH), an enthalpic parameter utilizing the phase transition temperature (Tm) of the lipid, was determined by equation (3-1) (86): cal p m vHH C RTH max 24 (3-1) Lipid cooperativity was assessed by both measuring the peak width at half height ( T) and by calculating the cooperativity unit based on th e ratio of the vant Hoff enthalpy to the calorimetric enthalpy. Solid state NMR sample preparation: A methanol solution of KL4 was added to chloroform solutions of 4:1 DPPC(d-62) :POPG, 4:1 DPPC:POPG(d-31), 3:1 POPC(d31):POPG, 3:1 POPC:POPG(d-31), DPPC(d-62) POPC(d-31), and POPG(d-31) to make a series of samples with final peptide to lipid ratios ranging from <1:1000 to >1:50. The MeOH and CHCl3 were evaporated under nitrogen and the peptide/lipid film was dissolved in cyclohexane and lyophilized overnight to remove residual solvent. Approximately 25mg of each sample was then placed in a 5mm diameter NM R tube and 300L of bu ffer containing 5mM HEPES buffer at pH 7.4,140 mM NaCl, and 1mM EDTA in 2H depleted water (Cambridge Isotope Laboratories, Andover MA) was added. NMR samples were then subjected to 3-5 freeze-thaw cycles to form MLVs.

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85 Phosphorous (31P) NMR: 31P NMR data were collected on a 600 MHz Bruker Avance system (Billerica, MA) using a standard 5mm BBO probe and a st andard pulse-acquire sequence with 25 kHz proton decoupling during acquisition. Spectra were acquired at 3 temperatures (39, 44 and 49oC) with 1024-2048 scans for each spectrum and a 5 second recycle delay between scans to minimize RF heating of the samples. The transform of a powder lineshape to an or iented spectrum at a single frequency is termed dePaking. The dePaking of NMR spectra was performed using an algorithm provided by Professor Edward Sternin. The algorith m employed a Tikhonov regularization method ( 102, 103, 105) to simultaneously determine the extent of macroscopic ordering in partially aligned lipid spectra and the dePaked frequencies (103 ). Deuterium (2H) NMR: 2H NMR data were collected on a 500MHz Bruker Avance System (Billerica, MA) using a standard 5m m BBO probe and quad echo sequence with a B1 field of 40 kHz. Spectra were acqui red at 3 temperatures (39, 44 and 49oC) with 1024-2048 scans and 0.5 second recycle delay. The dePaking of 2H NMR data was performed using the same algorithms as for the 31P NMR spectra. Individual assignments of peaks were made based on work by Petrache, et al ( 104, 117, 124) and Seelig ( 104). KL4 Affects Lipid Phase Behavior The dynamic air-fluid interface in the lung requ ires lung surfactant to possess specific attributes to lower surface tension and allow rapi d respreading. Lipid-ri ch surfactant relies on relatively low levels of lung surfactant proteins B and C (<0.2m ol%) to alter the structure, dynamics, and phase properties of the lipids to achieve the characterist ic properties of lung surfactant. The peptide KL4 similarly affects the macroscopic properties of the lipids and thus has been pursued as a replacement for SP-B in therapeutic formulations ( 137, 140).

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86 The size and shape of the 31P chemical shift anisotropy in phospholipid headgroups can identify the existence of hexagonal, lamellar, or other lipid phases ( 108 ), and in lamellar phases the size of the anisotropy is primarily dependa nt on the average orientation of the phosphate headgroups relative to the bila yer normal. Furthermore, 2H NMR spectra of lipid systems where the palmitoyl chains of either the phospha tidylcholines or the phosphatidylglycerol are deuterated can determine the e ffects and insertion depth of KL4. The role of KL4 in lipid phase behavior can also be observed using 31P NMR. Calorimetric data at increasing concentrations of KL4 also assesses the effect of the peptide on the thermodynamics and cooperativity of the L to L lipid phase transition in DPPC:POPG vesicles. The DSC data for 4:1 DPPC:POPG vesicles containing KL4 show a concentration dependent effect of the peptide on the main pha se transition (Figure 31). Adding POPG to DPPC in a 1:4 ratio shifts the Tm for DPPC from 42oC to below 36oC due to the monounsaturated fatty acid group in POPG. Addition of the pe ptide shifts the Tm back to higher temperatures in a concentration dependant manner. Small amounts of peptide increase the Tm slightly by 2-3oC; Hcal, and HvH remain relatively the same except for a slight increase at 0.75 mol% of peptide. At 0.75 mol % KL4, the shape of the thermogram noticeably bifurcates, providing indirect evidence of lipid domain separation. Th is bifurcation becomes more pronounced at higher concentrations of peptide, most noticeably 1.5 and 2.2 mol%, agreeing well with the observation of domain formation via Langmuir trough studies of KL4 interacting with fluorescently labeled DPPC in a DPPC:POPG:PA system ( 141 ). This bifurcation has also been seen by Saenz, et al at a lower peptide ratio (0.5 mol%) in 3:1 (w/w) DPPC:POPG MLVs ( 142). This correlates well with postulated domain fo rmation mediated via electrostatic interactions between anionic phospholipid s (such as POPG) and cationic peptides (such as KL4) ( 85). This

PAGE 87

87 behavior may be important to the function of the peptide in vivo during the lipid resorption/adsorption cycle (23 ). Table 3-1 summarizes the thermodynamic behavior of DPPC:POPG as a function of peptide concentration. 31P NMR: Addition of KL4 Leads to Changes in Orientation of the PG Headgroups 31P NMR spectra of 3:1 POPC:POPG and 4:1 D PPC:POPG preparations show orientation of the lipid bilayers in the magnetic field lead ing to distorted axially symmetric spectra with higher than expected intensities at the perp endicular edges (Figure 3-2 and Figure 3-4). Overlapping lineshapes resulting from the PC and PG headgroups are easily distinguished at this field. Lipid alignment in high magnetic fields has been observed previously and is caused by the negative diamagnetic susceptibil ity inherent to phospholipids ( 129, 130, 143 ). In lipid bilayers, the cooperative alignment of the magnetic moments of individual lipid molecules leads to a large bulk magnetic susceptibility and an overall macroscopic ordering of the sample within a magnetic field ( 132). Increasing amounts of KL4 disrupts this alignmen t in a concentration dependent manner, as evidenced by the grad ual increase in the parallel edges of the 31P spectra. The reduction in alignment of the lipid lamellae on addition of KL4 suggests the peptide disrupts lipid-lipid interactions and dissipates the ellipso idal distortion of the vesicles in the magnetic field; the degree of alignment seen is similar for all lipids within the sample. When dePaking the NMR spectra the ellipsoidal parameter ( ) provides a measure of decreases in alignment with increasing KL4. DePaking of the 31P NMR spectra for 3:1 POPC(d-31) :POPG and 4:1 DPPC(d-62):POPG clearly show changes in the PG headgroup orienta tions based on the shifts in the POPG peak on addition of KL4 while the DPPC anisotropy remains consta nt (Figure 3-3 and Figure 3-5). When the POPG acyl chains are deuterated, the results are the same as expected since the deuteration

PAGE 88

88 should have little effect on the headgroup of th e lipid. These results strongly support a concentration dependant ionic in teraction between the cationic KL4 peptide and electronegative PG moiety. Table 3-2 lists the 31P CSA span for the headgroups and a general reduction in span with increased KL4 concentration is seen for POPG in both lipid systems. Figures 3-6 and Figure 3-8 show the static 31P NMR spectra of lipids with deuterated POPG and Figure 3-7 and 3-9 show the corresponding dePaked spectra. The shift in the POPG parallel edge frequency is consistently seen throughout all lipid systems studied regardless of which lipid was deuterated. Figure 3-10 shows the (change in CSA) for DPPC and POPG in DPPC:POPG(d-31). To assess the underlying causes of the changes in POPG CSAs and th e ordering of the acyl chains in the binary lipid mixtures, 31P and 2H NMR spectra were collect ed on individual lipids alone and with 1.5mol% KL4. 31P NMR spectra are shown in Fi gure 3-11 and the corresponding dePaked spectra are shown in Figure 3-12. Only small changes in CSA are seen on addition of KL4. However, the CSA for POPG alone is si gnificantly smaller than observed for 4:1 DPPC:POPG or 3:1 POPC:POPG mixtur es prior to the addition of KL4. However, for the binary lipid mixtures with higher amounts of KL4, the 31P CSAs are more similar to those expected based on the spectra of the neat lipids. Thus, KL4 is clearly affecting the interactions of the PC and PG lipids as well as their miscibility. This is in agreement with the DSC data showing phase separation or lipid seque stration on addition of KL4. Hence one important role possibly mediated by the peptide is lipid demixing via interactions of KL4 with the PG headgroups. KL4 Effects on Lipid Acyl Chain Ordering Dependent on the Saturation of the Acyl Chains Deuterium solid state NMR can serve as a se nsitive non-perturbing probe for investigating dynamic processes of proteins and lipids. The size of the 2H quadrupolar interaction is wellmatched to the time scale of many dynamic processes occurring in membranes and proteins, particularly for fatty acid chains in lipid systems ( 144). The extent of peptide interaction with

PAGE 89

89 the bilayer can easily be monitored by determ ining the time averaged order parameters, , for each position in the fatty acid chain and monito ring their changes with addition of peptide. The order parameter encapsulates motions of the lipids, including axial rotation around the lipid long axis, undulations or reor ientations of the lip ids with respect to the bilayer normal known as wobbling, and trans-gauche isomerization at individual me thylene positions along the acyl chains ( 119). The order parameter reflects the collective averaging of these motions on the NMR timescale (10-5 seconds), which also includes latera l diffusion of the lipid molecule. Typical values are on the order of 0.2 for fatty acyl chains in individual lipids near the headgroup ( 145). The effect of KL4 on motions at each individual C-D bond methylene position, and on lipid motions in general can thus be quantitatively determined. NMR spectra taken for binary lipid systems w ith palmitoyl chains deuterated on different lipids are shown in Figures 3-13 to 317 along with their dePaked spectra. As with 31P NMR data, some lipid alignment in the static field is seen. Spectra taken with different deuterated acyl chains were taken to determine whether and how the peptide interacts with the acyl region of a particular lipid. A recent reevaluation of deuterium order pa rameters on a series of disaturated PC headgroups was performed by Petrache, et al at different temperatures ( 124 ) which were extrapolated to 44oC to assign the sn-1 and sn-2 positions in DPPC spectra. Using this and the acyl chain assignments from Seelig ( 104) as a guide, assignments and order parameter calculations were made. The calculated or der parameters (using Equation 2-14) from q values for each lipid system studied can be found in the Appendices. Furthermore, 2H NMR spectra of neat lipids DPPC(d-62), POPC(d-31), and POPG(d-31) with and without 1.5mol% KL4 were also collected, dePaked and assi gned (Figure 3-18) unfortunately, the poor signal to noise ratio

PAGE 90

90 obtained from these measurements prevented cl ear assignments for each methylene position after dePaking. The order parameters profiles obtained fo r 4:1 DPPC(d-62):POPG 4:1 DPPC:POPG (d-31), 3:1 POPC(d-31):POPG and 3:1 POPC:POPG( d-31) demonstrate that the peptide interacts with the two lipids in a manner that is dependant on the degree of saturation in the fatty acid chains. Figure 3-19 shows the calculated orde r parameters as a function of carbon number for the sn-1 and sn-2 chains of DPPC from spectra of 4:1 DPPC(d-62):POPG. KL4 clearly affects the acyl chain dynamics of DPPC at positions 9-15 in a concentration dependant manner and increases their ordering. Similar concentratio ns of peptide added to 3:1 POPC(d-31):POPG MLVs lead to only small changes in order para meters (Figure 3-20). Smaller, negative changes in order are seen for corresponding regions in the sn-1 chain of POPC in 3:1 POPC(d-31):POPG bilayers suggesting a more peripheral interaction with the lipids or preferential interaction with POPG as was found from 31P NMR. Hence, the degree of saturation and lipid system has an effect on mode of binding of KL4. The data shown above are more pronounced when the observations are viewed as the percent change in order parameter at each position along the acyl chain. Shown are the changes in at a particular methylene i for 4:1 DPPC(d-62):POPG (Figure 3-21) and 3:1 POPC(d-31):POPG (Figure 3-22). Changes in the order parameter, relative to lipids alone, are shown for C-D bond representing the plateau re gion (carbon 3), middle of the acyl chain, and the terminal ends. Significant changes in occur for both sn-1 and sn-2 chains of DPPC, with increases of up to 10% for carbon 10 and 12 and 8.5% for carbon 8. This high degree of ordering suggests KL4 is penetrating deep into the bilayer and decreasing the mobility of the acyl chains. This increase in orderi ng at the middle and ends of the acyl chains from deuterium data

PAGE 91

91 for DPPC(d-62):POPG argue for a deep penetra tion of the peptide, but the lack of similar changes in positions 2-3 suggest the peptide does not adopt a transmembrane orientation. Thus KL4 lodges into the hydrophobic region of the bi layer, while maintaining a perpendicular orientation to the bilayer normal. Quantitatively, the calculated for 3:1 POPC(d31):POPG, decrease in magnitude by up to 4-6% indicating the acyl chains have increased mobility. The order parameter shifts are not obvious in the order parameter profiles shown in Figure 3-22 because the difference in their orde r parameter values are very small, but their percent changes make this dist inction possible (Appendix A-D). In experiments where the palmitoyl chain of POPG were deuterated; 3:1 POPC:POPG (d-31) and 4:1 DPPC:POPG(d-31), show an ove rall decrease in orde ring of the POPG acyl chains on addition of KL4 (Figure 3-23 and Figure 3-24). Decr eases in the order parameters are seen at all the acyl positions in POPG. These re sults further corroborate 31P NMR findings that show an association with KL4 and the PG headgroup. KL4 in Relation to other Peptides of Similar Size, Composition and Length A possible explanation for how KL4 inserts into the bilayers is that it may form a structure in which the lysines lie on one side of a helix. The snorkeling of these lysine sidechains would allow an amphipathic helical conformation of KL4 to deeply penetrate the bilayer while still having electrostatic interactions between the lysine sidechains and the lipid phosphate groups. The snorkeling of lysine residues have previously been postulated to provide negative curvature strain in the context of the tran smembrane WALP and KALP peptides ( 146 ). Of particular interest to the snorkeling model is a molecu lar simulation study of the 22 amino acid peptide KKLLKLLLLLLLLLLKLLLLKK which was found to have a transmembrane orientation and snorkel in POPC membranes ( 147 ). This peptide bears striking homology to KL4 in both

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92 amino acid content and hydrophobic to hydrophilic ra tio, but the lysines are distributed more toward the ends of the peptide leading to a transmembrane configuration. If KL4 adopts a helical structure in the lipid environment whic h is perpendi cular to the membrane normal, it cannot form a canonical -helix as this would place the lysines evenly around the helix rather than in an amphipathic co nfiguration. Nonetheless, FT-IR measurements indicate KL4 is helical in lipid bilayers, and sits on top of the membrane ( 148, 149). Lysine and leucine rich peptides have been known to adapt to different amphi phathic secondary structures at the air-water interface and on pol ymer surfaces based on the ami no acid pattern of the residues ( 149, 150). Similar driving forces would exist at the lipid bilayer surface a nd peptides of varying ratios of lysine and leucine residues have b een used to modulate the peptide helicity and topology in bilayer systems ( 93). Amphipathic -helices with specific ratios of L to K that generate a greater hydrophobic face to the helix have been demonstrated to perturb lipid bilayers and aggregate, causing micelle formation ( 151). No indication of micelle formation by KL4 addition has been found again suggesting its st ructure and mechanism is different from amphipathic -helices. The structure of KL4 will be further discussed in Chapter 4. The ability of peptides to affect lipid magne tic field alignment has previously been noted ( 130, 132, 143, 152 ), particularly for amphipathic peptides. One example is the synthetic alamethicin derivatives. In these studies 14 and 21 residue helical peptides with crown ether side chains were studied by 31P NMR to probe peptide induced lipid polymorphism. These findings indicate the peptide changing the elastic proper ties of the membrane and causing a deformation of the bilayer. Synthetic peptides such KIGAKI, designed to be an amphiphillic beta-sheet, have also been found to change the ratios of the perpendicular to parallel edges in static 31P spectra of POPC:POPG MLVs ( 153). The opposite phenomenon--alignment of lipid bilayers on addition

PAGE 93

93 of peptide--has also been observed by 31P NMR. Studies of the effect of melittin on DPPC/cholesterol bilayers s how the ordering of the lipid s on addition of peptide ( 154), high amounts of magainin antibiotic s was found to magnetically orient POPC bilayers ( 112 ), and the opioid peptide dynorphin was also demonstrated to increase alignment of DMPC MLVs ( 155). Furthermore, the antimicrobial peptides f ound in Australian tree frogs, caerin 1.1 and maculatin 1.1 have been demonstrated to increas e molecular order in bicelles, but the shorter peptides aurein 1.2 and citropin 1.1 do not ( 156). Hence, the influence of peptides on the magnetic properties of lipids has been shown to exist across a broad spectrum of peptides of similar size and length. Though the primary am ino acid sequence of th ese peptides differs markedly, the results indicate th at the decrease in macroscopi c lipid alignment mediated by KL4 may not be entirely unique. The differences in alignment properties are suggestive of different effects on the lipid organizational properties. Un fortunately, the diversity in lipid composition, peptide concentration and experi mental conditions precludes a systematic evaluation of how peptide sequences and length affects lipid organization. A recent study does show antimicrobial activity for KL4 in hypoxic-injured mice infected with lipopolysacharride ( 72). Cationic, leucine-rich amphipathic -helical peptides have been pursued as antimicrobial agents since their positiv e charge leads to preferential targeting of the anionic rich membranes typical to prokaryotes ( 80, 146 ). These peptides are designed to be amphipathic -helices based on naturally occurring antimicrobial pep tides which are thought to disrupt the membranes on binding through the form ation of toroidal or barrel stave pores structure( 79, 157 ). However, these peptides have significantly higher percentage of charged residues (>50% compared to <25% for KL4) and in all probability a different secondary structure.

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94 KL4 Shares Many Properties with Cholesterol and Transmembrane Helices in DPPC Ordering of DPPC by KL4 interacting with bilayers is a phenomenon commonly associated with molecules that penetrate the hydrophobic region of the bilayer, such as cholesterol, or span it, such as transmembrane helices ( 127, 158-160). Much like KL4, cholesterol has also been shown to decrease magnetic alignment of DMPC lipids at 20 mol% levels ( 159). 2H NMR studies also indicate increased order of DPPC acyl chains, decreas ed area per lipid molecule, and domain separation mediated by choleste rol on the range of roughly 15-25mol%, ( 161-163). Raman spectroscopy studies reveal that higher am ounts of cholesterol induce a liquid-ordered (lo) phase on DPPC, a state which has limited acyl chain flexibility but a greater degree of lipid mobility (164 ). This raises the interesting possibility of KL4 acting in a similar capacity to cholesterol but at significantly lower mol%. The values for DPPC alone from the literature or in DPPC:POPG ve sicles are very similar ( 124); addition of KL4 increases SCD values in a manner that has been seen for cholestero l. The resultant ordering seen could be the consequence of phase separation as suggested by the DSC thermograms (Figure 3-1), but complete segregation is unlikely given that SCD of DPPC with KL4 >SCD of neat DPPC. In comparison to 4:1 DPPC(d-62):POPG MLVs, the 2H spectra of 3:1 POPC(d-31):POPG MLVs only show small downward shifts in the order parameter profiles on KL4 addition, suggesting only an electrostatic interaction of KL4 with POPC and a more sh allow interaction with the bilayer. Molecular Model of KL4 with POPC:POPG and DPPC:POPG Based on the assumption of helicity for KL4, with the lysines on one side of the helix, a molecular model of its potential orientation and penetration based on our data is shown in Figure 3-25. The peptide was modeled based on data pres ented in Chapter 4 and PDB files to simulate a lipid bilayer milieu were based on published values from Professor Scott Feller et al (71).

PAGE 95

95 Based on the thickness of the bilayer and the length of the peptide, a transmembrane orientation of the peptide is not likely due to the periodic placement of the lysi ne residues as well as the lack of a shift in parameters for the lipid plateau region on addition of KL4 ( 146, 165). This peripheral orientation has been verified by in frared reflection-absorp tion spectroscopy (IRRAS) taken on KL4 with DPPC and 7/3 DPPC:POPG bilayers. In these measurements, a beta-sheet structure at the membrane interface in a Langmuir trough arrangement was determined (166). However, our data suppor ts the conclusion that KL4 is helical with the lysine side chains aligned to allow them to snorkel to the phosphate headgroups in the DPPC:POPG bilayer. Lysine snorkeling is supported by the values we observed, particularly at acyl chain positions 815. This maximizes electrostatic interacti ons between the amino groups and the phosphate headgroups. Such snorkeling, which has been re ported for many short, lysine capped peptides, can allow for a much greater penetration of the peptide while allowing it to remain perpendicular to the bilayer normal. The thermodynamic penalty imposed by placing KL4 in a transmembrane orientation would be prohi bitive since it would result in charge d amino acids partitioned into the hydrophobic core. Also, a transmembrane orientati on of the peptide would cause more ordering of the positions 2-8 in the acyl chains than we see, as has been shown for more hydrophobic helical peptides with a high distribution of leucines (167). The binding of KL4 to model POPC:POPG lipids are more peripheral based on 2H NMR derived values and 31P NMR. Derivatives of magainin antibiotics, peptides wh ich have an orientation perpendicular to the bilayer normal, also cause a decrease in order parameters that decrease upon addition of 3mol% of peptide, though the downward shifts in orde r parameter seen are significantly higher than what is seen in this work ( 79). This is probably due to the to roidal pore mechanism of cell lysis attributed to these peptide types. Order parameters seen for KL4 with POPC(d-31):POPG

PAGE 96

96 similarly decrease indicating a mostly periphe ral interaction with the lipid; however, the comparatively smaller decrease in values presumably reflects KL4 facilitating lipid-lipid interaction, and not cellular de gradation as would be the case for the magainin family of peptides. The snorkeling model of KL4 within DPPC:POPG lipid multila mellar vesicles could have important consequences for membrane structure and function. For the most part, this represents a novel peptide-lipid interaction that is unique in comparison to what has been seen for amphipathic -helical antimicrobial peptides. Most antimicrobial peptides tend to bind peripherally and destroy bacteria l cell membranes via a toroidal pore or carpet mechanism ( 79, 113). While the binding of KL4 to POPC:POPG vesicles suggest it could bind to these lipids in a manner similar to antibiotic peptides, its ability to snorkel in DPPC:POPG lipids would have a different effect on lipid biophysics. Some of the thermodynamic co sts of peptide penetration can be relieved by the formation of alternative non -lamellar structures, su ch as hexagonal phases ( 160, 165). The inverted HII phase, in which the headgroups invert and follow an aqueous channel can form if peptide lipid hydrophobic mismat ch occurs. Such inverted isotropic phases have been shown to exist in phosphatidylcholine membranes at concentrations greater than 3mol% ( 168 ), the maximum concentration used in these studies. The formation of alternative lipid geometries, specifically, the inverted HII phase, has been found to be important in the proper formation lung surfactant ( 139 ). The model depicted suggests that KL4 could facilitate formation of non-lamellar lipid pha ses and/or induce a curvature strain as its role in lung surfactant. Alternatively, lipid geometry coul d be critical during fusion of lipids to the airsurfactant interface and KL4 snorkeling could be essential fe ature for the proper shuttling of lipids.

PAGE 97

97 In conclusion, DSC, 31P and 2H data indicate KL4 binds peripherally to 3:1 POPC:POPG lipids through electrostatic intera ction of the lipid phosphates with the positively charged lysines. A different interaction is seen with 4:1 DPPC:P OPG lipids suggesting the peptide penetrates to a far greater depth in the bilayer. Thus, both peptide structure a nd acyl chain saturation play an important role in determining the insertion level of KL4. The DSC and 31P NMR results show KL4 having similar attributes to c holesterol in terms of lipid or dering and DSC peak broadening, as well the peptide displaying an electrostatic interac tion with the PG headgroups when POPG is added to DPPC and POPC lipid systems. 31P NMR spectra indicate spont aneous lipid alignment to the magnetic field which is abrogated by the addition of increasing levels of peptide. A decrease in lipid-lipid associ ation is also seen from our 31P NMR data as peptide is added. With the findings presented here, a mo re thorough structural model can be established for how this small peptide mimics lung surfactant protein B a nd drive the development of future mimetics. ACKNOWLEDGMENTS: The authors of this pape r thank Dr. Alfred Chung for synthesis of peptide KL4, and the Molecular Structure Facility at University of California, Davis for AAA analysis. The research here in was funded by NIH 1R01HL076586 awarded to Dr. Joanna R. Long and UF, MBI.

PAGE 98

98 Figure 3-1. Differential scanning calorimetry on KL4 with 4:1 DPPC:POPG vesicles with KL4 at the indicated molar percentages. Shown is clear phase separation on addition of peptide. DSC scans on each sample were performed in triplicate at a scan rate of 1 degree per from 10-70oC.

PAGE 99

99 Table 3-1. Thermodynamic parameters obtained from DSC thermograms. cooperativity unit ( HvH/ Hcal) ** sample only run once in the DSC instrument Tm Hcal HvH S Cp max Main Transition (oC) (kcal/mol) (energy/mol) (cal/mol/K) T1/2 CU* kcal/mol/oC36.4 0.2 DPPC:POPG 10.0 1.3 115 12 32 4 6.0 0.4 12 3 1.6 0.1 0.1% KL4 36.9 0.1 9.7 0.3 124 2 31 1 5.2 0.1 13 1 1.6 0.1 0.2% KL4 36.70.2 8.0 .9 133 1 26 3 5.5 0.1 17 1 1.4 0.1 36.8 0.2 0.4% KL4 9.0 .7 121 2 29 2 5.5 0.2 14 1 1.4 0.1 0.8% KL4 ** 37.1 6.7 113 4 22 5.6 17 1.1 1.5% KL4 39.20 0.03 9.5 0.4 115 3 30 1 5.84 0.04 12 1 1.4 0.1 2.3% KL4 39.50 0.04 7.9 0.1 122 1 25 1 5.5 0.1 12 2 1.2 0.1

PAGE 100

100 Table 3-2. CSA span for phos phate headgroup in 3:1 POPC:POPG and 4:1 DPPC:POPG MLVs with and without KL4. The greatest change occurs in PG headgroups in 4:1 DPPC:POPG MLVs indicating a preferential inter action of this lipid with the peptide. A change in the PG 31P CSA span is also seen in the 3:1 POPC:POPG MLVs on addition of peptide. 3:1 POPC:POPG 4:1 DPPC:POPG KL4 POPC POPG DPPC POPG concentration 0 -33.9 -26.9 -42.2 -33.2 0.1 -35.1 -26.9 -42.2 -32.3 0.2 -35.1 -27.6 -41.4 -32.4 0.4 -28.2 -20.7 -43.0 -32.4 0.8 -36.4 -27.6 -43.7 -30.9 1.5 -35.8 -26.3 -43.0 -28.6 2.3 -33.9 -23.2 -42.4 -26.4

PAGE 101

101 -40-30-20-10010203040 Chemical Shift (ppm) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 0% KL4-40-30-20-10010203040 Chemical Shift (ppm) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 0% KL4 Figure 3-2. Static 31P NMR spectra of 3:1 POPC(d-31) :POPG MLVs with the increasing addition of KL4. As peptide levels increase, signal increases at the parallel edge suggesting KL4 reduces macroscopic alignment of the lipids. Spectra taken with 1024 scans in a 600MHz Bruker instrument at 44oC.

PAGE 102

102 -60-50-40-30-20-100102030Chemical Shift (ppm) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4POPC POPG -60-50-40-30-20-100102030Chemical Shift (ppm) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4POPC POPG Figure 3-3. Static 31P dePaked NMR spectra of 3:1 POPC(d-31):POPG with increasing amounts of KL4. The movement of the POPG peak upon addition of peptide is clearly visible.

PAGE 103

103 -40-30-20-10010203040 Chemical Shift (ppm) 2.3% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4 Figure 3-4. Static 31P NMR spectra of 4:1 DPPC(d-62):POP G MLVs with increasing addition of KL4. As peptide levels increase, signal increases at the parallel edge as seen for 3:1 POPC:POPG MLVs.

PAGE 104

104 -50-40-30-20-100102030Chemical Shift (ppm) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4DPPC POPG -50-40-30-20-100102030Chemical Shift (ppm) 2.5% 1.6% 0 0 0.1% DPPC POPG -50-40-30-20-100102030Chemical Shift (ppm) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4 -50-40-30-20-100102030Chemical Shift (ppm) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4DPPC POPG -50-40-30-20-100102030Chemical Shift (ppm) 2.5% 1.6% 0 0 0.1% -50-40-30-20-100102030Chemical Shift (ppm) 2.5% 1.6% 0 0 0.1% DPPC POPG Figure 3-5. Static 31P dePaked NMR spectra of 4:1 DPPC(d-62):POPG with increasing amounts of KL4. The movement of the POPG peak upon addition of peptide is clearly visible.

PAGE 105

105 -40-30-20-100102030 3% KL4 1% KL4 0.2% KL4 3:1 POPC(d-31):POPGChemical Shift (ppm) Figure 3-6. Static 31P NMR spectra of 3:1 POPC:POPG(d-31) MLVs with increasing amounts of KL4.

PAGE 106

106 -60-50-40-30-20-100102030 Chemical Shift (ppm) 3% KL4 1% KL4 0.2% KL4 3:1 POPC:POPG(d-31)POPC POPG-60-50-40-30-20-100102030 Chemical Shift (ppm) 3% KL4 1% KL4 0.2% KL4 3:1 POPC:POPG(d-31)POPC POPG Figure 3-7. DePaked 31P NMR spectra of 3:1 POPC:POPG( d-31) with incr easing amounts of KL4. The movement of the POPG peak upon a ddition of peptide is clearly visible.

PAGE 107

107 -30-20-100102030 Chemical Shift (ppm) 3% KL4 1% KL4 0.2% KL4 4:1 DPPC:POPG(d-31) Figure 3-8. Static 31P NMR spectra of 4:1 DPPC:POPG(d-31) MLVs with increasing amounts of KL4.

PAGE 108

108 -60-50-40-30-20-100102030 Chemical Shift (ppm) 3% KL4 1% KL4 0.2% KL4 4:1 DPPC:POPG(d-31) Figure 3-9. DePaked 31P NMR spectra of 4:1 DPPC:POPG( d-31) with incr easing amounts of KL4. The movement of the POPG peak upon addition of peptide is clearly visible DPPC POPG

PAGE 109

109 0.00 5.00 10.00 00.511.522.53 mol% KL4 (ppm) DPPC POPG Figure 3-10. The shift in dePaked frequency in ppm ( ) for DPPC and POPG (in ppm) by KL4 in 4:1 DPPC:POPG(d-31).

PAGE 110

110 -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol%KL4 -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% KL4 -60-40-200 204060 ppm POPG(d-31) POPG(d-31) with 1.5 mol%(A) (B) (C) -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol% -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% -60-40-200 204060 ppm POPG(d-31) POPG(d-31) with 1.5 mol% KL4 -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol% -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% -60-40-200 204060 ppm POPG(d-31) (A) (B) (C) -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol%KL4 -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% KL4 -60-40-200 204060 ppm POPG(d-31) POPG(d-31) with 1.5 mol% -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol%KL4 -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol%KL4 -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% KL4 -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% KL4 -60-40-200 204060 ppm POPG(d-31) POPG(d-31) with 1.5 mol% -60-40-200 204060 ppm POPG(d-31) POPG(d-31) with 1.5 mol%(A) (B) (C) -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol% -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol% -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% -60-40-200 204060 ppm POPG(d-31) POPG(d-31) with 1.5 mol% KL4 -60-40-200 204060 ppm POPG(d-31) POPG(d-31) with 1.5 mol% KL4 -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol% -60 -40 -20 0 20 40 60 Chemical Shift (ppm) DPPC(d-62) DPPC(d-62) 1.5mol% -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% -60-40-200204060 ppm POPC(d-31) POPC(d-31) with 1.5 mol% -60-40-200 204060 ppm POPG(d-31) -60-40-200 204060 ppm POPG(d-31) (A) (B) (C) Figure 3-11. Static 31P NMR spectra of single lipid and lipid with 1.5 mol% KL4 A: DPPC(d-62), B: POPC(d31) and C: POPG(d-31).

PAGE 111

111 -40 -30 -20 -10 0 Chemical shift (ppm) DPPC(d-62) DPPC(d-62) with 1.5 mol% -40 -30 -20 -10 0 Chemical Shift (ppm) POPC(d-31) POPC(d-31) with 1.5 mol%KL4 -40 -30 -20 -10 0 Chemical shift (ppm) POPG(d-31) POPG(d-31) with 1.5 mol%KL4 -40 -30 -20 -10 0 Chemical shift (ppm) DPPC(d-62) DPPC(d-62) with 1.5 mol% KL4 -40 -30 -20 -10 0 Chemical Shift (ppm) POPC(d-31) POPC(d-31) with 1.5 mol% -40 -30 -20 -10 0 Chemical shift (ppm) POPG(d-31) POPG(d-31) with 1.5 mol% -40 -30 -20 -10 0 Chemical shift (ppm) DPPC(d-62) DPPC(d-62) with 1.5 mol% -40 -30 -20 -10 0 Chemical shift (ppm) DPPC(d-62) DPPC(d-62) with 1.5 mol% -40 -30 -20 -10 0 Chemical Shift (ppm) POPC(d-31) POPC(d-31) with 1.5 mol%KL4 -40 -30 -20 -10 0 Chemical Shift (ppm) POPC(d-31) POPC(d-31) with 1.5 mol%KL4 -40 -30 -20 -10 0 Chemical shift (ppm) POPG(d-31) POPG(d-31) with 1.5 mol%KL4 -40 -30 -20 -10 0 Chemical shift (ppm) POPG(d-31) POPG(d-31) with 1.5 mol%KL4 -40 -30 -20 -10 0 Chemical shift (ppm) DPPC(d-62) DPPC(d-62) with 1.5 mol% KL4 -40 -30 -20 -10 0 Chemical shift (ppm) DPPC(d-62) DPPC(d-62) with 1.5 mol% KL4 -40 -30 -20 -10 0 Chemical Shift (ppm) POPC(d-31) POPC(d-31) with 1.5 mol% -40 -30 -20 -10 0 Chemical Shift (ppm) POPC(d-31) POPC(d-31) with 1.5 mol% -40 -30 -20 -10 0 Chemical shift (ppm) POPG(d-31) POPG(d-31) with 1.5 mol% -40 -30 -20 -10 0 Chemical shift (ppm) POPG(d-31) POPG(d-31) with 1.5 mol% Figure 3-12. DePaked 31P spectra for (Top) DPPC(d-62), (M iddle) POPC(d-31) and (Bottom) POPG(d-31) with and without 1.5mol% KL4.

PAGE 112

112 -20000-15000-10000-500005000100001500020000 Frequency (Hz) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4 Figure 3-13. 2H spectra of 3:1 POPC(d-31):POPG MLVs with increasing amounts of KL4. The 2H spectra overlap indicating very little change in this li pid system as a function of KL4.

PAGE 113

113 -30000-20000-100000100002000030000 Frequency (Hz) 2.5% KL4 1.6% KL4 0.8% KL4 0.4% KL4 0.2% KL4 0.1% KL4 no KL4 Figure 3-14. 2H NMR spectra of 4:1 DPPC(d-62):P OPG with increasing amounts of KL4.

PAGE 114

114 -35.00-25.00-15.00-5.005.0015.0025.0035.00 Frequency (kHz) 2.5% KL4 0.8% KL4 0.1% KL4 no KL4 -35.00-25.00-15.00-5.005.0015.0025.0035.00 Frequency (kHz) 2.5% KL4 0.8% KL4 0.1% KL4 no KL4(A) (B) -35.00-25.00-15.00-5.005.0015.0025.0035.00 Frequency (kHz) 2.5% KL4 0.8% KL4 0.1% KL4 no KL4 -35.00-25.00-15.00-5.005.0015.0025.0035.00 Frequency (kHz) 2.5% KL4 0.8% KL4 0.1% KL4 no KL4(A) (B) Figure 3-15 DePaked 2H spectra for (A ) 4:1 DPPC(d-62):POPG MLVs and (B) 3:1 POPC(d-31):POPG MLVs with increasing amounts of KL4.

PAGE 115

115 -40-30-20-10010203040 Frequency (kHz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4 -20 -15 -10 -5 0 5 10 15 20Frequency (Hz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4(A) (B) -40-30-20-10010203040 Frequency (kHz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4 -20 -15 -10 -5 0 5 10 15 20Frequency (Hz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4(A) (B) Figure 3-16 Deuterium NMR spectra for (A) 3:1 POPC:POPG(d-31) MLVs and (B) DePaked spectra with increasing amounts of KL4.

PAGE 116

116 (A) (B) -30-25-20-15-10-5051015202530 Frequency (kHz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4 -20-15-10-50 5101520Frequency (kHz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4(A) (B) -30-25-20-15-10-5051015202530 Frequency (kHz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4 -20-15-10-50 5101520Frequency (kHz) 3.0% KL4 1.0% KL4 0.2% KL4 no KL4 Figure 3-17. Static 2H spectra for (A) 4:1 DPPC:POPG(d-31) MLVs and (B) DePaked spectra with increasing amounts of KL4.

PAGE 117

117 -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPG(d-31) 1.5 mol% KL4 -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPC(d-31) 1.5 mol% KL4 -35-30-25-20-15-10-505101520253035 Frequency (kHz) DPPC(d-62) 1.5 mol% KL4(A) (B) (C) -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPG(d-31) 1.5 mol% -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPC(d-31) 1.5 mol% -35-30-25-20-15-10-505101520253035 Frequency (kHz) DPPC(d-62) 1.5 mol% (A) (B) (C) -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPG(d-31) 1.5 mol% KL4 -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPG(d-31) 1.5 mol% KL4 -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPC(d-31) 1.5 mol% KL4 -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPC(d-31) 1.5 mol% KL4 -35-30-25-20-15-10-505101520253035 Frequency (kHz) DPPC(d-62) 1.5 mol% KL4 -35-30-25-20-15-10-505101520253035 Frequency (kHz) DPPC(d-62) 1.5 mol% KL4(A) (B) (C) -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPG(d-31) 1.5 mol% -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPG(d-31) 1.5 mol% -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPC(d-31) 1.5 mol% -35-30-25-20-15-10-505101520253035 Frequency (kHz) POPC(d-31) 1.5 mol% -35-30-25-20-15-10-505101520253035 Frequency (kHz) DPPC(d-62) 1.5 mol% -35-30-25-20-15-10-505101520253035 Frequency (kHz) DPPC(d-62) 1.5 mol% (A) (B) (C) Figure 3-18. Static 2H NMR spect ra of single lipid MLVs with and without 1.5mol% KL4. Blue spectrum is single lipid, pink spectrum is lip id with 1.5mol% KL4. (A) DPPC(d-62), (B) POPC(d-31) and (C) POPG (d-31).

PAGE 118

118 0 0.05 0.1 0.15 0.2 0.25 024681012141618 carbon numberOrder parameter no KL4 0.8% KL4 2.3% KL4 0 0.05 0.1 0.15 0.2 0.25 024681012141618 carbon numberOrder parameter no KL4 0.8% KL4 2.3% KL4 0 0.05 0.1 0.15 0.2 0.25 024681012141618 carbon numberOrder parameter no KL4 0.8% KL4 2.3% KL4 0 0.05 0.1 0.15 0.2 0.25 024681012141618 carbon numberOrder parameter no KL4 0.8% KL4 2.3% KL4 0 0.05 0.1 0.15 0.2 0.25 024681012141618 carbon numberOrder parameter no KL4 0.8% KL4 2.3% KL4 0 0.05 0.1 0.15 0.2 0.25 024681012141618 carbon numberOrder parameter no KL4 0.8% KL4 2.3% KL4 Figure 3-19. Order parameter profiles for (4 :1) DPPC(d-62):POPG MLVs with and without KL4. Top:sn-1 chain Bottom: sn-2 chain

PAGE 119

119 0 0.05 0.1 0.15 0.2 0.25 246810121416 carbon numberOrder parameter no KL4 0.8% KL4 2.3%KL4 0 0.05 0.1 0.15 0.2 0.25 246810121416 carbon numberOrder parameter no KL4 0.8% KL4 2.3%KL4 Figure 3-20. Order parameter profile for the sn -1 chain of 3:1 POPC(d-31):POPG MLVs with and without KL4

PAGE 120

120 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15(A) (B) 3 8 10 12 15 3 8 10 12 15 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15(A) (B) 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15(A) (B) 3 8 10 12 15 3 8 10 12 15 3 8 10 12 15 Figure 3-21. Three dimensional plot of change in order para meter for DPPC(d-62):POPG MLVs as a function of mole percentage of KL4. The change in order parameter is colorcoded for individual carbons. The top graph is for the sn-1 chain and the bottom graph is for the sn-2 chain.

PAGE 121

121 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 -6% -5% -4% -3% -2% -1% 0%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 3 8 10 12 15 0 0.1 0.2 0.4 0.8 1.5 2.3 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 -6% -5% -4% -3% -2% -1% 0%% change in mol% KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 3 8 10 12 15 Figure 3-22. Three dimensional plot of change in time averaged order parameter for 3:1 POPC(d-31):POPG MLVs as a function of mole percentage of KL4. The change in order parameter is color-c oded for individual carbons.

PAGE 122

122 (A) (B) 0.00 0.05 0.10 0.15 0.20 0.25 2345678910111213141516carbon numberorder parameter no KL4 1% KL4 3% KL4 0.00 0.05 0.10 0.15 0.20 2345678910111213141516 carbon numberorder parameter no KL4 1% KL4 3% KL4(A) (B) 0.00 0.05 0.10 0.15 0.20 0.25 2345678910111213141516carbon numberorder parameter no KL4 1% KL4 3% KL4 0.00 0.05 0.10 0.15 0.20 2345678910111213141516 carbon numberorder parameter no KL4 1% KL4 3% KL4 0.00 0.05 0.10 0.15 0.20 2345678910111213141516 carbon numberorder parameter no KL4 1% KL4 3% KL4 Figure 3-23. Order parameter profile for A) 4:1 DPPC:POPG(d-31) MLVs and B) 3:1 POPC:POPG(d-31) MLVs with varying amounts of KL4.

PAGE 123

123 0 1 3 carbon 3 carbon 10 carbon 15 -9% -4% 1% % KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.2 1 3 carbon 3 carbon 10 carbon 15 -15.0% -10.0% -5.0% 0.0% 5.0% % KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 carbon 16(A) (B) 0 1 3 carbon 3 carbon 10 carbon 15 -9% -4% 1% % KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.2 1 3 carbon 3 carbon 10 carbon 15 -15.0% -10.0% -5.0% 0.0% 5.0% % KL4 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 carbon 16(A) (B) Figure 3-24. Change in order parameter values on (A) POPC:POPG(d-31) and (B) DPPC:POPG(d-31) upon addition of KL4.

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124 Figure 3-25. Model of KL4 penetration in two lipi d environments. Based on 2H NMR data, KL4 appears to snorkel in 4: 1 DPPC:POPG lipid vesicles. This snorkeling may have consequences in the lung, where these lipids are the most prevalent, since it may help facilitate lipid shuttling and/or promote formation of different lipid phases. In contrast, in 3:1 POPC:POPG MLVs lipids, a more peripheral interaction of KL4 with the headgroup region is seen

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125 CHAPTER 4 STRUCTURAL STUDIES OF KL4 This chapter describes structural measurements of KL4 in a heterogeneous lipid environment using CD and ssNMR. This work was done in collaboration with Dr. Douglas Elliot and Professor Joanna Long. Characterization of KL4 Secondary Structure In vitro and clinical studies show that KL4 minimizes surface tension and provides for relief from respiratory distress. The linear charge distribution of KL4 is based on charge distribution in the C-terminus of SP-B with the presumption that both SP-B59-80 and KL4 form amphipathic helices. In Chapter 3, the effects of KL4 on lipid dynamics and order were measured using 31P and 2H NMR. 31P NMR spectroscopy indicated the peptide predominantly interacting with the PG h eadgroup and having small negative effects on acyl chain order parameters in POPC:POPG. The opposite e ffects were found in DPPC:POPG LUVs where deuterium-derived order parameters indicate KL4 ordering the fatty acyl ch ains. We postulated this increase in deuterium orde r parameters as being due to KL4 penetrating deeper into DPPC:POPG bilayers as opposed to POPC:POPG b ilayers. While the different effects on one lipid system compared to the other is interest ing, the question remains as to the secondary structure of the peptide is when bound to D PPC:POPG and POPC:POPG and how its structure allows shallower or deeper penetration. In Figure 3-25, it is assumed in the molecular models that KL4 is helical and that it is superficial in one lipid system while embedded in the other. The assumption of KL4 being helical in both membrane milieus is an assumption based on findings that SP-B and SP-C are helical; particularly, SP-B59-80, which has been purported to be an amphipathic alpha helix ( 11). However, even though SP-B59-80 is presumed helical, it does not necessarily implicate KL4 as being an -helix in a lipid environment. Furthermore, the structure

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126 of KL4 in phospholipid bilayers has not been definitively resolved and may be contingent on lipid composition. Infrared studies (148, 166) involving the orientati on of the peptide in different lipid environments gi ve conflicting secondary struct ure adaptations. One IR study shows KL4 being a transmembrane helix in 7:3 DPPC:PG bilayers ( 148 ); the other study, combining IR measurements with surface pressure isotherms, postulated the peptide to be an anti-parallel beta sheet in a 7:3 DPPC:DPPG membrane environment ( 166). Thus, even when the lipid composition is constant, disagreements exists in the liter ature. The simple assumption of KL4 being helical, based on sequence periodicity and charge distributi on, cannot be readily accepted and other biophysical methods need to validate the IR measurements and higher resolution measurements could provide even more insight. In this chapter, circular dichroism an d ssNMR studies are described which were undertaken to determine the structure of KL4 in the lipid environments of 3:1 POPC:POPG and 4:1 DPPC:POPG LUVs as well as neat lipids. Ci rcular dichroism represents the simplest and most effective qualitative measure of global seco ndary structure in solution and in the presence of LUVs. Under optimized CD conditions, one can rule out -sheet or -helical structures for a peptide interacting with differe nt lipid moieties. We undertook a systematic CD study of KL4 in the presence of differing concen trations of POPC:POPG and D PPC:POPG LUVs. These are the two lipid systems where different topologies of KL4 were postulated in Chapter 3 based on NMR studies examining phospholipid dynamics. As pa rt of these experiments characterizing the structure of the peptide, CD expe riments were also performed to assess the influence of aqueous buffer or helix-inducing solvents on the gross secondary structure of the peptide in the absence of lipids.

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127 In collaboration with Professor Joanna Long and Dr. Douglas Elliot, ssNMR studies were performed on 13C-labeled KL4 peptides using the DQ-DRAWS pulse sequence (detailed in Chapter 2). 13C labels were enriched in adjacent carbonyl leucines of KL4 to determine backbone torsion angles phi ( ) and psi ( ). Each 13C-labeled peptide was complexed to lipids and the DQ-DRAWS sequence was used to first determine the torsion angle and then the torsion angle The technique provides for a more detailed structural ch aracterization of KL4 in the lipid environments. These structural studies can provide insight in to the IR results and possibly explain their discrepancies. They also demonstrate the utility of ssNMR in resolving peptide secondary structures in complex environments; in this case, mixtures of phospholipids are used to simulate eukaryotic me mbranes and membranes from the lung. Materials and Methodology Peptide synthesis: KL4 was synthesized via automated solid-phase peptide synthesis (ABI 430, ICBR, UF) on a Wang resin. Peptide wa s cleaved from the resin with 90% TFA/5% triisopropyl-silane/5% water a nd ether precipitated. The crude product was purified by RPHPLC using an acetonitrile/water grad ient and purity was presence of KL4 was verified by mass spectrometry with a m/z ratio of 2471. Finally the product was lyophilized and stored at oC until used in sample preparation. Preparation of KL4 in buffer: Dried peptide was weighed and solublized in 1:1 methanol:water to an estimated stock concentra tion of ~200M. This ensured full dissociation of the peptide and prevented any unwanted form ation of aggregates. The solution of 1:1 methanol:water was placed under a stream of nitrogen to remove the methanol and then concentrated HEPES buffer was added to ach ieve a final concentration of ~100M of KL4 in 5mM HEPES, pH 7.4.

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128 Preparation of POPC:POPG and DPPC:POP G Vesicles For CD Measurements with KL4: DPPC, POPC and POPG lipids dissolved in chloroform were purchased from Avanti Polar Lipids (Alabaster, AL). The lipids were mi xed at molar ratios of 3:1::POPC:POPG and 4:1:: DPPC:POPG and the CHCl3 was removed under a nitrogen stream. The dried lipid films were dissolved in cyclohexane and lyophilized to remove residual chloroform. The lipids were then reconstituted in 5mM HEPES, pH 7.4, and s ubjected to 3 freeze-thaw cy cles to facilitate the formation of multilamellar vesicles (MLVs). Larg e unilamellar liposomes (LUVs) were prepared by extrusion of the MLVs through 100 nm polycar bonate filters (Avanti Polar Lipids) at room temperature. Extrusion of DP PC:POPG LUVs took place above the mixtures phase transition temperature. Preparation of solution CD samples: CD spectra were collected on 150M KL4 in 1:1 methanol:water as well as hexafluroisopr opanol. Since the helical content of KL4 is unknown in solution, a spectrum of 40M KL4 in the helix-inducing solvent trifluoroethanol (TFE) ( 81) was also collected to generate a baseline CD spect rum for the purely helical peptide. In this preparation, stock KL4 (498M +/3M) in methanol solution was dried under compressed nitrogen gas and the peptide film was solubilized in 1mL TFE to a final pe ptide concentration of 40M. The spectrum for KL4 in TFE was run at 45oC and subtracted from a spectrum of only TFE. Preparation of KL4/lipid samples in organic solvent for CD: A stock concentration of KL4 in methanol (498M +/3M) was added to ch loroform solution of lipids at 1, 2 and 3mol% peptide relative to lipids. The peptide-lipid mixtures were allowe d to dry overnight before being dissolved with cyclohexane and lyophilized overnight. Samples were hydrated in 10mM HEPES

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129 pH 7.4, 140mM NaCl and freeze-thawed 3-5 times over the course of 48 hours before extrusion. Extrusion to generate LUVs we re done as described above. Preparation of CD samples with peptides and lipids mixed in buffer: 100M KL4 in HEPES buffer was added to extruded DPPC, POPC, POPG, 4:1 DPPC:P OPG, 3:1 POPC:POPG vesicles in the same HEPES buffer. For samp les containing DPPC, samples were extruded at 50oC, and 100M KL4 stock, prepared as above, was adde d to freshly extruded vesicles to achieve a final peptide:lipid ratio of 1:33. The final concentra tion of peptide in these samples was 40M and the final concentration of lipid wa s 1.33mM. CD spectra on these samples were recorded at 45oC. Collection of CD spectra: CD experiments were performed on an Aviv Model 215 at a wavelength range 195-260nm with a step size of 1nm for 10-20 s cans. The averaging time for each sample was 1 second. The settling time for each time was 0.3-0.6 seconds. All CD spectra were buffer subtracted. CD shows KL4 to be helical Circular dichroism provides a simple qualitati ve method for determin ing peptide secondary structure in solution as well as for peptide bound to lipid. As an initial assessment of global secondary structure, CD was performed on KL4 in 5mM HEPES pH 7.4. At three different concentrations in 5mM HEPES buffer at pH 7.4, the peptide displa yed spectra that are characteristics of a helical peptide, although some other secondary structure elements can also be seen (Figure 4-1). CD performed on KL4 at 150M, still show a minimum at 208nm, however the appreciable loss of signal at 222nm may indicat e the onset of aggregation of the peptide. Given the highly hydrophobic nature of the peptide, aggregation or higher order peptide structures is highly possible. In the presence of the helix-inducing organic solvent hexafluoroisopropanol (HFIP), the CD spectrum of 150M KL4 indicates helical structure

PAGE 130

130 (Figure 4-2). These initial CD experiments indicate that KL4 is helical in buffer and HFIP induces further helicity. At higher concentrations in buffer, peptide aggregation most likely occurs. In methanol and water a reduced heli cal signature is seen. We then undertook CD experiments to examine the secondary structure of KL4 when it is interacting with LUVs to answer the question of whether lipid bilayers can serve as a substrate to induce secondary structure formation. Figure 4-3 shows CD measur ements taken of 40M KL4 interacting with 1.33mM lipid LUVs in which both peptide and LUVs were reconstituted separately in 10mM HEPES buffer and then mixed. Helicity was assessed by examining the ellipticity at 208 and 222nm. KL4 added to neat POPG and DPPC LUVs yielded spectra with adsorption minima at 208nm and 222nm indicative of helical secondary structure. The mixture of 4:1 DPPC:POPG LUVs with 3mol% peptide also showed a pronounced helical CD spectrum (Figure 4-3). CD recorded of KL4 in the presence of neat POPC lipids result ed in a noisy spectrum with poor signal; however when the same amount of peptide was added to 3:1 POPC:POPG LUVs, signa l with clear helical tendencies was recaptured (Figure 4-3). Th e following observations from CD supports 31P NMR data that reflect an affinity of the peptide w ith the anionic phosphatidyl glycerol lipid. Since KL4 shows helical propensity in solution and is clear ly not unstructured (Figure 4-1), the role of POPG may not necessarily involve folding of the peptide, yet the presence of the lipid clearly results in changes in ellipticity at 222nm strongly implicating th e modulation of helical content by this phospholipid species. Solution CD of KL4 in 10mM HEPES buffer, pH 7.4 and 140mM NaCl show a double minima that is characteristic of some helical qualities of the peptide, but the CD spectrum indicates the peptide is not 100% helic al, especially when compared to peptide in TFE (Figure 4-4).

PAGE 131

131 Ellipticity at 222nm ( 222) has been found to increase linearly with the extent of helix formation ( 169, 170). Shown in Table 4-1 are the 222 values obtained by adding peptide to preformed LUVs. Negative elliptic ities at 222nm were found when KL4 was interacting with DPPC, POPG and DPPC:POPG LUVs. When viewed as the dichroic ratio of 222/ 208, the two wavelengths that yield double minima in an alpha helical CD spectrum, it is clear that the helical signatures of the peptide are changing relative to the peptide in solution. The CD data strongly implicate PG headgroups and saturated PC lipid s in enhancing the helic al propensity of the peptide. Peptide and lipid mixed together in organic so lvent and reconstituted also result in helical CD spectra typified by the minima at 208nm a nd 222nm. Shown in Figure 4-4 are spectra for 40M KL4 in the presence of POPC:POPG and DPPC:P OPG LUVs at a peptide molar ratio of 1%. To determine if we can di scern structural changes in KL4 as a function of concentration, samples of 40M KL4 which was either at 2mol% and 3mol % with respect to the lipids were also run. At 2mol% peptide, the double mi nima in both DPPC:POPG and POPC:POPG can be seen, although the signal is degraded by light scattering from th e lipids (Figure 4-5). 40M KL4 with POPC:POPG LUVs shows a CD spectrum that displays si gnificant overlap in signal to 40M KL4 in HEPES/NaCl solution. In an environm ent of POPC:POPG LUVs, the peptide is in a state that is helical but less so than in TFE. In comparis on, the double minima at 208nm and 222nm is prominent at 2mol% peptide in DPPC:POP G LUVs. (Figure 4-5). At 3 mol% peptide, the CD signal from KL4 is barely discernable when interacting with POPC:POPG LUVs, suggesting it is disturbing the lipid phase properties leading to light scattering, but the double minima is still prominent in DPPC:POPG LUVs (Figure 4-6). The data clearly indicate concentration and lipid dependence in CD signal and can help explain the differing dynamics and

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132 lipid ordering seen from 2H and 31P NMR based on KL4 studied in POPC:POPG versus DPPC:POPG vesicles. Figure 4-7 shows the CD si gnal generated as peptid e levels are increased in DPPC:POPG LUVs and POPC:POPG LUVs The helical signature seen for KL4 in DPPC:POPG bilayers, where we postulated the peptide is more embedded, is seen in Figure 4-7. The signals at 208nm and 222nm seen in DPPC :POPG LUVs could also be a result of contributions from other t ypes of helices such as a -helix that do not make such contributions in POPC:POPG LUVs. Table 4-2 shows the raw CD ellipticity (in millidegrees) of DPPC:POPG and POPC:POPG at different molar percentages of KL4. Comparing the ellipticity values amongst the lipid systems, we find that helica l signatures in DPPC:POPG at higher peptide levels than in POPC:POPG. However, it is no t known if the changes seen in ellipticity are representing changes at the residue level of KL4 or entire sections of the peptide are assuming different helical conforma tions as concentration increases rela tive to the lipids. If the spectrum of KL4 in TFE assumes complete helicity, then th e CD data indicate th at both lipid systems increase helical content of KL4 relative to that in solution. Comparing KL4 in buffer relative to that in TFE solvent, it is clear that KL4 contains a mixture of seconda ry structures rather than a purely -helical motif. These experiments were performed with 1020 scans for signal averaging; it is not known if an increase in th e number of scans woul d allow better comparison of KL4 samples in an embedded bilayer relative to bound to the headgroup regions. It can be seen however that differences can be seen when KL4 binds DPPC:POPG versus POPC:POPG LUVs. While the typical double mi nima characteristic of -helices are seen in CD spectra of KL4 both with and without lipid, it is important to realize that such a method is only sensitive to average properties of a molecule and the distribution of helical and non-helical residues cannot

PAGE 133

133 be determined ( 169). Additionally, CD does not allow clear distinction between different types of helices. The peptide in combination with lipids may have signal du e to formation of nonstandard helices. Our data do show, however, that KL4 has helical characteristics both in solution and when interacting with DPPC, POPG, DPPC:POPG and POPC:POPG LUVs. CD indicates that KL4 is helical in solution; however, the periodic placement of lysines would make an -helical conformation favorable in buffe r, but not at a lipid interface where amphipathic structures ar e favored. Placement of KL4 in helical wheel diagrams for different types of helices show that in order for the peptid e to bind to an amphipathic substrate, such as a lipid interface, the peptide might form i, i+5 hydrogen bonds (Figure 4-8). Such a helical wheel diagram suggests that a helix has a favorable alignment of the hydrophobic leucine residues and hydrophilic lysine residues for snorkeling into a lipid bilayer. Whereas modeling KL4 as a i i+4 helix shows hydrophilic lysines around the periphery of the helical wheel. This secondary structure should be favored in th e solution-state, where interactions occur isotropically, but is disfavored in a lipid environment. This begs the question whether the peptide adapts a canonical i i+4 helix or another helix with a different hydrogen bonding pattern in the context of a lipid environment. Torsion angle measurem ents using ssNMR were used to answer this question. Different Types of Helices KL4 May Adapt in a Lipid Bilayer A standard i i + 4 helix has 3.6 residues per turn provi ding for the most stable atomic arrangement thermodynamically, accounting for r oughly 30% of secondary structure found in proteins ( 171 ). Torsion angles used to defi ne the backbone conformation at C-N bond (defined as the angle psi: ), at the C-C bond (defined as the angle phi: ) are -57o ( ) and -47o ( ) for a classic -helix. However, variations do exist, and on average values of =-65o and =-45o are seen in x-ray structures of crystalline proteins ( 172 ). Given the nature of lysine side chains

PAGE 134

134 spaced every five residues in KL4, hydrogen bonds to residues in an i i+5 arrangement cannot be disqualified as a resulting helical fold in the lipid environment. This can give the helix a tighter coil which may help the peptide interact with heterogeneous environments and facilitate the snorkeling model envisaged in Chapter 3. An i i+5 hydrogen bond pattern results in a helix with 4.4 residues per turn and is termed a helix ( 171) with 16 atoms between the hydrogen bonds. This type of helix is consider ed to be thermodynamically unfavorable in free solution ( 173 ). Solid-State NMR Studies of KL4 in POPC:POPG and DPPC:POPG Work in determining the helical nature of KL4 was undertaken by Professor Joanna Long and Dr. Douglas Elliott. In these studies, KL4 was synthesized with 13C enrichment at specific leucine C positions in the peptide and the peptide was complexed with 3:1 POPC:POPG MLVs for CPMAS experiments. Using the DRAWS pu lse sequence, the double quantum (DQ) state between the 13 C spins was excited during mixing times in the pulse sequence. The mixing time it takes to generate the DQ coherence is de pendant on the distance between the spins. Distances between adjacent 13C spins were determined by a least squares fit of DQ buildup curves to numerical simulations (Figure 4-9). As can be seen in this figure, the build-up curve is consistent with a angle of -100o to -105o. Using a 2D-DRAWS e xperiment, the relative orientations of the amide planes were m easured and compared to simulations. The 2-fitting of the torsion angle based on comparison of experiment al data to numerical simulations is shown and the best fit torsion angles from these simulations were = -105o = -26o and = -105o, = 132o (Figure 4-10). Using torsion angles of -105o and -26o in the molecular graphics program PYMOL renders a helix where all the charged lysines lie on one side of the helix (Figure 4-11). Such a configuration would indeed have biological impli cations; particularly in terms of the peptide

PAGE 135

135 being able to embed to the membrane. However, the splaying of the lysines suggest it would not be deeply embedded. From the deuterium NMR data performed in Chapter 3, POPC and POPG acyl chain order parameters decreased slightly in the presence of KL4 corroborating this model. Similar experiments performed by Professor Joanna Long on KL4 in DPPC:POPG LUVs reveal backbone torsion angles of =-65 and =-78. Placement of these torsion angles into the molecular graphics program PYMO L also yields a helix (Figur e 4-12). However, unlike what was seen for the peptide bound to POPC:POPG vesicl es, it is found that the lysine residues are more aligned along one side of the helix. Comp aring Figure 4-11 and Fi gure 4-12 one sees that in POPC:POPG MLVs, the lysine residues have a more radial distributi on around the axis of the helix as compared to in DPPC:POPG MLVs. In POPC:POPG MLVs, the side chain lysines face preferentially one side of the helix, but the first and last lysine residue are particularly out of phase. In DPPC:POPG MLVs, the first and fifth lysine side chains are more parallel and in phase with each other. The argument presented for lysine snorkeling in Chapter 3 from deuterium NMR data corroborates nicely with the structure Professor Long found for the 13C labeled peptide in DPPC:POPG. The helical structure obtained from DQ-DRAWS aids in explaining the increase in deuterium NMR order pa rameters found in the middle to the end of the acyl chain. If the lysine residues of KL4 are more aligned in DPPC:POPG, the peptide can bury itself deep into the bilayer, while the long side chains of lysine can exte nd out into the interface and form a favorable electrostatic interaction with the phosphate headgroups. In POPC:POPG, where the acyl chains become less ordered in the presence of peptide, the lack of lysine alignment seen in the structure found from DQ-DRAWS meas urements strongly implicate a peripheral interaction of the peptide with the headgroup only. This is seen from our CD data where in POPC:POPG the helical signature was lost at 3mol% peptide but can still be discerned

PAGE 136

136 in DPPC:POPG. The CD data also rules out =-105 and =132 as one of the torsion angles obtained from Professor Longs 2D-DRAWS simu lations since such backbone torsion angles would predict a -sheet for the peptide. Hence, the findings from Professor Long and Dr. Elliott show that headgroup conformation and acyl chain dynamics clearly affect the type of helix KL4 adapts in a lipid bilayer setting. Our deuter ium NMR and CD data with the ssNMR data from Professor Long show that in DPPC:POPG, KL4 adapts a structure that has more qualities of a helix. Based on CD and ssNMR torsion angle measurements, in POPC:POPG, KL4 adapts into a structure that is helical but intermediate between an -helix and a -sheet. Fodje and Karadaghi recently re-eva luated the proteins in the PDB ( 171), and according to their criterion, have found an unde restimation in the amount of helices accounted for. Based on their algorithms, the authors have come up with mean dihedral angles ( ) of -76o and -41o, which are in stark contrast to previous measurements of -57o and -70o ( 171, 172) but in good agreement with the DPPC:POPG structure. Furthermore, Fodje and Karadaghi found significant differences in dihedral angles from the i-4 to the i+4 residues within the helix. Hence it seems probable that KL4 has characteristics (particularly in th e middle of the sequence) similar to a helix, offering the advantages of partitioning the charged lysine side chains to adapt to a lipid environment and reducing the surface area and volume occupied by the peptide ( 171) which would be entropically favorable. helix formation may be modulated by amphilicity and may also help to stabilize lipid-w ater or air-water interfaces (174). Thus, the types of helix resulting from ssNMR data of KL4 represent non-canonical structures due to residues changing alignment in the presence of amphiphilic lipid substrates such as 3:1 POPC:POPG and 4:1 DPPC:POPG and could indicate how KL4 modulates lipid dynamics in the l ung. The CD data show different secondary structure signals con tingent on the different lipid sy stem which agree nicely with 31P

PAGE 137

137 and 2H NMR data. Professor Longs structural meas urements of the pep tide in DPPC:POPG and POPC:POPG clearly indicate th e peptide has a different structure in each environment underscoring the observation that th e peptides structure, orientat ion, and depth penetration is dependant on the saturation level of the acyl chains.

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138 -40 -20 0 20 40 60 80 195205215225235245255 Wavelength (nm)Ellipticity (mdeg) 15uM KL4 30uMKL4 60uM KL4 -300 -200 -100 0 100 200 300 195205215225235245255 Wavelength (nm)Ellipticity (mdeg) 150uM KL4 -40 -20 0 20 40 60 80 195205215225235245255 Wavelength (nm)Ellipticity (mdeg) 15uM KL4 30uMKL4 60uM KL4 -300 -200 -100 0 100 200 300 195205215225235245255 Wavelength (nm)Ellipticity (mdeg) 150uM KL4 Figure 4-1. CD Spectra of KL4 in 5mM HEPES at pH 7.4. Below is a spectrum of 150M KL4 indicating potential aggregation.

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139 Wavelength (nm) 190200210220230240250260270 Molar Ellipticity (mdeg/ aa [M] cm) -20000 -15000 -10000 -5000 0 5000 10000 15000 Figure 4-2 CD spectra of KL4 in organic solvents. Red is a spectrum of peptide in 150M hexafluroisopropanol and black is a spectrum in 50:50 MeOH:dH20.

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140 190200210220230240250260270 -60 -40 -20 0 20 40 60 80 100 120 Ellipticity (mdeg)Wavelength (nm) POPC POPG 3:1 POPC:POPG DPPC 4:1 DPPC:POPG 100M KL4 Figure 4-3. CD Spectra of 40M KL4 added to 1.33mM LUVs. DP PC containing samples were run at 45oC. Remaining samples were run at room temperature. Samples were in 10mM HEPES pH 7.4 and 140mM NaCl.

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141 190200210220230240250260270 -30 -20 -10 0 10 20 30 40 Ellipticity (mdeg)Wavelength (nm) DPPC:POPG LUVs with 1% KL4 POPC:POPG LUVs with 1% KL4 40M KL4 in HEPES/NaCl 40M KL4 in TFE Figure 4-4. CD spectra of 40M KL4 reconstituted in 4mM 4:1 DPPC:POPG and 3:1 POPC:POPG LUVs. Shown in comparison is 40M KL4 in 10mM HEPES, 140mM NaCl and in TFE.

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142 190200210220230240250260270 -30 -20 -10 0 10 20 30 40 DPPC:POPG LUVs with 2% KL4 POPC:POPG LUVs with 2% KL4 40M KL4 in HEPES/NaCl 40M KL4 in TFEEllipticity (mdeg)Wavelength (nm) Figure 4-5. CD spectra of 40M KL4 reconstituted in 2mM 4:1 DPPC:POPG and 3:1 POPC:POPG LUVs. Shown in comparison is 40M KL4 in 10mM HEPES, 140mM NaCl and in TFE.

PAGE 143

143 190200210220230240250260270 -30 -20 -10 0 10 20 30 40 Ellipticity (mdeg)Wavelength (nm) DPPC:POPG LUVs at 3% KL4 POPC:POPG LUVs at 3% KL4 40M KL4 in HEPES/NaCl 40M KL4 in TFE Figure 4-6. CD spectra of 40M KL4 recons tituted in 1.33mM 4:1 DPPC:POPG and 3:1 POPC:POPG LUVs. Shown in comparison is 40M KL4 in 10mM HEPES, 140mM NaCl and in TFE.

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144 -30 -20 -10 0 10 20 30 40 190200210220230240250260 Wavelength (nm)Ellipticity (mdeg) 4:1 DPPC:POPG with 1% KL4 4:1 DPPC:POPG with 2% KL4 4:1 DPPC:POPG with 3% KL4 -30 -20 -10 0 10 20 30 40 190200210220230240250260 Wavelength (nm) Ellipticity (mdeg) POPC:POPG with 1% KL4 POPC:POPG with 2% KL4 POPC:POPG with 3% KL4 -30 -20 -10 0 10 20 30 40 190200210220230240250260 Wavelength (nm)Ellipticity (mdeg) 4:1 DPPC:POPG with 1% KL4 4:1 DPPC:POPG with 2% KL4 4:1 DPPC:POPG with 3% KL4 -30 -20 -10 0 10 20 30 40 190200210220230240250260 Wavelength (nm) Ellipticity (mdeg) POPC:POPG with 1% KL4 POPC:POPG with 2% KL4 POPC:POPG with 3% KL4 Figure 4-7. CD Spectra of (Top) POPC:POP G LUVs and (Bottom) DPPC:POPG LUVs with increasing mol% of KL4.

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145 Table 4-1. Ellipticity (in mdeg) of KL4 at 222nm and 208nm and ratio of helical signatures 222nm and 208nm 208nm 222nm 222nm/ 208nm 40M KL4 in POPC -5.4 -4.8 0.9 POPG -6.4 -14.1 2.2 3:1 POPC:POPG -5.2 -6.7 1.3 DPPC -17.9 -17.9 1.0 4:1 DPPC:POPG -16.3 -17.3 1.1 100M KL4 in solution -45.7 -9.5 0.2

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146 Table 4-2. Ellipticity (in mdeg) of 40M KL4 reconstituted in LUVs from organic solvent. 208nm 222nm 222nm/208nm 4:1 DPPC:POPG 1% KL4 -22.5 -16.1 0.7 2% KL4 -15.4 -13.5 0.9 3% KL4 -17.4 -15.2 0.9 3:1 POPC:POPG 1% KL4 -20.6 -16.2 0.8 2% KL4 -11.2 -9.4 0.8 3% KL4 -10.9 -9.6 0.9 40M KL4 -8.9 -9.0 1.0 (in 10mM HEPES, 140mM NaCl) 40M KL4 -24.9 -16.5 0.7 (in TFE)

PAGE 147

147 Figure 4-8. Helical wh eel projections of KL4 as: (left) 310 helix, (middle) standard -helix, and (right) helix.

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148 0.0 0.2 0.4 0.6 0.8 1.0 02468 Mixing time (msec)Normalized Intensit y KL4-L2,L3 KL4-L4,L5 KL4-L7,L8 KL4-L9,L10 phi = -65 phi = -120 Figure 4-9. DQ-DRAWS buildup curv es generated from spectra on 13C labeled KL4 with POPC:POPG (3:1). Data taken with pe rmission from Professor Joanna Long.

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149 -110 -108 -106 -104 -102 -100 -98 -96 -94 -92 -90 150 100 -50 0 50 100 150 (-105o, -26o) -110 -108 -106 -104 -102 -100 -98 -96 -94 -92 -90 150 100 -50 0 50 100 150 (-105o, -26o) Figure 4-10. Ramachandran plot showing a 2 minimum at =-105 and =-26 for KL4 in 3:1 POPC:POPG. Contours shown in blue, gr een and brown are 1,2, and 3 standard deviations away from the minima. Data ta ken with permission from Professor Joanna R. Long.

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150 Figure 4-11. Model of KL4 based on torsion angles obtained fr om the 2D-DRAWS experiments. The charged lysines (shown in blue) line up along one side of the helix.

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151 Figure 4-12. KL4 with torsion angles of = -65, = -78 obtained from ssNMR studies of KL4 in a DPPC:POPG lipid environment. Different views of the peptide indicate the lysine side chains line up along one side of the helix. In comparison, in POPC:POPG the lysine side chains splay radially along th e helical axis. Data taken with permission from Joanna R. Long.

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152 CHAPTER 5 COMPARATIVE BIOPHYSICAL STUDIES OF SP-B59-80 This chapter describes 2H and 31P NMR studies of 4:1 DPPC(d -62):POPG and 3:1 POPC(d31):POPG lipid systems on incorporation of SP-B59-80, the C-terminus of SP-B. Fragments of SP-B have biophysical activityWhile the entire 80 amino acid SP-B protein is essential for lung surf actant organization, lung dynamics and respiration, subfragments of the native sequen ce have also shown significant biophysical function, particularly peptides correspondi ng to the N-terminal and C-terminal 20-25 amino acids. While both ends of protein have in teresting biophysical properties, to date, the Nterminal region of the peptide has been more exte nsively studied in terms of functional properties and possible structural adaptation in different lipid environments. The N-terminal 25 residues of SP-B (SP-B1-25) have been shown to interact with specific anionic lipids to facilitate squeeze-out of lipids based on surface-film studies (52 ). SP-B1-25 has also been shown to mediate mixing of lipid vesi cles. Fourier-transform infrared measurements and CD studies of the N-terminal peptide in methanol, SDS micelles, and egg yolk lecithin (phosphatidylcholine) indicate the pe ptide has a high helical content. Spin-labeling of the first phenylalanine residue for electron spin resonance st udies show that the N-terminus of the peptide retains mobility when bound to lipid ( 175). Recent molecular dynamics simulations of the peptide in DPPC monolayers indi cate that the most likely eq uilibrium conformation is an -helix parallel to the interface ( 176). However, the N-terminal fragment of SP-B has not been used in any formulations of artificial lung surfactants in clinical use indicating that wh ile the role of the N-terminus may be of importance, it does not co nvey all the functionality needed for the protein component of lung surfactant.

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153 The C-terminal fragment of SP-B, specifically residues 59-80 (SP-B59-80), has shown potential in in vitro assays measuring surface tension such as pulsating bubble surfactometry. Isotherms relating surface pressure to surface area show that this peptide imparts lipid monolayer stability under compression, preventing collapse at pressures where monolayers of lipids alone collapse (133 ). Of particular clinical interest, the last 22 amino acids of SP-B served as the template for designing KL4 as discussed in Chapters 3 and 4 ( 67, 133 ). The basis of the design for KL4 was modeling the charge distribution and hydrophilic/hydrophobic rati o of the primary sequence of SP-B59-80. As with the N-terminus, the C-termin al peptide is believed to form an amphipathic helix involved in headgroup ordering, but direct structural m easurements in varying lipid contexts have to date not been document ed. Nonetheless, a solution NMR study of the C-terminus of SP-B (residues 63-78 ) reconstituted in either SDS micelles or the organic solvent HFIP was recently published. This study was unable to see structure in th e first five residues, but established that the rest of the sequence formed a helix in both SDS micelles and organic solvent (177 ). Unlike SP-B1-25, FTIR or CD studies of SP-B59-80 interacting with lipids are not documented and molecular dynamic simulations of SP-B59-80 do not exist. A previous CD study ( 178) using TFE and SDS micelles a nd the solution NMR study are the only current structural assessment of the C-terminal region of SP-B. Despite the scarcity of literature pertaining to residues 59-80 of SP-B, it is widely believed that many in vivo activities of SP-B are fulfilled by the C-terminal end based on in vitro studies ( 134, 177 ). Interestingly, both N and C-terminal ends of th e peptide have been repo rted to be cationic, amphipathic helices which could serve in a functi onal role by interacting with anionic lipids, particularly PG headgroups ( 11). Placement of SP-B59-80 in helical wheels based on standard helical geometries as well as a 310 and helix are shown in Figure 5-1. While solution NMR

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154 measurements indicate, and the helical wheel di agrams depicted in Figure 5-1 assume regular periodicity to the helix, it should be noted that this may not be its exact structural adaptation in a lipid environment. Also, an -helical conformation leads to unfavorable placement of the charged residues in a lipid milieu Of particular interest to us, the KL4 sequence originates from the primary sequence residues 59-80 of SP-B. However, if KL4 provides an essential f unction to artificial lung surfactant, as the clinical studies suggest, a significant question that is raised is whether the peptide behaves like SP-B59-80 or SP-C. From infrared studies, SP-C was found to be a transmembrane helix that can perfectly span the width of a DPPC bilayer and an FTIR study of KL4 in lipids yielded similar results ( 5, 11, 148). However, our data described in Chapter 3 show that KL4 is not transmembrane in DPPC:POPG and POPC:POPG bilayers, and therefore would not orient in the same manner as SP-C. Nonetheless, a significant question remaining is whether KL4 and SP-B59-80 act similarly, despite clear deviations at the primary amino acid level. One key to answering this is to determine if both peptides orient similarly in model lipid membranes with similar effects on their dynamic properties using 2H and 31P NMR experiments. Additionally, one can compare their effects on lipid phase transitions with DSC. The following studies were undertaken to determine whether the biophysical activity of the native sequence closely followed that of KL4. Materials and Methodology Synthesis of SP-B residues 59-80: SP-B59-80, (sequence from the N to C-terminus: DTLLGRMLPQLVCRLVLRCSMD) was synthesized via solid-phase peptide synthesis by Dr. Alfred Chung at the University of Florida a nd cleaved from the resin with 90% TFA/5% triisopropyl-silane/5% water a nd ether precipitated. The cl eaved product was purified via a HPLC with C18 Vydac column using a wate r/acetonitrile gradient with 0.3% TFA

PAGE 155

155 (trifluoroacetic acid). The fractions corresponding to SP-B59-80 were collected and purity of the product was verified by mass spectrometry with a mass to charge ratio of (m/z) of 2533. DSC on 4:1 DPPC(d-62):POPG LUVs with SP-B59-80: DSC measurements were taken on DPPC(d-62):POPG LUVs with varying levels of SP-B59-80. Samples were prepared by drying peptide:lipid complex from chloroform:methanol mixtures at a specific molar ratio, drying and dissolution in cyclohexane. Af ter lyophilization overnight, samp les were hydrated with 5mM HEPES buffer pH 7.4 containing 1mM EDTA and 140mM NaCl in a water bath maintained at 50oC. The samples underwent multiple freeze-thaw cycles to facilitate MLV formation and then were extruded through 100nm filters a minimum of 15 times to generate LUVs Each sample was run in triplicate at a temperature range of 10-70oC and a scan rate of 1oC/min. Solid-state NMR sample preparation: Samples for ssNMR were prepared in an identical manner to KL4 detailed in the Materials section of Chapter 3. Briefly, SP-B59-80 peptide was dissolved in MeOH to a stock concentration of 1mM. DPPC(d-62), POPC(d-31), and POPG were purchased from Avanti Polar Lipids (Alabaster, AL) in chloroform solutions and mixed to the desired molar ratios. 4:1 DPPC(d-62):POPG and 3:1 POPC(d-31):POPG solutions were mixed with the peptide in MeOH solution to create a range of samples containing 0-3mol% SPB59-80. After mixing corresponding amounts of pep tide and lipids to the desired molar percentage, the samples were reconstituted by drying peptide/lipid mixture to a film under compressed nitrogen gas, solubilizing them in 2mL cyclohexane and freeze-drying overnight. The next day, approximately 20-30 mg each of dried sample was packed into a standard 5mm NMR tube (Wilmad, Buena NJ) and hydrated with 150-200L 5mM HEPES buffer pH 7.4, containing 1mM EDTA, and 140mM NaCl. NMR samples were freeze-thawed 3-5 times to

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156 facilitate MLV formation. For samples containi ng DPPC:POPG, the samples were thawed in a water bath maintained at 50oC. The 31P NMR data were collected on a 600 MH z Bruker Avance system (Billerica, MA) using a standard 5 mm BBO probe, and 25 kHz proton decoupling during acquisition. Spectra were acquired at 3 temperatures with 1024-2048 s cans for each spectrum and a 5 second recycle delay between scans to minimize RF heating of the samples. 2H NMR data were collected on a 600 MHz Bruker Avance System (Billerica, MA) using a standard 5mm BBO probe and quad echo sequence with a B1 field of 40 kHz. Spectra were acquired at 3 temperatures with 10242048 scans and 0.5 second recycle delay. Depaking of 31P and 2H NMR spectra was performed using a Tikhonov regularization met hod that takes into account m acroscopic lipid alignment. This algorithm was provided by Professor Edward Sternin ( 103, 124). DSC Studies on SP-B59-80 with DPPC:POPG LUVs DSC thermograms for DPPC(d-62):POPG LUVs with varying amounts of SP-B59-80 are shown in Figure 5-2. Overall, in a concentration dependant manner, SP-B59-80 increases Cp max and Hcal for the phase transition from the L to the L state in DPPC:POPG LUVs. In comparison to Figure 3-1, where DSC thermograms performed on similar 4:1 DPPC:POPG LUVs with varying levels of KL4 indicate domain formation, a clear form ation of 2 lipid phases on addition of SP-B59-80 is not seen. However, it is evident that SP-B59-80 causes an ordering of the lipids by the increased amplitude of the transition seen in the DSC curves. This becomes manifest when comparing Hcal, the integration of each DSC peak, for no peptide and with 3% peptide. Hcal is approximately 2-3 times greater with 3% SP-B59-80 than without any peptide (Table 5-1). The phase transition temperature is not dramatically influenced by SP-B59-80, staying relatively constant at 32oC.

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157 Table 5-1 displays a thermodynamic evaluation of the effects of SP-B59-80 on the DPPC(d-62):POPG phase transition. The most interesting and unexpected finding stemming from these DSC studies is that, unlike KL4, SP-B59-80 does not produce phase separation in 4:1 DPPC:POPG. In a concentration dependant manne r we see an increase in the enthalpy of the DSC curves upon more addition of peptide. DSC studies on KL4, at a similar concentration range, show a drop in enthalpy at concentrations where phase separation or domain formation became obvious, which were at 1.5 and 2.2mol%. At 1.5mol% KL4, the DSC curve bifurcated and was characterized by two Tm values, with one Tm value shifted towards the phase transition of DPPC (Figure 3-1) suggesting lipid sequestration or phase separation by the peptide. No such bifurcation or change in th e appearance of the DSC curve occurred with addition of SP-B59-80 in similar concentration ranges. Th e increase in enthalpies (both Hcal and HvH) seen as more peptide is added, suggest SP-B59-80 orders the lipids and stabilizes the DPPC:POPG L state. This is corroborated by the finding that H>> S indicating that the phase transition is enthalpic, not entropic in nature. While addition of peptide seems to order the lipids and stabilize a particular state, it has no substa ntial effect on cooperativity other than at 0.5% of peptide (Table 5-1). We cannot explain the sudden increase in peak-width at half height seen only at 0.5mol% of peptide. However, the broadened lineshapes s een in the DSC curves strongly indicate that the phase transition of the lipids in the presence of SP-B59-80 is not a highly cooperative process. However, with the exception of 0.5mol% peptide, SP-B59-80 seems to decrease the cooperativity of lipid motions and no phase separation is seen when the peptide is added to 4:1 DPPC:POPG LUVs. This is also verified from DSC calcula tions that measure indirectly the extent of cooperativity by comparison of the Van Hoft and cal orimetric enthalpies wh ere it is seen that their ratio decreases slightly on addition of peptide.

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158 It is known that the addition of monouns aturated lipids markedly reduces the Tm when added to saturated phospholipid dispersions, such as DPPC, thus aiding such mixtures to be more fluid. This is important in lung surfactant b ecause without such molecules, the enriched amount of DPPC in the lungs would cause the monolayer to exist as rigid ge l. In our DSC results with SP-B59-80, we see an increase in the energy required for 4:1 DPPC:POPG to shift to any other state or transition. By keep ing the thermodynamic barrier high for any such temperature dependant transitions of 4:1 DPPC:POPG it is possibl e to hypothesize that SP-B59-80 is destabilizing the fluidizing prope rties of the monounsaturated lipid s, particularly POPG. This would be beneficial in situati ons where DPPC needs to be selectively enriched, such as would occur when the alveolar lipid monolayer need s to be compressed. However, the exact implication of ordering of lip ids by residues 59-80 of SP-B, as seen by DSC, is complex especially with regard to the complicated lipid environment in the lung as well as in DPPC:POPG. Still unanswered is whether the ordering seen is due to SP-B59-80 interacting with DPPC, with POPG, or by stabi lizing the mixed state of both species. Since there was no significant shift in phase transition temperature (Tm) of the binary lipid system with SP-B59-80, the exact role of the C-terminus of the peptide in alte ring lipid biophysics is not easy to interpret based on DSC data. 31P NMR of Lipid MLVs Containing SP-B59-80 Show POPG Interacting with the Peptide Natural abundance phosphorous NMR for 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs with varying levels SP-B59-80 were collected to assess the effect of this peptide on lipid phases and headgroup dynamics in comparison to KL4 (Chapter 3). Shown in Figure 5-3 are 31P NMR spectra for the DPPC(d-62):POPG samples. Only lamellar phases are observed and the resonance for the POPG lipids moves with additio n of peptide. Like the experiments with KL4, macroscopic lipid alignment is seen and the extent of alignment was accounted for by the

PAGE 159

159 dePaking algorithm. As with KL4, SP-B59-80 reduces this macroscopic alignment and generates spectra that are more powder-like in nature. Th is is apparent by the concentration dependent change in the intensity of the double-singularity seen in the compiled spectra (Figure 5-3). DePaking the series of spectra (Figure 5-4) yiel ds the parallel component of the powder spectra for DPPC and POPG and allows a quanti tative measurement of the change in 31P CSAs. Plotting these changes relative to lipids alone reveals th at addition of peptide causes a decrease in the POPG 31P CSA by up to 10% but does not affect the DPPC 31P CSA (Figure 5-5). This is similar to behavior seen for DPPC:POPG MLVs on addition of KL4 and argues strongly for a similar interaction of the two peptides with the anionic POPG headgroups. The 31P spectra for 3:1 POPC(d-31):POPG MLVs show that a lamellar phase is also observed (Figure 5-6). Although the spectrum at 3% peptide potential ly shows the onset of other phase behavior; however, it has not been fully characterized. Examination of the dePaked 31P spectra show that changes in the POPG peak occur upon increasing levels of peptide (Figure 57). The dePaked spectra stacked together show that the PC headgroup remains largely invariant while significant ppm shifts are seen in the PG headgroup (Figure 5-8). Interpretation of the dePaked spectrum for the sample with 3mol% SP-B59-80 is not straightforward and the shape of the spectrum indicates a drastic ch ange in headgroup dynamics at this specific concentration. As with KL4, we did not see an inverted HII phase upon addition of peptid e. However, at a higher percentage of SP-B59-80, this might be observed. Also, sim ilar to the non-natural peptide analog, there seems to be a preferential electrostatic interaction with the PG headgroup. However, we also see SP-B59-80 affecting the dynamics of POPC at higher c oncentrations, though the physiological amount of SP-B believed to be in th e lung is much lower (~0.2mol% relative to the lipids) than the levels where these dynamics are seen by our 31P NMR measurements.

PAGE 160

160 2H NMR Indicates that the Properties of KL4 are Similar to SP-B59-80.Deuterium NMR was performed on DPPC(d -62):POPG and POPC(d-31):POPG MLVs with varying levels of SP-B59-80 (Figure 5-9) to assess how the peptide affects the acyl chain dynamics in the two lipid systems. If increasing order is seen, then it can be assumed that SPB59-80 penetrates deeply into the lipid bilayer much like the findings reported with KL4. Time averaged deuterium order parameters , calculated after dePaking (Figure 5-10) and assigning each C-D bond in the acyl ch ain, show that, just as with KL4, SP-B59-80 increases order in acyl chains of DPPC in 4:1 DPPC(d-62):POPG MLVs (Figure 5-11). These large scale effects in ordering are not equal over the entirety of the acyl chain, which can be seen from the change in order parameter profiles depicted in Figure 5-11; the largest shifts in are seen from the middle to the end of the acyl chains. Regions corresponding to the plateau region (carbons 2-8) ( 145) have order parameter values changi ng on the order of 4% even at SP-B59-80 levels of 2 mol%. Positions 9-15 show ordering even at 0.1 mol% peptide. In 4:1 DPPC(d-62):POPG MLVs, the largest change in time averaged order parameter < SCD> occurs in C-D bonds at positions 10-13 which show increases up to 10% in C-D bonds 10-12, and up to a 15% increase for the C-D bond at position 13. This is seen in bo th the sn-1 and sn-2 chains of DPPC(d-62) in the context of 4:1 DPPC:POPG MLVs. Similar experiments were run on 3:1 PO PC(d-31):POPG MLVs containing SP-B59-80 (Figure 5-12) and dePaked (Figure 5-13) for calc ulation of order parameters. Looking at the same sn-1 C-D positions 10-13 for POPC(d-31) in 3:1 POPC:POPG, we find that changes in order parameters are negative and the ordering goes down by as much as 13-16%, with a 16% decrease in ordering seen for position 13 (Figure 5-14). This strongl y correlates to SP-B59-80 having minimal to little interact ion with the acyl chains of POPC in 3:1 POPC(d-31):POPG MLVs. Conversely, the peptide displays prof ound ordering of DPPC(d-62) in 4:1 DPPC(d-

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161 62):POPG MLVs. Since only the middle carbons s eem to be ordered on addition of peptide (Figure 5-11), a transmembrane orientation of SP-B59-80 is unlikely. If SP-B59-80 were indeed transmembrane in any of our lipid systems, we would expect ordering of the plateau carbons as well as the middle and tail ends of acyl chains. Th is is what is seen for helical transmembrane peptides including the WALP and KALP synthetic peptides ( 79, 127). More intriguing is the finding that, like KL4, we see that when acyl chains of the PC lipids are deuterated, SP-B59-80 causes changes in order of the acyl regions dependent on whether the lipid is saturated or monounsaturated. The derived order parameters for each acyl chain C-D bond from 2H NMR spectra seem to contradict literature 2H data studying the effects of full-length SP-B on DPPG ( d62) and DPPC ( d-62) lipids. These studies concluded that there was little e ffect of SP-B on the orientational order parameters for perdeuterated acyl chains examined in the liquid crystalline state (179, 180). In these papers, it was argued that SP-B perturbation was not localized at a particular depth along the bilayer at concentrations of up to 11% by weight. However, they used concentrated, non-physiological levels of SP-B to de termine its effects and orientation in various lipid environments. More recent findings by the same group indicate that full length SP-B can reduce chain order in DPPC(d-62) ( 181 ) which even further contradicts our 2H data which shows an ordering of the lipid in the presence of POPG. However, there are several plausible explanations for these discrepancies. It should be noted that in these published works, order parameters are not reported for each carbon alon g the acyl chain, which correspond to a more discrete, quantitative measure of order derived from the dePaked spectra. Instead, a first spectral moment (M1) is reported as a function of temperature. While the spectral moment reported is proportional to the overall average orientational order parameter, it does not yield a thorough

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162 analysis of the change in the order at each carbon of the acyl chain, and thus changes at particular positions would be averaged out, or not seen, by such an analysis. It also cannot be discounted that the sample prepar ation choices may have given rise to these conflicting findings. Although Morrow, et al and our preparations both involve reconstitution from organic solvents, we used substantially lower concentrations of peptide in our experiments, which more closely reflect physiologic levels and prev ent aggregation of the peptide. Their findings that SP-B has no effect on acyl chain order in DPPC and DPPG may stem from the amount of SP-B used in these experiments. In one study, concentrations of 6-17 weight percent of full length SP-B were used ( 182). At these levels, protei n aggregation is highly possi ble, especially given the hydrophobicity inherent within the full length prot ein. Higher order in termolecular complexes such as dimers, tetramers and multimers cannot be ruled out at these concentration ranges used for their NMR experiments. This may explain th e little to no perturbation caused by the peptide on DPPC acyl chains. The potential of fu ll-length SP-B at these high, non-physiologic concentrations to form higher order aggregates has not been fully addressed by the authors and any minimal interaction seen with the acyl chain may thus reflect this. In our studies, we are seeing dramatic effects on acyl chai n order in DPPC with as little as 0.5mol% of the C-terminal end of the peptide. Aggregation of SP-B59-80 at the levels of peptide we use is unlikely given the concentration dependent effects on lipid ordering and disordering in this range; if aggregation were indeed a concern we would expect minimal pe rturbation of these chai ns in a concentration dependant manner. However, we cannot completely reconcile their findings with ours since our studies used SP-B59-80 in binary lipid systems mi micking the lung and Morrow, et al used full length protein SP-B in neat lipid systems. Also, we cannot rule out the regulation of activity by the C-terminal end of SP-B by the full length protein; it also provide s evidence that SP-B is

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163 sensitive in its function to the lipids it is in complex with. Thus our studies as well as those of Morrow et al and numerous others clearly underscore the vital notion that membrane protein form and function is highly contingent on the lipid environment used to study it. Evidence of Alternative Dynamics at High Concentrations of SP-B59-80? In 3:1 POPC(d-31):POPG vesicles, the deuter ium NMR order parameters decrease in a concentration dependent manner (F igure 5-14). The downward sloping < SCD> values seen in Figure 5-14 are similar to the trends found for KL4. However, the changes seen with SP-B59-80 are larger. In conjunction with 31P NMR, these findings indicate that the SP-B59-80 is clearly stationed at the headgroup region of these lipids. However, at the highest concentration used in these studies, we found a clear deviation in the typical powde r spectrum in 3:1 POPC(d-31):POPG. With 3mol% SP-B59-80 complexed to 3:1 POPC(d-31):POPG, the 2H NMR spectrum shows a large coalesced peak in the center of the spectrum (Figure 5-12). The dePaked spectra for 3:1 POPC(d -31):POPG MLVs are shown in Figure 5-13, and while all spectra were readily dePaked, the spectrum taken with 3% SP-B59-80 has less definition. The 31P NMR spectrum for the sample with this concentra tion of peptide also re sulted in a poor quality dePaked spectrum that was uninterpretable (Fig ure 5-7). One possibility emerging from the 2H NMR and 31P NMR data is that at 3mol% SP-B59-80, the formation of a second, non-bilayer lipid phase is occurring. This averaging seen at this peptide concentration is due to additional fast motions of the lipid acyl chains which could occu r in a non-lamellar phase, such as an inverted HII phase, addressed briefly in Chapter 2 or due the formation of small vesicles which tumble quickly on the NMR timescale. These results indicate that higher concentrations of SP-B59-80, the dynamics and motion of the acyl chain are significantly a ffected. Not only is the finding shown in Figure 5-12 a possible indicator of an inverted HII phase, but it may also implicate SP-B59-80 in lipid shuttling, lysis or

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164 degradation. Lipid lysis and de gradation has been reported to be one of the putative functions attributed to SP-B that could be necessary for effective surfactant recy cling and remediation. Some reports implicate SP-B in anti-microbi al function which could help explain and corroborate the striking dynamics seen here, wh ich should be of no surprise since SP-B is classified into the saposin family of proteins ( 23, 30, 33, 71 ). However, further studies are needed to differentiate between a role for SP-B59-80 in lipid degradation or alternative phase formation. Surprisingly, we only saw this effect in 3:1 POPC(d-31):POPG, and not in DPPC(d-62):POPG at 3 mol% SP-B59-80. If the above finding is indeed reproducible, it would represent an exciting discovery of an activity for SP-B59-80 that can discriminate based on saturation level of the acyl chain. Comparisons of SP-B59-80 and KL4 Even though the sequence of KL4 is based on SP-B59-80, there are some differences seen from our data. First, the most striking difference is seen in the DSC data. In the same concentration ranges, SP-B59-80 does not seem to influence the phase properties of DPPC(d-62):POPG as markedly as KL4. While SP-B59-80 clearly affects the thermodynamic properties of these LUVs, we saw clear affects of KL4 in the lipid miscibility and overall mixing properties in DPPC(d-62):POPG LUVs and MLVs. Our data showed that during the initial stages of lipid sequestration, KL4 lowered the Cp max and Hcal of the peptide-lipid system, seen at 1.5 and 2.2mol% peptide in MLVs. Clear phase separation was also seen with KL4 added to 4:1 DPPC(d-62):POPG LUVs (Figure 3-1). SP-B59-80 does not cause similar behavior. 31P and 2H static NMR data show that KL4 and SP-B59-80 behave similarly in 4:1 DPPC(d-62):POPG lipid environments. 31P NMR data show that, after dePaking, very little change occurs in DPPC parallel edge frequency, while some modest changes occur in POPG parallel edge frequency. While an interaction of SP-B59-80 with anionic PG headgroup occurs is

PAGE 165

165 seen, the placement of lysines in KL4 could allow for a more enhan ced interaction with PG than with SP-B59-80, which does not possess the distinct charge periodicity found in KL4 and whose peptide sequence contains negative charges as we ll. The most striking changes seen on addition of either peptide is in deuterium order parame ters which indicate significant changes in the middle to the tail end of the acyl chain correspon ding to carbons 8-15. In creases in ordering of these regions while a lack of ordering in the ca rbons 2-8 were seen on addition of both peptides to DPPC:POPG MLVs. These findings discount a transmembrane orientati on of either peptide and predispose them to adapting a more peripheral orientation. In this regard, our observations implicate KL4 and SP-B59-80 to orient similarly in DPPC(d-62):POPG lipid environments. The 2H NMR data also show the KL4 and SP-B59-80 behave similarly in 3:1 POPC(d-31):POPG MLVs up to a concentration ra nge of 3mol%. The substantial decrease in time averaged order parameters indicate very li ttle effect on the motional freedom of the POPC sn-1 acyl chains mediated by both KL4 and SP-B59-80. In 3 mol% peptide, SP-B59-80 seems to facilitate additional lipid dynamics which need s to be addressed for reproducibility by 31P and 2H NMR. If indeed the observati ons at 3 mol% are valid, SP-B59-80 changes the dynamics of lipid systems based on the saturation of the PC acyl chains. This was seen with KL4 as well, however, the possibility of peptide-mediated change in lipid properties at high molar percentages of SP-B59-80 is a novel distinguishi ng feature relative to KL4. Static 31P NMR data reveal that in 4:1 DPPC:POPG MLVs, both SP-B59-80 and KL4 interact with the PG headgroup. Both peptid es displayed no predilection for an inverted HII phase though as-of-yet uncharacterized dynamics were seen at 3 mol% SP-B59-80. In KL4, we reconciled interaction with DPPC acyl chains and the POPG headgroup with domain formation that stemmed from our DSC studies with the pep tide. However, since we did not see any phase

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166 separation from our DSC studies with SP-B59-80, we have no data correlating SP-B59-80 interacting with DPPC acyl chains at the same time interacting with the PG headgroup. One way to reconcile these findings is to conduct experiments with deuterated PG acyl chains, i.e 4:1 DPPC:POPG(d-31) and 3:1 POPC:P OPG(d-31). If the deuterium order parameters for each C-D bond increase, clearly the peptide is buried deep within the bilaye r of the two lipids. If the values decrease, then a more complicated molecular picture must be occurring whereby the peptide somehow is buried with DPPC without facilitating de-mixing of the lipids. While KL4 has proven to be a clinically functional and potent peptide for re placing SP-B in treating respiratory distress and conventi onal lung surfactant formulations contain SP-B in full form, no formulation currently used contains either the N or C-terminal pept ide as a substitute for SP-B. This raises the possibility that while SP-B59-80 and KL4 may have similar stru ctural topologies in lipid environments, KL4 may have additional functions that SP-B59-80 lacks due to the flexibility imparted by using only leucines and lysines in the peptide. Preliminary Molecular Model of SP-B59-80 with Lipids With 31P NMR data looking at headgroup dynamics and 2H NMR data examining acyl chain ordering, a low resolution molecular picture of SP-B59-80 interacting with lipid systems can be hypothesized derived from NMR spectroscopy a nd DSC. It should be noted the following model contains the import ant assumption that SP-B59-80 is helical in a lipid environment. This estimation is valid given the findings in the liter ature pertaining to the helical nature of SP-B particularly at the C-terminal region; models describing this region as being helical are well documented in the literature ( 87, 133, 134, 177 ). However, as seen with KL4, the type of helix and the derived torsion angles may be non-canon ical and deviate from those seen for an i, i+4 helix. To answer the question of the type of helix SP-B form s in a lipid environment, MAS NMR experiments on 13C labeled peptide in complex with lipids need to be performed in

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167 conjunction with the DQ-DRAWS pulse sequence. The laboratory of Professor Joanna Long is currently testing various conditi ons for the expression of SP-B59-80 in a DNA vector for production by E. coli using recombinant molecular biology techniques. Once optimal conditions for expression are found, bacteria expressing the construct can be grown in isotopically enriched 13C medium for incorporation in to the growing polypeptide chain for NMR experiments With the assumption that SP-B59-80 is helical in lipid system s, Figure 5-15 displays a preliminary orientation of the peptide in lipid envi ronment. It is important to realize that the following model is theoretical and is only used to display the pres umed orientation of the peptide based on 2H and 31P NMR data. Since we saw an increase in ordering in 4:1 DPPC:POPG, we assume the peptide buries in DPPC acyl chains. In 3:1 POPC:POPG, the disordering in the acyl chains and increased ordering of the headgroup region indicates that the peptide is peripheral in this lipid system. Unlike KL4, which had its leucine cont ent buried in a hydrophobic environment, we predict that the ac idic aspartic acid residues in SP-B59-80 point toward the aqueous interface away from the phosphate headgroups. With the valid assumption of helicity for SP-B59-80, we predict that the peptide is mo re peripheral in POPC:POPG than KL4. The results of SP-B59-80 in DPPC:POPG may indicate snorkeling, which we postulated occurs for KL4. The snorkeling hypothesis as predicted for KL4 appears valid given its periodicity in primary amino acid sequence. However, despite the periodic charge distribution inherent in SP-B59-80, we cannot account for snorkeling for th e peptide unless we assume that the long chain of aspartic acid, glutamine, and argini ne extend to the interface to interact with the phosphate headgroup. As of this moment, more experiments are needed utilizing NMR and EPR on 13C labeled and spin labeled peptide in complex with lipid. This will yield more accurate information in terms of orientation and depth penetration of the peptide.

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168 31 0 31 0 Figure 5-1 Putative helical wheel for each type of helix rendered for the C-terminal residues 5980 of SP-B.

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169 102030405060 -200 0 200 400 600 800 1000 1200 Heat Capacity (cal/mol/oC)Temperature oC 3% SP-B(59-80) 2% SP-B(59-80) 1% SP-B(59-80) 0.5% SP-B(59-80) 0.2% SP-B(59-80) 0.1% SP-B(59-80) no SP-B(59-80)Figure 5-2. DSC thermograms of 4:1 DPPC:POP G LUVs with varying molar percentages of residues SP-B59-80.

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170 Table 5-1 Thermodynamic parameters derived from DSC on residues on 4:1 DPPC (d -62):POPG LUVs with SP-B59-80. Tm Hcal HvH S Cp max %SP-B (59-80) (oC) (kcal/mol) (energy/mol) (cal/mol/K) T1/2 CU* kcal/mol/oC no SPB(59-80) 32.1 0.4 2.50 0.04 102 6 8.1 0.1 6.7 0.4 39 2 0.34 0.02 0.1% 32.6 0.3 5.5 1.2 98 13 17.9 3.9 6.6 1.4 18 6 0.70 0.06 0.2% 32.8 0.3 5.9 .3 106 6 19.2 4.4 6.3 0.7 19 4 0.8 0.2 0.5% 32.44 0.03 7.1 .3 84 2.6 23.2 1.1 8.6 0.3 12 1 0.80 0.02 1% 32.0 0.2 6.7 0.2 117 3 21.6 0.7 5.8 0.1 18 1 1.042 0.008 2% 32.32 0.06 7.5 0.6 123 26 24.4 2.1 6.1 0.1 17 5 1.2 0.2 3% 32.44 0.03 7.01 0.07 137 29 23.0 0.2 6.0 0.3 20 4 1.3 0.3

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171 -50-40-30-20-10010203040 Frequency (ppm) 4:1 DPPC:POPG 0.1% SP-B(59-80) 0.2% SP-B(59-80) 0.5% SP-B(59-80) 1% SP-B(59-80) 2% SP-B(59-80) 3% SP-B(59-80) Figure 5-3. 31P NMR data of 4:1 DPPC:POPG MLVs with va rying molar percentages of SP-B59-80

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172 -50 -40 -30 -20 -10 0 10 Frequency (ppm) 4:1 DPPC:POPG 0.1% SP-B(59-80) 0.2% SP-B(59-80) 0.5% SP-B(59-80) 1% SP-B(59-80) 2% SP-B(59-80) 3% SP-B(59-80) Figure 5-4. DePaked 31P NMR data of 4:1 DPPC:POPG MLVs with varying molar percentages of SP-B59-80

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173 -2.0% 0.0% 2.0% 4.0% 6.0% 8.0% 10.0% 12.0% 00.511.522.533.5 mol% SP-B59-80% change (in ppm) DPPC POPG Figure 5-5. Change in 31P CSAs for 4:1 DPPC:POPG MLVs as a result of increasing molar percentages of SP-B59-80

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174 -50-40-30-20-10010203040 Frequency (ppm) 3:1 POPC:POPG 0.1% SP-B(59-80) 0.5% SP-B(59-80) 1% SP-B(59-80) 2% SP-B(59-80) 3% SP-B(59-80) Figure 5-6. Phosphorous NMR da ta of 3:1 POPC:POPG MLVs with varying molar percentages of SP-B59-80

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175 -50-40-30-20-10 0 1020 Frequency (ppm) 3:1 POPC:POPG 0.1% SP-B(59-80) 0.5% SP-B(59-80) 1% SP-B(59-80) 2% SP-B(59-80) 3% SP-B(59-80) Figure 5-7. DePaked 31PNMR da ta of 3:1 POPC:POPG MLVs with varying molar percentages of SP-B59-80

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176 -2.0% 0.0% 2.0% 4.0% 6.0% 8.0% 10.0% 12.0% 0 0.5 1 1.5 2 2.5 mol% SP-B59-80% change (in ppm) POPC POPG Figure 5-8. Change in 31P CSAs for 3:1 POPC:POPG MLVs as a result of increasing molar percentages of SP-B59-80

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177 -30000-20000-10000 0 100002000030000Frequency (Hz) 4:1 DPPC:POPG 0.1% SP-B(59-80) 0.2% SP-B(59-80) 0.5% SP-B(59-80) 1% SP-B(59-80) 2% SP-B(59-80) 3% SP-B(59-80) Figure 5-9. Deuterium NMR spectra of 4: 1 DPPC(d-62):POPG MLVs with SP-B59-80.

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178 -40-30-20-10010203040 Frequency (Hz) DPPC(d-62):POPG 0.1% SP-B(59-80) 0.2% SPB(59-80) 0.5% SPB(59-80) 1% SPB(59-80) 2% SPB(59-80) 3% SPB(59-80) Figure 5-10. Stacked dePaked spectra of 4:1 DPPC(d-62):POPG MLVs with SP-B59-80.

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179 0 0.1 0.2 0.5 1 2 3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% SP-B59-80 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.1 0.2 0.5 1 2 3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% SP-B59-80 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.1 0.2 0.5 1 2 3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% SP-B59-80 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 0 0.1 0.2 0.5 1 2 3 carbon 3 carbon 10 carbon 15 0% 2% 4% 6% 8% 10% 12%% change in mol% SP-B59-80 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 Figure 5-11. Percent change in time averaged order parameter < SCD> for DPPC (d-62) in 4:1 DPPC(d-62):POPG MLVs on addition of SP-B59-80. Top: change shown in the sn-1 and Bottom: the sn-2 chain.

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180 -30000-20000-100000100002000030000 Frequency (Hz) 3:1 POPC:POPG 0.1% SP-B(59-80) 0.5% SP-B(59-80) 1% SP-B(59-80) 2% SP-B(59-80) 3% SP-B(59-80) Figure 5-12. 2H NMR spectra of 3:1 POPC(d-31):POPG MLVs with SP-B59-80.

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181 -40-30-20-10010203040 Frequency (Hz) POPC(d-31):POPG 0.1% SP-B(59-80) 0.5% SP-B(59-80) 1% SP-B(59-80) 2% SP-B(59-80) 3% SP-B(59-80) Figure 5-13. Stacked dePaked spectra of 3:1 POPC(d-31):POPG MLVs with SP-B59-80.

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182 0 0.1 0.5 1 2 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 -10% -5% 0%% change in mol% SP-B59-80 carbon 3 carbon 8 carbon 10 carbon 12 carbon 15 Figure 5-14. Percent change in time averaged order parameter < SCD> for POPC (d-31) in 3:1 POPC(d-31):POPG MLVs on addition of SP-B5 9-80. Shown is the change in the sn-1 chain.

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183 DPPC:POPG POPC:POPG Figure 5-15. Hypothesized or ientational model of SP-B59-80 in two different lipid MLV systems. SP-B59-80 is assumed helical based on previous li terature findings and is shown as a yellow ribbon. The following models are preliminary and are based on 2H and 31P solid state NMR spectroscopy results.

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184 CHAPTER 6 CONCLUSIONS AND FUTURE EXPERIMENTS Our data a r gue that KL4 has a different bi nding orientation in 3:1 POPC:POPG and 4:1 DPPC:POPG lipid systems. Deuterium NMR is a powerful tool to study the dynamics of the lipid acyl chains based on the quadrupole splittings and the orientational order parameters calculated therefrom. From th ese data, it is clear that KL4 has a perturbing effect on DPPC(d-62):POPG. The addition of KL4 increases the ordering of the acyl chains near the middle and the tail ends indicati ng that it is these regions of the fatty acyl chai ns undergoing the most interaction with KL4. KL4 is modeled after the last 21 am ino acids of SP-B, particularly residues 59-80, in terms of hydrophilic and hydro phobic distribution of residues. Preliminary measurements done in our lab on SP-B59-80 have shown that in DPPC(d-62):POPG, a similar increase in order parameters is seen upon additi on of peptide. It is believed that both KL4 and SP-B59-80 lodge similarly in DPPC(d-62):POPG lipids. When the POPG sn-1 acyl chain is deuterated, we find that KL4 decreases the order parameters for both DPPC:POPG and POPC:POPG lipid systems, arguing for very little interaction with the PG acyl chains. In conjunction with the deuterated DPPC data, a possible scenario can be envisioned of phase separation, with KL4 snorkeling with the DPPC acyl chai ns, and interacting with the phosphate headgroups of POPG. The DSC da ta strongly argue for the case of lipid phase separation at concentration ranges of 1.5 mol%---such a concentration reported here is dependent on DSC sample preparation. While the orientation of these peptides may be the same, the question then becomes, what is that orientation? For KL4, a transmembrane orientation ha s been proposed based on FT-IR data, but the preparation of the sample wa rrants some doubt. In those experiments, KL4 was dried with 7:3 DPPC:DPPG bilayers in nonphysiological amounts. The transmembrane

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185 orientation hypothesis contradicts many previous reports that postulated that the evenly spaced charged lysine residues can i onically interact with the phosphate headgroup, thus imparting a more peripheral orientation to the peptide. A second study using IR absorption spectroscopy showed the peptide adapting an an ti-parallel beta sheet structure in DPPC/DPPG bilayers while being alpha-helical in DPPC. That work did predict KL4 to be neither transmembrane nor helical. In light of thes e findings, our data show KL4 being helical by circular dichroism, at concentration ranges that negate the effect of potential aggregation. Solid state NMR experiments taken in conjunction with Prof essor Joanna Long and Dr. Doug Elliott on 13C labeled KL4 were performed in POPC:POPG and DPPC:POPG lipids. 13C -13C distances and torsion angle measurements show KL4 to be helical albeit in a non-canonical form as mentioned in Chapter 4. Using DQ-DRAWS, the peptide displayed torsion angles ( =-105, =-26) that were helical but had the lysines partially aligned along one side of the helix. Similar experiments of 13C labeled KL4 in DPPC:POPG show that the peptide follows a more -helical fold with the lysines fully aligned along one face of the helix. In our final model depicted in Chapter 3 and in the submitted manuscript, we model KL4 as a helix in both DPPC:POPG and POPC:POPG lipids based on the input of the torsion angles generate d into the molecular graphics program PYMOL. In light of the data borne out from DSC, and solid-state NMR experiments, it seems that the preferred orientation of KL4 in 4:1 DPPC:POPG bilayers is to be buried within the bilayer beneath the headgroups. This re presents a unique binding orientation of the peptide in lipid compositions modeled after the lung. In mode l 3:1 POPC:POPG MLVs, we can state with some degree of confidence that KL4 is bound peripherally to this lipid system at a shallower depth. 31P solid state NMR on static peptide-lipid samples show the peptide decreasing spontaneous macroscopic orientatio n of the lipids, giving rise to more spherical shapes of the

PAGE 186

186 MLVs. This was found in both lipid systems. As an unintended byproduct of samples run at 14.1Tesla field strength, lipid samples exhibited spontaneous alignment, leading to a loss of the parallel edge of the powder pattern. At a macroscopic level, this can be depicted as normal spherical liposomes being deformed into ellipsoid al shaped vesicles. As part of Professor Edward Sternins dePaking algorithm, a parameter that reports the extent of orientation caused by magnetic field alignment is reported. Adding KL4 primarily decreases this parameter indicating the peptide affects the macroscopic ordering of lipids and converting ellipsoidal deformed liposomes into spherical ones. Conversion of an ellipsoidal form of liposomes, as mediated by KL4, to spherical ones may help in potenti ating surface tension but this hypothesis remains to be tested. The interesting aspect of this work is what KL4 is doing in the lung. The snorkeling hypothesis presented shows two seemingly conflicting points arising from the NMR data interaction with POPG headgroup a nd interaction with DPPC acyl chai ns at the interfacial region of the tail. One way to reconcile these two results stemming from the 31P and 2H NMR data is phase separation or lipid sequestration. This is seen in the DSC data on DPPC:POPG vesicles, regardless of the method of sample preparation. With phase separation, we envisage a scenario where one population of the peptide can sequest er DPPC into a microdomain (of size which cannot be determined from these experiments) and at the same time a second population of the peptide can be interacting with the phosphates of POPG in a POPG-rich domain. Similar 31P and 2H studies were also performed on SP-B59-80, the native sequence after which KL4 is modeled, to see if the two peptides or ient similarly. Our data show that SP-B59-80 presence increases acyl chain ordering in D PPC(d-62):POPG while decreasing it in POPC(d31):POPG. Similar to KL4, the ordering or disordering of th e acyl chains is dependant on the

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187 saturation status of the phospholipids. 31P NMR data also show SP-B59-80 interacting with PG headgroups as was seen for KL4. Despite these similarities seen from ssNMR spectroscopy, DSC measurements at increasing levels of SP-B59-80 show no indication of phase separation. This finding implies that the way the two peptid es partition between the lipids is different. Hence, SP-B59-80 seems to orient similarly to KL4 but conveys a different biophysical function to the lipid systems studied in this dissertation. It was hoped that an inverted HII phase would be seen in our data when KL4 was added to both our lipid systems. Such a phase can clearly be characterized by 31P NMR and a lipid polymorphism of that arrangement was believed to be important for lung surfactant functioning. Though we did not see such a lipid geometry in both our lipid systems, we believe that snorkeling of KL4 in DPPC:POPG can induce a curvature stra in on the lipids that can make these lipids more prone to alternative geometries (like an inverted HII phase). However, because we did not see an inverted HII phase, we can only speculate that this could be one advantage of lysine snorkeling. Of interesting note, our findings show that at 3mol% SP-B59-80 a potential alternative lipid phase was seen in 3:1 POPC(d-31):POPG lipids but not in 4:1 DPPC(d-62):POPG lipids. This discovery needs to be further investigated to see if indeed the peptide facilitates a concentration dependant change in the geometry that is sensitive to the level of saturation of the lipid chain. The spectroscopy and calorimetry experiments delineated above were performed on simple binary lipid systems of defined molar ratio. Obviously, lung surfactant is much more heterogeneous than just DPPC and POPG, and a whole host of other minor constituents exist which even include diand tri-unsaturated fatt y acids. Future experiments should involve addition of more layers of comp lexity to lung surfactant that t ypify what is found endogenously.

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188 Such experiments have been performed on more native bovine lung surf actant extracts which have been spiked with DPPC(d-62) to measure effect of albumin on phase separation and lipid dynamics ( 183). The studies presented here can also be performed with the addition of palmitic acid to our DPPC:POPG lipid mix to more closel y simulate the clinical version marketed by Discovery Labs and Tanakas original work ( 18). Finally, the studies presented here can be extended to other peptides systems such as RL4 (lysines replaced with arginine residues), SP-B1-25, and to Mini-B, a surface active fusion peptide of SP-B1-25 and SP-B59-80 ( 184 ). By such work, it can be clarified whether peptide mimics based upon SP-B act vi a a common structural intermediate and interact with lipids in a simila r manner. It could also be possible that these peptide mimics can have a substantially different effect of lipid thermodynamics and lipid NMR time-scale dynamics. If so, it w ould imply more than one mechanism for simple peptides to achieve surface tension lowering capab ilities in the lung. Also, it w ould be interesting to note if the helical secondary structure as reported here for KL4 (in solution and by solid-state) is essentially the overriding secondary structure ne eded for proper lung su rfactant function. It would be interesting to see whether even more si mple peptides that follow the alpha-helical fold can be rationally designed for the treatment of RDS. These short, small molecular weight peptides may also be of aid in solving other problems involving interfacial biology or surface tension minimization.

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189 APPENDIX A CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS 4:1 DPPC(D-62):POPG WITH KL4 (MOLAR PERCENTAGES) Table A-1 Order Parameters for sn -1 and sn-2 chain of DPPC(d-62) sn-1 chain Order Parameter Carbon number no KL4 0.09% KL4 0.19% KL4 0.38%KL4 0. 76% KL4 1.5% KL4 2.3% KL4 2 0.21 0.22 0.22 0.22 0.22 0.22 0.21 3 0.21 0.21 0.21 0.21 0.21 0.21 0.21 4 0.21 0.22 0.22 0.22 0.22 0.22 0.21 5 0.21 0.21 0.21 0.21 0.21 0.21 0.21 6 0.21 0.21 0.21 0.21 0.21 0.21 0.21 7 0.21 0.21 0.21 0.21 0.21 0.21 0.21 8 0.21 0.21 0.21 0.21 0.21 0.21 0.21 9 0.19 0.19 0.19 0.19 0.19 0.20 0.20 10 0.18 0.18 0.18 0.18 0.19 0.19 0.19 11 0.16 0.16 0.17 0.17 0.17 0.18 0.18 12 0.15 0.15 0.15 0.15 0.15 0.16 0.16 13 0.13 0.13 0.13 0.13 0.13 0.14 0.14 14 0.11 0.11 0.11 0.11 0.11 0.12 0.12 15 0.08 0.08 0.08 0.08 0.08 0.09 0.09 16 0.02 0.02 0.02 0.02 0.02 0.03 0.03 sn -2 chain Carbon number no KL4 0.09% KL4 0.19% KL4 0.38% KL4 0.76% KL4 1.5% KL4 2.3% KL4 2 0.21 0.22 0.22 0.22 0.22 0.22 0.21 3 0.21 0.21 0.21 0.21 0.21 0.21 0.21 4 0.21 0.22 0.22 0.22 0.22 0.22 0.21 5 0.21 0.21 0.21 0.21 0.21 0.21 0.21 6 0.21 0.21 0.21 0.21 0.21 0.21 0.21 7 0.21 0.21 0.21 0.21 0.21 0.21 0.21 8 0.21 0.21 0.21 0.21 0.21 0.21 0.21 9 0.19 0.19 0.19 0.19 0.19 0.20 0.20 10 0.19 0.19 0.19 0.19 0.19 0.20 0.20 11 0.18 0.18 0.18 0.18 0.19 0.19 0.19 12 0.16 0.16 0.17 0.17 0.17 0.18 0.18 13 0.15 0.15 0.15 0.15 0.15 0.16 0.16 14 0.12 0.12 0.12 0.12 0.12 0.13 0.13 15 0.09 0.10 0.10 0.10 0.10 0.10 0.10 16 0.02 0.02 0.02 0.02 0.02 0.03 0.03

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190 APPENDIX B CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNFICANT FIGURES) FOR DEUTERATED LIPIDS 4:1 DPPC:POPG(D-31) WITH KL4 (MOLAR PERCENTAGES) Table B-1 Order Parameters for deuterated sn-1 chain of POPG palmitoyl chain Order Parameter carbon No KL4 1.0% KL4 3.0% KL4 2 0.200.190.18 3 0.200.190.18 4 0.200.190.18 5 0.200.190.18 6 0.200.190.18 7 0.200.190.18 8 0.200.190.18 9 0.180.180.17 10 0.170.170.17 11 0.160.160.16 12 0.140.150.14 13 0.120.130.13 14 0.100.110.11 15 0.080.080.08 16 0.020.020.02 0.160.160.15

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191 APPENDIX C CALCULATED 2H ORDER PARAMETERS (TO TWO SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS 3:1 POPC(D-31):POPG WITH KL4 (MOLAR PERCENTAGES) Table C-1 Order Parameters for deuterated sn-1 chain of POPC palmitoyl chain Order Parameter Carbon number no KL4 0.09% KL4 0.19% KL4 0.38% KL4 0.76% KL4 1.5% KL4 2.3% KL4 2 0.20 0.19 0.19 0.19 0.19 0.19 0.19 3 0.20 0.19 0.19 0.19 0.19 0.19 0.19 4 0.20 0.19 0.19 0.19 0.19 0.19 0.19 5 0.20 0.19 0.19 0.19 0.19 0.19 0.19 6 0.20 0.19 0.19 0.19 0.19 0.19 0.19 7 0.20 0.19 0.19 0.19 0.19 0.19 0.19 8 0.20 0.19 0.19 0.19 0.19 0.19 0.19 9 0.17 0.17 0.17 0.17 0.17 0.17 0.17 10 0.16 0.16 0.16 0.16 0.16 0.16 0.16 11 0.14 0.14 0.14 0.14 0.14 0.14 0.14 12 0.13 0.12 0.12 0.12 0.12 0.12 0.12 13 0.11 0.11 0.11 0.11 0.10 0.11 0.11 14 0.09 0.09 0.09 0.09 0.09 0.09 0.09 15 0.07 0.07 0.06 0.06 0.06 0.07 0.07 16 0.02 0.02 0.02 0.02 0.02 0.02 0.02

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192 APPENDIX D CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS IN 3:1 POPC:POPG(D-31) WITH KL4 (MOLAR PERCENTAGES) Table D-1 Order Parameters for deuterated sn-1 chain of POPG palmitoyl chain Order Parameter Carbon no KL4 0.2% KL4 1.0% KL4 3.0% KL4 2 0.19 0.18 0.18 0.17 3 0.19 0.18 0.18 0.17 4 0.19 0.18 0.18 0.17 5 0.19 0.18 0.18 0.17 6 0.19 0.18 0.18 0.17 7 0.19 0.18 0.18 0.17 8 0.18 0.18 0.18 0.17 9 0.17 0.16 0.16 0.15 10 0.15 0.15 0.15 0.14 11 0.14 0.13 0.13 0.12 12 0.12 0.12 0.12 0.11 13 0.10 0.10 0.10 0.09 14 0.08 0.08 0.08 0.08 15 0.06 0.06 0.07 0.06 16 0.02 0.02 0.02 0.01

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193 APPENDIX E CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS IN 4:1 D PPC(D-62):POPG WITH SP-B(59-80) (MOLAR PERCENTAGES) Table E-1 Order parameters for sn-1 and sn-2 chain of DPPC with SP-B(59-80) Order Parameter sn-1 chain carbon no SP-B 0.1% SP-B 0.2% SP-B 0.5% SP-B 1.0 SP-B 2.0% SP-B 3.0% SP-B 2 0.22 0.22 0.22 0.22 0.23 0.23 0.23 3 0.21 0.21 0.21 0.21 0.22 0.22 0.22 4 0.22 0.22 0.22 0.22 0.23 0.23 0.23 5 0.21 0.21 0.21 0.21 0.22 0.22 0.22 6 0.21 0.21 0.21 0.21 0.22 0.22 0.22 7 0.21 0.21 0.21 0.21 0.22 0.22 0.22 8 0.21 0.21 0.21 0.21 0.22 0.22 0.22 9 0.19 0.19 0.19 0.20 0.20 0.20 0.20 10 0.18 0.18 0.18 0.19 0.19 0.20 0.19 11 0.16 0.16 0.16 0.17 0.18 0.18 0.18 12 0.15 0.15 0.15 0.16 0.16 0.16 0.16 13 0.13 0.13 0.13 0.14 0.14 0.14 0.14 14 0.11 0.11 0.11 0.12 0.12 0.12 0.12 15 0.08 0.08 0.08 0.09 0.09 0.09 0.09 16 0.02 0.02 0.02 0.02 0.03 0.03 0.03 sn-2 chain carbon no SP-B 0.1% SP-B 0.2% SP-B 0.5% SP-B 1.0 SP-B 2.0% SP-B 3.0% SP-B 2 0.22 0.22 0.22 0.22 0.23 0.23 0.23 3 0.21 0.21 0.21 0.21 0.22 0.22 0.22 4 0.22 0.22 0.22 0.22 0.23 0.23 0.23 5 0.21 0.21 0.21 0.21 0.22 0.22 0.22 6 0.21 0.21 0.21 0.21 0.22 0.22 0.22 7 0.21 0.21 0.21 0.21 0.22 0.22 0.22 8 0.21 0.21 0.21 0.21 0.22 0.22 0.22 9 0.19 0.19 0.19 0.20 0.20 0.20 0.20 10 0.19 0.19 0.19 0.20 0.20 0.20 0.20 11 0.18 0.18 0.18 0.19 0.19 0.20 0.19 12 0.16 0.16 0.16 0.17 0.18 0.18 0.18 13 0.15 0.15 0.15 0.16 0.16 0.16 0.16 14 0.12 0.12 0.12 0.13 0.13 0.13 0.13 15 0.10 0.10 0.10 0.10 0.10 0.10 0.10 16 0.02 0.02 0.02 0.02 0.03 0.03 0.03

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194 APPENDIX F CALCULATED 2H ORDER PARAMETERS (TO 2 SIGNIFICANT FIGURES) FOR DEUTERATED LIPIDS IN 3:1 PO PC(D-31):POPG WITH SP-B(59-80) (MOLAR PERCENTAGES) Table F-1 Order Parameters for deuterated sn-1 chain of PO PC with SP-B(59-80) Order Parameter Carbon no SP-B(59-80) 0.1% SP-B(59-80) 0.5% SP-B(59-80) 1.0% SP-B(59-80) 2.0% SP-B(59-80) 3.0% SP-B(5980) 2 0.19 0.19 0.19 0.19 0.19 0.18 3 0.19 0.19 0.19 0.19 0.19 0.18 4 0.19 0.19 0.19 0.19 0.19 0.18 5 0.19 0.19 0.19 0.19 0.19 0.18 6 0.19 0.19 0.19 0.19 0.19 0.18 7 0.19 0.19 0.19 0.19 0.19 0.18 8 0.19 0.19 0.19 0.19 0.19 0.18 9 0.16 0.16 0.16 0.17 0.15 0.15 10 0.15 0.15 0.15 0.15 0.14 0.13 11 0.13 0.13 0.13 0.13 0.13 0.13 12 0.12 0.12 0.12 0.12 0.11 0.11 13 0.10 0.10 0.10 0.10 0.10 0.10 14 0.08 0.08 0.08 0.08 0.08 0.08 15 0.06 0.06 0.06 0.06 0.06 0.06 16 0.02 0.02 0.02 0.02 0.02 0.02

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205 BIOGRAPHICAL SKETCH Vijay C. Antharam got his Bachelor of Scie nce degree in m icrobiology and cell science at the University of Florida, where he graduated in 3 years. He joined the PhD program in biomedical sciences at the Univer sity of Florida in the Fall of 2002 and joined the lab of Joanna R.Long in 2003. His interests include biophysics, structural biology, medicine, and playing chess.


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