A Multi-Frequency Electron Paramagnetic Resonance Spectroscopy Study of the Intrinsically Disordered Protein, IA3

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Title:
A Multi-Frequency Electron Paramagnetic Resonance Spectroscopy Study of the Intrinsically Disordered Protein, IA3
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1 online resource (179 p.)
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english
Creator:
Pirman,Natasha L
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University of Florida
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Fanucci, Gail E
Committee Members:
Smith, Benjamin W
Stewart, Jon D
Talham, Daniel R
Edison, Arthur S

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Subjects / Keywords:
epr -- ia3 -- idp -- protein -- sdsl -- tfe
Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
Intrinsically disordered proteins (IDPs) are proteins that contain little to no secondary or tertiary structure. IDPs are often functional proteins that are essential in biological systems and at times have been shown to undergo conformational changes where structure is induced upon binding to a target protein. Monitoring these conformational changes are typically difficult using traditional biophysical techniques such as X-ray crystallography, NMR spectroscopy or circular dichroism. Within this work, a multi-frequency approach to site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy was optimized to investigate and characterize the mobility and conformational changes of IDPs. We applied this method to IA3, which is a 68 residue IDP whose unstructured-to-alpha-helical conformational transition has been extensively characterized by various biophysical techniques. We monitored the chemically induced conformational change in the presence of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE), at both X-, and W-band frequencies. Analyses of the EPR spectral line shapes provided structural information on the conformational changes. It was shown that initial X-band data reported on global correlation time changes consistent with a two-state model of an unstructured system and the tumbling of a rigid helix; more detailed analyses of the X-band spectral line shapes provided site-specific information on the residue level. Analysis of the W-band EPR spectral line shapes, however, more directly revealed site-specific structural changes. Line shape simulations of the data at both frequencies appear to provide further information on the site-specific conformational changes occurring in the presence of TFE. Using IA3 as a model system, we show multi-frequency EPR can provide insight into structural changes occurring in IDP systems that are otherwise difficult to characterize.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Natasha L Pirman.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
Local:
Adviser: Fanucci, Gail E.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

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UFE0043144:00001


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1 A MULTI FREQUENCY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY STUDY OF THE INTRINSICALLY DISORDERED PROTEIN, IA 3 By NATASHA L. PIRMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIA L FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Natasha L. Pirman

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3 To my m other Patty S. Hurst

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4 ACKNOWLEDGMENTS First and foremost, I would like to express gratitude to God throug h which all things are p ossible. His guidance has led me to where I am today. All achievements, awards, and successes I have attained are attributed to Him. Secondly, I am extremely grateful to my parents Arvel and Patty Hurst for their continuous love an she has always expressed that I could do anything I set my mind to and that I should do every task to the best of my ability, has led me to truly believe in myself and was one of my main driving forces to complete such an extraordinary accomplishment of earning a Ph.D. I would also like to thank my entire family for always being there for me and helping support me by any means possible. I would like to especially thank my Aunt and Uncle, Shell a and Tim Franklin, for constantly encouraging me to do well in school, and taking such a vested interest in my education. I am very thankful to my mentor Dr. Gail E. Fanucci for giving me the opportunity to work in her research group and for guiding me throughout my graduate career. She provided me with the resources to develop and learn scientifically and her high expectations helped me to become a better scientist. Her generosity also made it possible for me to attend numerous regional and national s cientific conferences, and travel to various labs and collaborate with important people in our scientific field which certainly contributed to my growth and maturity as a scientist. I am also thankful for all the professors and teachers throughout my life that have inspired and encouraged me in my pursuit for higher education. My high school chemistry teacher, Dr. James Madsen, initially inspired my love for chemistry which

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5 eventually led me to declare a major in chemistry in college. I am also sincerely grateful to one of my undergraduate professors Dr. Johnny Evans, at Lee University, for his advice and mentoring throughout college. His persistent recommendation that I attend graduate school, and introducing me to his alma mater, ultimately led to my ap plication and admittance into the University of Florida. I would like to thank all the collaborators that were of assistance with my research project. I am grateful for Dr. Alex Smirnov, and Dr. Tatyana Smirnova at North Carolina State University for allo wing me to collect my initial W band data on their instrument, as well as providing me with simulation software that was used to calculate the rotational correlation time component in the local tumbling volume parameter used in my studies. I would like to thank Hans van Tol and Likai Song at the National High Magnetic Field Lab for providing time on the Bruker W band instrument and for helping me to collect additional W band data. I would also like to thank Dr. Wayne Hubbell and Dr. Christian Altenbach fo r allowing me to visit the lab at UCLA, and for teaching me how to use the Multi Component fit software developed by Dr. Altenbach. I would like to express my gratitude to all the members in my committee, Dr. Dan Talham, Dr. Jon Stewart, Dr. Alex Angerhofe r, Dr. Ben Smith, and Dr. Art Edison for many valuable discussions and their support. I would also like to thank the people in the graduate student office. Dr. Ben Smith and Lori Clark for all they do. I loved being a part of the graduate student recruit ing committee, and would have to say that it was one of my most memorable and favorite parts of graduate school.

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6 I wish to express my gratitude to both the current and past members of the Fanucci research group, especially Jordan Mathias, Jamie Kear, Mandy Blackburn, Luis Galiano, Jeff Carter, Austin Turner, Stacey Ann Benjamin, and Eugene Milshteyn for their friendship, patience, and help in the lab. Lastly, I need to thank the most important person of all, my husband David Pirman. His love, patience, enc ouragement, and support helped me to get through all the ups and downs of graduate school. This period in my life has been one of the most fulfilling times in my life, and I am so glad that I have had him to share in it with me. For all he has done for m e I am eternally grateful, and I do not know if I would have made it through without him.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 13 LIST OF AMINO ACIDS AND AMINO ACID ABBREVIATIONS ................................ ... 16 L IST OF ABBREVIATIONS ................................ ................................ ........................... 17 ABSTRACT ................................ ................................ ................................ ................... 19 CHAPTER 1 PROTEIN STRUCTURE AND FUNCTION ................................ ............................. 21 Protein Structure ................................ ................................ ................................ ..... 21 Protein Discovery ................................ ................................ ............................. 21 Structural Protein Components ................................ ................................ ........ 23 Primary structure ................................ ................................ ........................ 24 Secondary structure ................................ ................................ ................... 24 Tertiary structure ................................ ................................ ........................ 27 Quaternary structure ................................ ................................ .................. 27 Protein Structure Paradigm ................................ ................................ .............. 28 Intrinsically Disordered Proteins ................................ ................................ ............. 29 Discovery of Intrinsically Disordered Proteins ................................ .................. 30 The Protein Trinity Hypothesis ................................ ................................ ......... 32 Experimental Methods Commonly Employed to Characterize IDPs ................. 32 X ray crystallography ................................ ................................ ................. 33 NMR spectroscopy ................................ ................................ ..................... 33 Circular dichroism spectroscopy ................................ ................................ 34 Susceptibility to proteolysis ................................ ................................ ........ 34 Othe r experimental methods ................................ ................................ ...... 35 Utilizing SDSL EPR Spectroscopy as an Investigative Tool for Characterizing IDPs ................................ ................................ ...................... 37 The Intrinsically Disordered Protein, IA 3 ................................ ........................... 40 2 BACKGROUND AND THEORY OF MULTI FREQUENCY CONTINUOUS WAVE ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY ............. 53 Introduction to Electron Paramagnetic Resonance Spectroscopy .......................... 53 Zeeman Effect ................................ ................................ ................................ .. 54 Hyperfine Interaction ................................ ................................ ........................ 55 Spin Labeling ................................ ................................ ................................ .......... 56

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8 Site Directed Spin Labeling (SDSL) ................................ ................................ 57 Spin Label Structures ................................ ................................ ....................... 58 Spin Label Motion ................................ ................................ ............................. 59 EPR at Variable Frequencies ................................ ................................ .................. 60 Simulations of EPR Spectral Line Shapes ................................ .............................. 62 3 REDUCING THE EFFECTS OF THE OVERALL ROTATIONAL CORRELATION TIME OF PROTEINS ................................ ................................ ... 71 Materials and Me thods ................................ ................................ ............................ 75 Materials ................................ ................................ ................................ ........... 75 Protein Expression and Purification of IA 3 mutants ................................ .......... 75 Spin Labeling ................................ ................................ ................................ .... 76 EPR Sample Preparation ................................ ................................ ................. 76 Continuous Wave (CW) X Band EPR Spectra ................................ ................. 77 Results and Discussion ................................ ................................ ........................... 77 Qualitative Assessment of the EPR Spectral Line Shapes ............................... 77 Semi Empir ical Parameters ................................ ................................ .............. 79 Addition Method to Slow the Overall Correlation Time ................................ ..... 80 Summary ................................ ................................ ................................ ................ 82 4 CHARACTERIZATION OF THE DISORDERED TO ALPHA HELICAL TRANSITION OF IA 3 BY SDSL EPR SPECTROSCOPY ................................ ....... 86 Materials and Methods ................................ ................................ ............................ 88 Materials ................................ ................................ ................................ ........... 88 Protein Expression and Purification of IA 3 mutants ................................ .......... 89 Spin Labeling ................................ ................................ ................................ .... 89 EPR Sample Preparation ................................ ................................ ................. 90 Continuous Wave (CW) X Band EPR Spectra ................................ ................. 90 Data Analysis ................................ ................................ ................................ ... 91 Viscosity Measurements ................................ ................................ ................... 92 Results ................................ ................................ ................................ .................... 93 Effects of Spin Label Moiety o n Monitoring the Helical Conformational Change ................................ ................................ ................................ .......... 93 Quantitative Analysis of the TFE Induced Change Observed via SDSL ........... 94 Discussio n ................................ ................................ ................................ .............. 98 Effects of Spin Label Choice on Data Analysis ................................ ................. 98 Data Analysis via h (+1) /h (0) ................................ ................................ ................. 99 Comparison of V L to the Percent Change of h ( 1) ................................ ............ 100 Summary ................................ ................................ ................................ .............. 103 5 MULTI FREQUENCY EPR ANALYSIS OF THE C TE RMINUS OF IA 3 ............... 121 Materials and methods ................................ ................................ .......................... 123 Materials ................................ ................................ ................................ ......... 123 Protei n Expression and Purification of IA 3 mutants ................................ ........ 124

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9 Spin Labeling ................................ ................................ ................................ .. 125 Continuous Wave (CW) X Band EPR Spectra ................................ ............... 125 Continuous Wave (CW) W Band EPR Spectra ................................ .............. 126 X band Data Analysis ................................ ................................ ..................... 126 Viscosity Me asurements ................................ ................................ ................. 127 X and W band EPR Spectral Line Shapes Fitting ................................ ......... 128 Results and Discussion ................................ ................................ ......................... 129 Qualitative Analysis of the TFE Induced Change in the Spectral Line Shapes ................................ ................................ ................................ ........ 129 Quantitative Analysis of the TFE Induced Change in the Spectral Line Shapes ................................ ................................ ................................ ........ 129 Multi Frequency EPR Analysis ................................ ................................ ....... 130 Temperature control studies ................................ ................................ .... 131 Exp erimental multi frequency EPR spectral line shapes .......................... 132 Multi frequency fitting of the EPR spectral line shapes ............................ 133 Summary ................................ ................................ ................................ .............. 135 6 FUTURE WORK ................................ ................................ ................................ ... 149 Full Cysteine Scanning of IA 3 for Further X and W Band EPR Studies ............... 149 Additional High Frequency EPR Studies ................................ ............................... 150 SDSL EPR Study of IA 3 Variants Bound to YPRA ................................ ................ 150 APPENDIX A I A 3 WILD TYPE AND VARIENT PROTEIN AND AMINO ACID SEQUENCEs ..... 152 B CIRCULAR DICROISM AND x BAND AND W BAND CW EPR EXPERIMENTAL PARAMETERS ................................ ................................ ........ 155 C LOCAL TUMBLING VOLUME PARAMETERS AND EPR SPECTRAL INTENSITY VALUES AND RATIOS ................................ ................................ ..... 156 LIST OF REFERENCES ................................ ................................ ............................. 167 BIOGRAPHIC AL SKETCH ................................ ................................ .......................... 1 79

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10 LIST OF TABLES Table page 1 1 Common NMR Techniques for Characterizing Protein Structure and Flexibility ................................ ................................ ................................ ............. 51 1 2 Order/Disordered composition profile ................................ ................................ 52 2 1 Frequency bands in EPR spectroscopy ................................ .............................. 70 5 1 X band magne tic A tensor components determined from calculations ............. 146 5 2 W band magnetic A tensor components determined from calculations ............ 146 5 3 Best fit parameters of IAP labeled variants in 0% TFE buffer solution .............. 147 5 4 Best fit parameters of IAP labeled variants in 15% TFE buffer solution ............ 147 5 5 Best fit parameters of IAP labeled variants in 30% TFE buffer solution ............ 148 A 1 IA 3 Wild Type sequence ................................ ................................ ................... 152 A 2 I A 3 GC optimized wild type sequence ................................ .............................. 152 A 3 IA 3 GC optimized S14C sequence ................................ ................................ .... 152 A 4 IA 3 Y57C sequence ................................ ................................ .......................... 153 A 5 IA 3 N58C sequence ................................ ................................ .......................... 153 A 6 IA 3 K59C sequence ................................ ................................ .......................... 153 A 7 IA 3 L60C sequence ................................ ................................ ........................... 154 A 8 IA 3 K61C sequence ................................ ................................ .......................... 154 B 1 Circular dichroism experimental parameters ................................ .................... 155 B 2 Typical X band CW EPR parameters ................................ ............................... 155 B 3 Typical W band CW EPR parameters ................................ .............................. 155 C 1 Parameter values used to determine V L parameter at 27 C ............................ 156 C 2 Parameter values used to determine V L parameter at 5 C .............................. 156 C 3 Calculated local tumbling volume raw data for S14C IAP at 27 C .................. 157

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11 C 4 Calculated local tumbling volume raw data for S14C IAP at 5 C .................... 157 C 5 Calculated local tumbling volume raw data for N58C IAP at 27 C .................. 158 C 6 Calculated local tumbling volume raw data for N58C IAP at 5 C .................... 158 C 7 Calculated local tumbling volume raw data for Y57C IAP at 27 C .................. 159 C 8 Calculated local tumbling volume raw data for K59C IAP at 27 C .................. 159 C 9 Calculated local tumbling volume raw data for L60C IAP at 27 C ................... 160 C 10 Calculated local tumbling volume raw data for K61C IAP at 27 C .................. 160 C 11 EPR spectral line intensities, h ( +1) /h (0) and, h ( 1) values for S14C MTSL collected at 27 C ................................ ................................ .............................. 161 C 12 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for S14C MSL collected at 27 C ................................ ................................ .............................. 161 C 13 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for S14C IAP collected at 27 C ................................ ................................ .............................. 161 C 14 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) v alues for N58C MTSL collected at 27 C ................................ ................................ .............................. 162 C 15 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for N58C MSL collected at 27 C ................................ ................................ .............................. 162 C 16 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for N58C IAP collected at 27 C ................................ ................................ .............................. 162 C 17 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for Y57C IAP col lected at 27 C ................................ ................................ .............................. 163 C 18 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for K59C IAP collected at 27 C ................................ ................................ .............................. 163 C 19 EPR spe ctral line intensities, h (+1) /h (0) and, h ( 1) values for L60C IAP collected at 27 C ................................ ................................ .............................. 163 C 20 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for K61C IAP collected at 27 C ................................ ................................ .............................. 164 C 21 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for S14C IAP collected at 5 C ................................ ................................ ................................ 164

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12 C 22 EPR spectral line intensities, h (+1) /h (0) and, h ( 1) values for Y57C IAP collected at 5 C ................................ ................................ ................................ 164 C 23 EPR spectral line intensities, h (+1) /h (0) and h ( 1) values for N58C IAP collected at 5 C ................................ ................................ ................................ 165 C 24 EPR spectral line intensities, h (+1) /h (0) and h ( 1) values for K59C IAP collected at 5 C ................................ ................................ ................................ 165 C 25 EPR spectral line intensities, h (+1) /h (0) and h ( 1) values for L60C IAP collected at 5 C ................................ ................................ ................................ ............... 165 C 26 EPR spectral line intensities, h (+1) /h (0) and h ( 1) values for K61C IAP collected at 5 C ................................ ................................ ................................ 166

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13 LIST OF FIGURES Figure p age 1 1 The 20 standard amino acids that comprise proteins. ................................ ........ 44 1 2 Levels of structure in proteins ................................ ................................ ............. 45 1 3 Depiction of a peptide bond. ................................ ................................ .............. 46 1 4 Forbidden peptide bond configurations. ................................ ............................. 47 1 5 Ramacha nd ran plot for L Ala residues ................................ ............................... 47 1 6 The alternative hypothesis to the classical structure function paradigm ............. 48 1 7 Schematic of protein structure and functions ................................ ...................... 48 1 8 X ray crystal structure of IA 3 bound to YPRA. ................................ .................... 49 1 9 Depiction of the amphipathic nature the helical c onformation of the N terminal residues of IA 3 upon interaction with YPRA ................................ .......... 50 2 1 Energy diagram for a single free electron in an applied magnetic field. .............. 64 2 2 Energy diagram depicting the hyperfine splitting of an electron coupled to a nuclear spin of I=1 and the corresponding EPR lin e shape with three transitions ................................ ................................ ................................ ........... 65 2 3 Common ly us ed spin labels for EPR studies ................................ ...................... 66 2 4 Dependence of EPR spectral line shapes on motion ................................ .......... 67 2 5 Depiction of the three modes of mot ion affecting a spin label ............................ 68 2 6 Illustration of differences in EPR spectral line shapes from nitroxide spin labels change with frequency. ................................ ................................ ............ 69 3 1 Semi empirical parameters illustrated on IA 3 S14C IAP data ............................. 83 3 2 100G X band EPR spectra for S14C IAP and N58C IAP in 0% TFE and 30% TFE collected in buffer and 30% sucrose solution s at 27 C, and in buffer at 5 C. ................................ ................................ ................................ ...................... 84 3 3 Plot of the inverse second moment ( 1 ) versus inverse central line width ( H 0 1 ) ................................ ................................ ................................ ................. 84 3 4 Depiction of the tethering scheme. ................................ ................................ ..... 85

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14 3 5 100G X band EPR spectra for S14C IAP and N58C IAP in 0%, 15% and 30% TFE coll ected in buff er and tethered to Ni IDA resin ................................ .. 85 4 1 Sequence of IA 3 ................................ ................................ ............................... 105 4 2 Protein gel of S14C and N58C purification.. ................................ ..................... 106 4 3 Structures of spin labels used in this study and the resulting chemical modificat ion of the cysteine side chain ................................ ............................. 107 4 4 CD spectra of wild type IA 3 and IA 3 va riant S14C collected at 27 C. ............. 108 4 5 CD spectra of wild type IA 3 and IA 3 variant S14C collected at 5 C. ................ 109 4 6 CD spectra of wild type IA 3 and IA 3 va riant N58C collected at 27 C. ............. 110 4 7 CD spectra of wild type IA 3 and IA 3 variant N58C collected at 5 C ................. 111 4 8 Area normalized 100G X band EPR spectra of IA 3 variant S14C labeled with MTSL, MSL, and IAP in increasing TFE concentration. Spectra were collected at 27 C. ................................ ................................ ............................ 112 4 9 Area nor malized 100G X band EPR spectra of IA 3 variant N58C labeled with MTSL, MSL, and IAP in increasing TFE concentration. Spectra were collected at 27 C ................................ ................................ ............................. 113 4 10 Area normalized 100G X band EPR spect ra of IA 3 variants S14C and N58C labeled with IAP in increasing TFE concentration. Spectra were collected at 5 C. ................................ ................................ ................................ .................... 114 4 11 Area normalized 100G X band EPR spectra of IA 3 variants S14C and N58C l abeled with MTSL, MSL, and IAP in 0% TFE and 8M urea. Spectra were collected at 27 C. ................................ ................................ ............................ 115 4 12 Area normalized 100G X band EPR spectra of IA 3 variants S14C and N58C labeled with MTSL, MSL, and IAP in 0% TFE, 30% TFE, and 6% Sucrose. Spectra were collected at 27 C. ................................ ................................ ...... 116 4 13 100G X band CW EPR spectrum of IA 3 S14C IAP with labeled transitions indicating the peak to peak intensities of the low field, h (+1) center field, h (0) and high field, h ( 1) resonances ................................ ................................ ........ 117 4 14 Plots of h (+1) / h (0) as a function of TFE for S14C and N58C ................................ 118 4 15 The local tumbling volume ( V L ) of the protein as a function of increas ing percentage TFE for S14C IAP and N58C IAP ................................ .................. 119

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15 4 16 The absolute value of the percent change of the int ensity of the high field resonance, h ( 1) as a function of increasing percentage TFE for S14C and N58C labeled with IAP ................................ ................................ ...................... 120 5 1 Area normalized 75G X band EPR spectra of IA 3 variants Y57C, N58C K59C, L60C, and K61C labeled with IAP in increasing TFE concentration. ..... 137 5 2 The local tumbling volume ( V L ) of the protein as a function of increasing percentage TFE for Y57C, N58C, K59C, L6 0C, and K61C labeled with IAP collected at 27 C. ................................ ................................ ............................ 138 5 3 Bar graph plots of the local tumbling volume (V L ) of the protein as a function of 0%, 15%, and 30% TFE for Y57C, N58C, K59C, L60C, and K6 1C labeled with IAP collected at 27 C. ................................ ................................ .............. 138 5 4 180 G area normalized W band data of Y57C, N58C, K59C, L60C, and K61C labeled with IAP in 0%, 15%, and 30% TFE solutions ............................ 139 5 5 Plots of area normalized X band and W band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 0% TFE monitoring changes in spectral line shapes based on variations in temperature ................................ ............... 140 5 6 Overlaid plots of area normalized X band and W band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 0% TFE monitoring changes in spectral line shapes based on variations in temperature.. ................................ 141 5 7 Plots of area normalized X band and W band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 30% TFE monitoring changes in spectral line shapes based on variations in temperature. ................................ .............. 142 5 8 Overlaid plots of area normalized X band and W band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 30% TFE monitoring changes in spectral line shapes based on variations in tem perature.. ................................ 143 5 9 Experimental and simulated EPR spectra collected at 27 C ........................... 144 5 10 W band spectrum of free IAP spin label in aqeous solution collected at 150 K 145

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16 LIST OF AMINO ACIDS AND AMINO ACID ABBRE VIATIONS Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Hist idine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

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17 LIST OF ABBREVIATION S BME Mercaptoethanol CD Circular dichroism CW EPR Continuous wave electron paramagnetic resonance DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EPR Electron paramagnetic resonance GHz Gigahertz h (+1) Low field EPR transition h (0) Center field EPR tranisiton h ( 1) High field EPR transtion his6 tag C terminal addition to IA3 with the amino acid sequence LEHHHHHH IA3 Inhibitor of YPrA IAP 3 (2 Iodoacetamido ) PROXYL IDP Intrinsically disordered protein ITC Isothermal titration calorimetry IPTG Isopropyl D thiogalactoside kDa Kilodalton MOMD Microscopic order macroscopic disorder MSL 4 Maleimido TEMPO MTSL (1 Oxyl 2,2,5,5 Tetramethyl 3 Pyrroline 3 Methyl) Methanethiosulfonate NCSU North Carolina State University NHMFL National High Magnetic F ield Lab NMR Nuclear magnetic resonance

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18 OD Optical density PDB Protein DataBank SDSL Site directed spin labeling SVD Singular value decomposition TFE 2,2,2 trifluoroethanol WT Wild type YPRA Yeas t proteinase A

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19 Abstract of Dissertation Presented to th e Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy A MULTI FREQUENCY ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY STUDY OF THE INTRINSICALLY DISORDERED PROTEIN, IA 3 By Natasha L. Pirman August 2011 Chair : Gail E. Fanucci Major: Chemistry Intrinsically disordered proteins (IDPs) are proteins that contain little to no secondary or tertiary structure IDPs are often functional proteins that are essential in biol ogi cal systems and at times have been shown to undergo conformational change s where structure is induced upon binding to a target protein. M onitoring these conformational changes are typically difficult using traditional biophysical techniques such as X r ay crystallography, NMR spe ctroscopy or circular dichroism. Within this work, a multi frequency approach to site directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy was optimized to investigate and characterize the mobility an d conforma tional changes of IDPs. We applied this method to IA 3 which is a 68 residue IDP whose unstructured to helical conformational transition has been extensively characterized by various biophysical techniques. We monitored the chemically induced conformational change in the presence of the secondary structural stabilizer 2,2,2 trifluoroethanol (TFE), at bot h X and W band frequencies. A nalyses of the EPR spectral line shapes provided structural information on the conformational changes. It was shown that initial X band data report ed on global

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20 correlation time changes consistent with a two state model of an unstructured system and the tumbling of a rigid helix ; more detailed analys es of the X band spectral line shapes provide d site specific information on the residue level. A nalysis of the W band EPR spectral line shapes however, more directly reveal ed site specific structural changes Line shape simulations of the data at both frequencies appear to provide further information on the site specific conformational changes occurring in the presence of TFE. Using IA 3 as a model system, we show multi frequency EP R can provide insight into structural changes occurring in IDP systems that are otherwise difficult to characterize.

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21 CHAPTER 1 PROTEIN STRUCTURE AN D FUNCTION Protein Structure Protein Discovery dies on various albumins, mainly from large animals, revealed that the presence of the elements carbon, hydrogen, oxygen and nitrogen, and occasionally sulfur and phosphorus ( 1, 2 ) This finding led Mulder to spec ulate that all albumins had similar compositions and were likely synthesized by one host, most likely from some form of plant, supplying animals with these intact large molecules for nutrition. Mulder corresponded this idea with the prolific scientist J n Jakob Berzelius, where it is believe that the term protein, which originates from the Greek pr tos, was coined when Berzelius claimed that proteins prepare for the herbiv ( 1 ) This would mark the beginning of the study of one of the most essential compone nts in all living cells. Nearly thirty years later, two scientists Heinrich Hlasiwetz and Josef Habermann observed that by adding strong acidic solutions to casein, which is now known to be a phosphoprotein, the protein would be hydrolyzed into smaller s ubunits such as glutamate, aspartate, leucine, tyrosine, and ammonia. The study led to the recognition that proteins were made up of various amino acids, which are molecules that contain an amine group, a carboxylic acid group, and a side chain attached a t the Carbon position ( 2 ) A depiction of the common 20 amino acids is given in Figure 1 1. Soon after, Franz Hofmeister proposed that amino acids could be linked together, by what are

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22 now known as peptide bonds to create polypeptide chains; thus, revealing that amino acid subunits could join to form proteins ( 2 ) In the 1930s, Linus Pauling and Robert protein backbone structure. It was shown that the six atoms that comprise a peptide bond are in a planar configuration due to the partial double bond nature of the peptide bond. This enables electrons to resonate between the carbonyl oxygen and the amide nitrogen. Steric hindrance promotes preferential bond angles, where the oxyge n atom of the carbonyl group is trans to the hydrogen atom attached to the amide nitrogen. These findings led to the conclusion that polypeptide chains were comprised of a series of rigid planes where rotation can only occur about the bonds connected to t he carbon ( 3 ) Nobel Laureate, Emil Fis c her, also made various contributions to the enhancement of the understand ing of protein structure. His work with polypeptides also link together to form proteins, and his work with enzymes led to the lock and key model which indicated that enzymatic proteins had specific structure and interaction with their substrates ( 4 ) With emerging understanding of the importance of proteins in living organisms, protein studies began to grow rapidly. Throughout the first half of the 20 th century, many technological advances were made which allowed protein stru ctures to be investigated and more thoroughly understood. One important technological advance occurred with the accidental discovery of X rays in 1895 by Wilhelm R ntgen. Although a full understanding of X rays would take time and much debate, they would eventually prove to be of great usefulness in the scientific world. In 1915, William and Lawrence Bragg revealed that

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23 the structure of sodium chloride could be determined by monitoring the diffraction of X rays by crystals ( 5, 6 ) structures, and this technique was rapidly implemented into protein structural analysis. In 1958, Max Perutz and John Kendrew used X ray crystallography to obtain the structu re of sperm whale myoglobin ( 2 ) After the elucidation of the structure of myoglobin, the structures of the large multiple subuni t protein hemoglobin, as well as, the first enzyme lysozyme were obtained ( 7 ) Many new techniques and advances have been made in determining protein structures; however, X ray crystallography is still one of the main avenues for elucidating protein structures. Structural Protein Components Vast amounts of knowledge about protein structures and functio ns have been gained over the past 175 years. Proteins are now understood to be macromolecular structures that are the most abundant biological entities in all living cells ( 3 ) Proteins have a wide range of functions, such as enzymes that catalyze reactions, cytoskelatal proteins that comprise the very structures of cells, receptor proteins that recognize targets, an d chaperones that are accessory proteins that assist in folding other proteins, just to name a few ( 2 ) The variety of proteins a nd their functions are almost infinite, with new proteins and functions being discovered quite often. An extensive variety of protein sizes (ranging anywhere from a few amino acids to proteins that are upwards of 35,000 amino acids) as well as various pr otein structures are needed to perform the multitude of functions in living cells. These necessary protein traits can cause proteins to be rather complex. In order to adequately depict each part en devised to describe the assortment of components that make up protein structure.

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24 Primary structure Proteins have a distinct amino acid sequence that dictates its function and d sequences, as shown in the example given in the left most portion of Figure 1 2. Substitutions of not alter the overall structure of the protein. A substitution tha t does not alter structure or function is termed conservative. Conservative substitutions occur when a particular amino acid is replaced with another amino acid with similar chemical properties such as hydrophobicity and molecular bulk. Non conservative amino acid substitutions occur when the amino acid is replaced with another amino acid with different chemical properties ( 8 ) Non conservative amino acid substitutions often times lead to improper folding of proteins and can r esult in a variety of protein misfolding diseases ( 2 ) Secondary structure Peptide bonds link the various amino acid residues th primary structure. As discussed previously, the peptide bond has a planar configuration, with rotation allowed around only two bonds: the nitrogen/ carbon bond and the carbon/carbonyl carbon bond, as depicted in Figure1 3 ( 2 ) Due to the rigidity of the peptide bond, polyp eptide chains have a limited range of conformations that they can adopt. The two rotatable backbone dihedral angles have been denoted as for the nitrogen/ carbon bond angle and (psi) for the carbon/carbonyl carbon bond angle. Theoretically, each angle can range in value from 180 to +180 and w hen both the phi and psi angles are 180 the peptide bond is defined to be in a fully extended conformation, w here the peptide groups are in the same plane. Steric hindrances prohibit many possible a ngle combinations because certain angles cause interferences

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25 between atoms from the polypeptide backbone and atoms in the side chain groups ( 3 ) as seen in the depiction in Figure 1 4. In the early 1960s, G.N. Ramachandran and V. Sasisekharan developed a way to evaluate protein structure by graphically analyzing protein phi and psi values, now commonly referred to as a Ramachandran p lot. It was shown that by plotting the backbone dihedral angles distributions of allowed angle values tend to cluster within certain regions of the plot ( 9 ) As stated above, many phi and psi angle combinations are prohibited due to steric interferences; subsequently, the corresponding regions within R amachandran plots are usually scarcely populated ( 10 ) It has been observed that particular protein structures give rise to similar phi/ psi angle combinations, and typically fall within given regions in a Ramachandran plot, providing a means for monitoring structural properties in proteins. Figure1 5 shows a standard Ramachandran plot where the dark grey regions designate angle combinatio ns that have no steric interferences, light grey regions designate angle combinations that are at the extreme limits for allowable conformations, and white regions are areas that are prohibited due to steric hindrance ( 3 ) Local ized amino acid conformations that occur within various regions of the polypeptide chain can typically be grouped into one of three categorie s: helices, sheet s, and turns or loops secondary structure, and an example of helical secondary structure is depicted in Figure1 2. Secondary structures usually arise when certain amino acid resi dues are found in a particular consecutive order.

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26 Segments of consecutive amino acid residues with the same phi/psi angles of 60 and 50 respectively, have been shown to have helical structure ( 11 ) The residues involved in helices are hydrogen bonded at all NH and CO groups except at the terminal residues and due to hydrogen bonding. Certain residues have been shown to preferentially form helices while others have been shown not to favor helical formation. The follow ing amino acids are thought to be good helix forming residues Ala, Glu, Leu, and Met, while Pro, Gly, Tyr, and Ser are poor helix forming residues ( 12 ) sheet, which consists of a strands. strand s are normally comprised of 5 10 amino acid residues whose phi/psi angles are almost in the fully extended conformation Due to strands are normally very unstable, and to overcome the instability, two o r more strand s will typically come sheet secondary structure ( 11 ) helices, sheets are also driven by hydrogen bonding where certain residues sheet structures. O ften times sheets consist of the aromatic residues Tyr, Phe and Trp as well as the branched amino acids residues Thr, Val, and Ile Pro residues are frequently found in the edge of sheets, most likely to prevent association s between proteins that might lead to aggre gation and amyloid formation ( 13 ) Structured proteins typically contain secondary structural elements of helices and sheets that are often connected by turns or loops. Turns and loops comprise the third type of secondary structural element, which typ ically contain amino acids with small side chains such as the residues Gly, Asp, Asn, Ser, Cys, and Pro, with very low

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27 frequency of large aromatic or branched amino acids. Turns and loops are generally very flexible allowing for the polypeptide backbone t o orient into numerous conformations permitting greater diversity in proteins structures and functions ( 2 ) Tertiary structure Fre quently, secondary structural elements will fold into distinct compact three dimensional structures. This is considered to be tertiary structure; an example can be seen in Figure1 2. The major driving force for p rotein folding is caused by what is known as the hydrophobic effect where hydrophobic amino acid residues cluste r in the interior of the protein shielding them from aqueous solvents, which typically positions hydrophilic residues on the surface of the protein ( 14 ) By m inimizing the number of hydrophobic side chains exposed to aqueous solution, the protein is stabilized in the most energetically stable form. A lthough the hydrophobic effect is the main cause of protein folding, various types of interactions have been shown to assist in stabilizing a proteins tertiary structure Covalent bonds such as disulfide bridges or salt bridges, as well as any sort of non covalent bond such as electrostatic i nteractions, van der Waals interactions, or hydrogen bonding effects can contribute to tertiary structure ( 2 ) Quaternary struct ure Many proteins are comprised of a single polypeptide chain and are considered to be monomeric, where their three dimensional structures can be described solely by their tertiary structure. However, some proteins function as a combination of several po lypeptide chains (subunits), and are considered to be multimeric. The arrangement of multiple subunits into three dimensional structures is referred to as quaternary structure.

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28 Protein Structure Paradigm In 1894, Nobel Laureate, Emil Fis c her hypothesized, as discussed previously, the correct size and shape substrate could fit in the active site pocket of the enzyme led to the long standing belief that there is a struc ture function paradigm, where the unique 3 D structure of a protein dictates its function. X ray crystallography seemed to validate the idea of a structure function paradigm, when the first enzyme, lysozyme, was crystallized with a bound inhibitor and the specific locations of certain amino acid side chains seemed to aid in catalysis ( 15 ) Since the time of the first crystal structures, t he field of structural biology has vastly expanded. At the end of 2010 more than 70,000 sets of atomic coordinates for proteins have been deposited in the Protein Data Bank with the majority of the structures being elucidated via X ray crystallography The extensive growth in structural knowledge has led to an increasing convic tion that the re is a central dogma where protein sequence dictates 3 D structure which subsequently dictates biological function. For many years, the structure function paradigm has been an exclusively accepted notion where it is thoroughly understood th at protein structure and function are closely associated with one another However, over time there has been an increased realization that not all biologically functional proteins fold spontaneously into stable compact structures; in fact, some proteins h ave been shown to be entirely unstructured or contain segments of unstructured regions ( 15, 16 )

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29 The existence of functional un structured proteins skews the accepted notion of a s tructure function paradigm The discovery of the functionality of unstructured proteins has led to the need for an updated theory on protein structures and function s where a new classification of functio nal unstructured proteins typically denoted as intrinsically disorder proteins (IDPs), are included ( 17 ) The discovery of functional unstructured proteins has provided a breakthrough for bioch emistry, biophysics and molecular biology, allowing for new greater comprehension of the complex ity of proteins and their functions within living cells Intrinsically Disordered Proteins Intrinsically disordered proteins (IDPs) are defined as proteins or p rotein segments of 50 or more residues that lack highly populated secondary and tertiary structure under physiological conditions ( 18 ) Proteins that contain intrinsic disorder have been described as dynamic, flexible ensembles that experience significant variability in their ph i/psi Ramachandran angles with no specific equilibrium values ( 15 ) IDPs differ from structured or ordered proteins whose 3 D structure is relatively stable and have fairly defined phi/psi Ramachandran angles and equilibrium positions ( 15, 19 ) The variability of IDP systems makes investigating their structure and function challenging, and could explain the gradual recognition of their importance in biological functions. is directly associated with its structure led to the idea that a protein was only functional in biological systems when it had a well defined 3 D structure encoded by its g iven amino acid sequence. However, within the last 20 years, many studies have shown that IDPs are vital functional proteins in biological systems. It is now understood that often times IDPs can play a variety of roles within living cells, of which typic ally fall into one of

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30 the following four categories: molecular recognition, molecular assembly, protein modification, and entropic chain activities ( 20 ) It took many years for the recognition of the significance of IDPs. In 2001, 29 genomes had been surv eyed and structural predications indicated that proteins from eukaryotes have more disordered segments than either bacteria or archaea with 30% of the eukaryotic proteins having disordered regions of 50 or more residues ( 18 ) It has also been shown that a number of IDPs have been linked to various human diseases. A few well known diseases associated with IDPs are: Alzheimer's disease ( 21 ) Down's syndrome ( 22 ) Parkinson's disease ( 23, 24 ) prion diseases ( 25 ) disease ( 26 ) The involvement of IDPs in various biological functions within living cells, as well as t heir association with various known diseases, demonstrates the importance of understanding their structural changes and functions, revealing a completely new class of proteins to be discovered and investigated. Discovery of Intrinsically Disordered Protein s In the early 1980s, researchers began to notice that several protein crystal structures lacked discernable electron density in particular protein regions despite the fact that biochemical studies had indicated that these specific regions were essential f or biological function s ( 27 ) It is now known that many f unctional proteins characterized by X ray crystallography have various amounts of missing electron density in their solved crystal structures. Although missing electron density can be a consequence of several various factors, quite often it is a result of atoms, side chains, residues, or regions of the protein failing to scatter X rays uniformly. This phenomenon is thought to be caused by variations in the positions of one of the aforementioned protein components which

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31 consequently results in non uniform unit cells. This implies that those regions could be flexible and disordered ( 28 ) The concept that flexible and disordered proteins belong to a unique classification took many years to take shape despite numerous accounts of these types of proteins reported in the literature. Before a common classification for functional disordered proteins had been established, reports regarding these types of proteins were published with various names such as intrinsically diso rdered ( 18 ) natively den atured ( 29 ) natively unfolded ( 30 ) intrinsically unstructured ( 31 ) mostly unstructured ( 32 ) and natively disordered ( 16 ) which seemingly caused literature. C onsequently protein s with highly unusual structural properties like extreme flexibility or biologic ally functionality despite a lack of secondary or tertiary structure was generally considered to be a n infrequent exception to the known structure function paradigm. In the latter half of the 1990s commonalities in various flexible and disordered proteins were discovered by four independent research groups ( 17, 18, 30, 31 ) Each of the research groups came to the same significant conclusion that flexible /disordered proteins were themselves a unique class of protei ns and not an infrequently observed phenomenon, using rather different investigative tools, namely: bioinformatics, N MR spectroscopy, protein folding/misfolding, and protein structural characterization From the information obtained by the various investigative tools, it became blatantly apparent that the classical structure function paradigm was an inadequate way to describe this new class of proteins; thus, leading to the suggestion of a mor e comprehensive view of

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32 the protein structure function relationships and the concept of the hypothesis of the protein trinity. The Protein Trinity Hypothesis The protein trinity hypothesis, as illustrated in Figure 1 intracellular p roteins or functional regions of such proteins can exist in any one of the ( 18 ) In 1994, the molten globule state was determined to be a third thermodynamic state, with properties that fall somewhere in between those of an ordered state and randomly coiled state. Most often molten globules have intrinsic secondary structure and overall architecture but contain very little if any intrinsic tertiary structure ( 33 ) The protein trinity hypothesis takes into account that proteins may function at any one of these three state s or by transition ing among st any of the three T his view allows for any of the states not just the ordered state, to be the n ative state of a protein. This new structure function paradigm expands upon the original notion that the protein sequence dicta tes structure which in turn dictates functions to include the function of disordered proteins as shown in Figure 1 7. Experimental Methods Commonly Employed to Characterize IDPs It is now somewhat understood that particular amino acid residues have tende ncies to form specific secondary structures ( 2 ) where some residues are order promoting while others are disorder promoting, as s hown in Table 1 2 With that notion many computational protein structure predication programs have been developed in an attempt to elucidate protein structures from amino acid sequences. Due to the vast amount of possible amino acid combinations, these programs at this point in time, are useful as supplementary tools for determining protein structures. Experimental

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33 techniques are still the primary method in determining protein structures. Due to the very nature of IDPs, even experimental structural st udies are often challenging. A few methods commonly used to study IDP systems are described in the sections below. X ray crystallography Although completely disordered proteins are not applicable for X ray crystallography studies, this method can still be used as a helpful guide in identifying disordered regions. Missing electron density in X ray crystallography structures can signify that a protein segment is disordered. However, a key concern regarding information obtain by this method is, without investi gation by a secondary method, there is uncertainty whether regions of missing electron density are a result of highly dynamic structured domains, intrinsic disorder, or the result of technical difficulties with the crystallization itself ( 16, 18 ) NMR spectroscopy Nuclear magnetic resonance (NMR) is an experimental spectroscopic technique that can be used to characterize protein structures and dynamics on a residue by residue level. Unfolded and partly folded protei n structures are also susceptible for investigation by various NMR techniques. Initial IDP NMR studies presented extreme difficulties due to a lack of resonance dispersion, especially for protons ( 34, 35 ) Since most IDP amino acid residues are solvent exposed, the NMR frequencies of each amino acid are similar and thus cause strong overlaps in 1 H resonances. With advances in multidimensional homonuclear and heteronuclear NMR experiments and the ability to unifor mly label protein samples with 15 N and 13 C, resolving and assigning individual resonance frequencies for ful l length IDPs became possible. Relaxation base d NMR methods can be used to characterize dynamics in protein systems.

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34 Solution NMR spectroscopy has become one of the most useful biophysical techniques for elucidating disordered regions in proteins, monitoring protein flexibility, and characterizing protein folding and unfolding mechanisms ( 36 ) Table 1 1 shows a variety of NMR techniques that have been developed that he lp in characterizing numerous proteins, including IDPs. Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy has also been shown to provide structural information for proteins in solution. Most commonly, fa r UV CD (180 nm 250 nm) spectrosc opy is used to monitor secondary structural components present in proteins i.e. helices, sheets, and random coils ( 37 ) Each secondary component has a distinct CD spectrum allowing for the various components to be observed. N ear UV CD (250 nm 350 nm) spectroscopy provides information on tertiary protein structures given that aromatic groups give rise to s harp peaks when the protein is ordered when irradiated with UV light within this wavelength range. However, near UV irradiation does not cause p eaks to appear in proteins that are in molten globular or random coil forms due to motional averaging and there fore do not provide any tertiary information about these types of protein structures ( 18 ) A collective far and near UV CD experiment can be used to distinguish between ordered, molten globular, and random coil forms of proteins This method is h owever, only semi quantitative an d cannot provide information on the residue level. Susceptibility to proteolysis It has long been understood that enzymes have the ability to catalyze the hydrolysis of protein bonds. Researchers, in as early as the 1920s, showed that unfolded proteins we re more likely to undergo protease cleavage than folded proteins

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35 ( 16 ) More recently, it has been shown that not only unfolded or denatured proteins are more susceptible to cleavage by various enzymes but flexible regions in proteins are also often times vulnerable to proteolysis ( 38 42 ) Protein regions that are prone to proteolysis are typically positioned within highly flexible loops, are often solvent exposed, and generally lack secondary or t ertiary structure with regions of disorder of at least 13 residues ( 28 ) A likely reason for the enhanced possibility of proteolysis in disordered regions is the propensity of those regions to protrude out into so lution making them easier targets for proteolysis as compared to more ordered protein regions that tend to be more compact and shielded. In combination with a secondary biophysical technique, the greater susceptibility for disordered protein regions to und ergo proteolysis can help to distinguish between ordered and disordered segments within proteins and therefore can be considered a valuable tool in identifying IDPs. Other experimental methods Besides the most commonly used methods listed above, numerous o ther biochemical and biophysical methods have been utilized to investigate disorder in proteins. The presence or lack of global tertiary structure has been evaluated by several various techniques. One of which is differential scanning microcalorimetry (D SC) which can be used to indicate a lack of tertiary structure by detecting an absence in cooperative thermal transitions ( 28 ) Another example of a technique that is used to monitor the lack of tertiary structur e is the use of extrinsic fluorescence compounds. Fluorescence compounds can penetrate into hydrophobic pockets within proteins helping to distinguish between the two intrinsically disordered protein forms: random coiled and molten globular. Molten globu lar proteins can consist of hydrophobic pockets since they contain some residual

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36 secondary or tertiary structures, while random coiled proteins are nearly fully extended therefore devoid of hydrophobic pockets. These distinctions between the two protein f orms allow for differentiation between the two intrinsically disordered forms by fluorescence. Another feature of IDPs that has been studied by a variety of techniques is the determination of the degree of extension and disorder within proteins. Hydrodyna mic properties of proteins can be used to indicate disorder in proteins, and therefore can be used to distinguish between ordered and disordered protein states. Size exclusion chromatography (SEC) is one such technique that can provide information on hydr odynamic properties. SEC separates proteins based on their hydrodynamic size due to the fact that typically an abnormally large stokes radius will indicate disorder, whi ch is how SEC is used to differentiate between various degrees of extension ( 42 ) Small angle X ray scattering (SAXS) is another method used to determine degrees of extension and disorder. This technique is considered to be one of the most powerful methods for determining protein dimensions and shapes ( 28 ) The SAXS technique provides such information by monitoring the intensity of the X ray scatter which is not only sensitive to the size of the pr shape and conformational proprieties. The conformational proprieties provide information on whether the protein is in a globular or random coiled state by evaluating the data using a Kratky plot. In this plot, the shapes of the plotted data indicate the conformational state of the protein ( 43, 44 )

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37 Each of the methods used for characterizing IDPs have advantages and disadvantages. Implementing new and various techniques m ay provide slightly different perspectives and provide a means for greater understanding of IDP systems. Given that each technique has certain limitations, and no one technique can fully characterize all IDP systems solely, it is without question that inve stigation by multiple techniques is the best approach in understanding and characterizing this diverse new class of proteins. Utilizing SDSL EPR Spectroscopy as an Investigative Tool for Characterizing IDPs As will be extensively discussed in chapter 2, si te directed spin labeling (SDSL) electron paramagnetic resonance (EPR) is a spectroscopic technique that has widely been used to investigate macromolecular structures and dynamics. In order to utilize the EPR technique, the system under study must contain an unpaired electron. Quite frequently, a cysteine residue is incorporated into the protein via site directed mutagenesis allowing for a paramagnetic nitroxide spin label to be chemically attached to the reactive sulfhydral at the chosen substitution sit e within the protein Previous SDSL EPR studies on structured proteins, such as Bacteriorhodopsin and T4 lysozyme, helped to esta blish that backbone secondary structure and various tertiary structural features were determinable from variations in nitroxid e spin label accessibility when located at different positions within the protein ( 45, 46 ) EPR spectral line shapes can also depict g eneral protein folds and orientations by monitoring motional dynamics of the side chain ( 45, 46 ) The dynamics of the spin label can provide information on conformational changes which mediate protein function on the millisecond time scale ( 47, 48 )

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38 SDSL EPR has been shown to be an extremely applicable technique for studying protein systems that are often difficult to characterize with other biophysical techniques such as membrane proteins, protein membrane interactions, and large protein complexes This technique is suitable for these types of proteins since it has no correlation time limit due to the tumbling of the protein molecules ( 49 ) and in the past has been primarily applied to these types of protein systems. The capability of SDSL EPR to monitor folding unfolding processes in structured proteins led to the belief that the technique should also be well suited for inv estigating IDPs, which often undergo induced folding events upon binding to their physiological partners. In 200 6, the first report regarding the investigation of an IDP via SDSL EPR was published ( 50 ) The Longhi laboratory used this technique to monitor the induced folding event s of the intrinsically disordered C terminal domain (N TAIL ) of the measles virus (MV) nucleoprotein in the presence of its physiological binding partner, the C terminus of the X domain (XD) of P protein, and in the presence of the secondary structure stabi lizer 2,2,2 trifluoroethanol (TFE). MV nucleoprotein had previously been studied by many of the aforementioned biophysical techniques. N TAIL was first identified to be a disordered region of more than 100 residues by structural prediction and the identif ication was verified by its high susceptibility to protease digestion as well as characterization via NMR and CD spectroscopy ( 51, 52 ) Cloning, expressing and isolating only the N TAIL segment allowed for characte rization of this region. Despite being separated from the rest of the protein, N TAIL was still shown to be biologically functional and able to bind with XD ( 52 )

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39 The reported SDSL EPR study investigated f our indep endently spin labeled sites within the N TAIL This technique was able to contribute various information regarding structural changes occurring within N TAIL that was difficult to attain with other methods. SDSL EPR distinguished that different regions wit hin the N TAIL had varying contributions in the induced folding process. I t was also shown that the protein binding partner, XD, induced a structural transition within certain regions within N TAIL that were indicative of an helical transition It establ ished that t he induced folding event in the presence of XD is a reversible process Lastly, it was concluded that information can be inferred about structural propensities by monitoring induced folding events in the presence of TFE, based on the results o f the four different spin labeled sites variability in the presence of TFE ( 50 ) From the results of the N TAIL study it was concluded that SDSL EPR spectroscopy could be an extremely valuable method for monitoring induced folding events and it is capable of p roviding information at the residue level. Given that valuable information can be acquired from this approach, it is only logical that it should be considered as an experimental method choice when study ing IDPs. However, to best utilize SDSL EPR to chara cterize IDP systems, more studies need to be conducted to establish the most beneficial line shape analyzes for obtaining information on a variety of IDP systems. The scope of this dissertation focuses on the optimization and utilization of multi frequen cy SDSL EPR experiments for characterizing small dynamic IDPs. For this purpose, the TFE induced helical conformational change in the intrinsically disordered protein, IA 3 was investigated by SDSL EPR. As discussed in detail in the section below, IA 3 is a relatively simplistic protein and had been extensively characterized in detail

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40 previously by C D and NMR spectroscopy Because an abundant quantity of information was available on IA 3 the protein was thought to be an excellent model system for further development of the SDSL EP R methodology for studying IDPs and was the basis for the choice of the system. The Intrinsically Disordered Protein, IA 3 IA 3 is a 68 residue intrinsically disordered protein found in the cytoplasm of Saccharomyces cerevisiae Previous studies have shown that IA 3 is unstructured in solution, and u pon binding to the active s ite pocket of its target protein, yeast proteinase A (YPRA), the N terminus of IA 3 undergoes a disordered to ordered transition adopting helical conformation. The remaining C terminal residues were unresolved ( 53 55 ) as shown in the crystal structure depiction in Figure 1 8 It has been postulated that IA 3 lacks intrinsic secondary structure in solution due to its amphipathic characteristics. When IA 3 binds in the active site pocket of YPRA it adopts an helical conformation where the hydrophobic residues face into the active site pocket, and the hydrophilic residues face into solution ( 56 ) as shown in Figure 1 9 It has long since been determined that IA 3 is an endogenous, potent inhibitor of YPRA, and has been shown that it does not inhibit any other aspartic proteinases with similar sequences/structures from a w ide variety of other species that have been tested ( 57 ) In fact, IA 3 was degraded as a substrate by the other tested aspartic proteinases and only escapes cleavage from YPR A b y being stabilized helical conformation upon interaction with the active site of the target ( 57 ) Studies revealed that IA 3 inhibitory activity is located with in the first 34 residues and the potent and specific i nteraction between IA 3 and YPRA has been shown to be caused by hydr ophobic interactions made by three key clusters in the inhibitory sequence ( 58 )

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41 Besides X ray crystallography studies, various other biophysical techniques have been employed to study the structure and function of IA 3 It is been shown by far UV circular dichroism (CD) spectroscopy experiments that in the presence of the secondary structural stabilizer 2,2,2 trifluoroethanol (TFE) a two state transition occurs from an unstructured to helical conformation ( 55 ) The CD analysis cannot, however, establish which residues within the IA 3 sequence became helical, or whether the two state transition was uniform throughout the protein. NMR spectroscopy has also been employed in c haracterizing IA 3 It is known that NMR chemical shifts are typically sensitive indicators of protein secondary structure as illustrated from reports in literature ( 59 ) In an effort to characterize IA 3 15 N HSQC NMR was collected on free IA 3 in solution which revealed that generally the protein had characteristics of an unfolded protein, given that all of the amide protons were highly accessible to the solvent ( 53 ) According to researchers in the field proteins that 1 H NMR experiment sole ly however, for IA 3 the peaks of the collected 1 H NMR spectr um were extremely overlapped, and could not be assigned with this technique In an effort to assign the resonance peaks three dimensional (3 D) 15 N based methods were utilized however, th e resonance peaks in the spectra were also fairly overlapped and no more than 20% of the assignments could be determined In order to determine the peak assignments for the majority of the protein a doubly labeled ( 13 C and 15 N) IA 3 sample was generat ed and 3 D 15 N TOCSY HSQC and HC(CO)NH TOCSY experiments were performed. These techniqu es coupled with other standard triple resonance data,

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42 allowed the assign ments of most of the backbone and determination of partial side chain for free IA 3 in solution to be attained ( 53 ) A 15 N HSQC spectrum of IA 3 bound in the active site pocket of YPRA was shown to be overtly different from the spectrum of free IA 3 in solution. Many of the resonances peaks either shifted or disappeared altogether, and the differences were attributed to the conformational change t hat IA 3 undergoes in the presence of YPRA to an helical conformation as observed by the X ray crystal structure. Resonance peaks could not be assigned for IA 3 when in complex with YPRA due to a lack of peak dispersion. Although peak assignments were un attainable, information about the system was still acquired. IA 3 YPRA titration NMR experiments alluded to the fact that the C terminal residues of IA 3 in the presence of YPRA were in intermediate exchange, which was an indication that the C terminal regi on of the protein was affected by the presence of YPRA even though those particular residues have been shown to have no contribution in the inhibition of the protease ( 53 ) In a pursuit to characterize the ordered state of IA 3 at the residue level, NMR studies were conducted in the presence of TFE, which provided information that was inaccessible in the presence of YPRA. TFE induced structural transitions occurring in IA 3 were monitored using 15 N HSQC in conjuncti on with singular value decomposition (SVD). The study revealed that the N terminal residues are undergoing a more pronounced TFE induced transition than the C terminal residues, thus indicating that the N terminus is undergoing a greater transition toward an helical structure than the C terminus as the TFE concentration increases ( 60 ) Although the NMR studies coupled with SVD provided great insight in the chemically induced transitions of IA 3 the ability to

PAGE 43

43 assign all the resonance peaks was still u nattainable. 40 out of 68 cross peaks were unable to be tracked due to ambiguity in particular peaks within the spectrum ( 60 ) Lastly, the kinetics of folding as function TFE concentrations as well as the interactions between IA 3 and YPRA were investig ated by laser temperature jump fluorescence spectroscopy (T jump) and fluorescence resonance energy transfer (FRET) experiments. These experiments provided folding and unfolding rate constants for IA 3 in TFE solution. They also provided a means for determ ining a folding model, where the results indicated that IDPs may interact with target proteins via template driven folding ( 61 ) As indicated above, IA 3 ha s been fairly well characterized b y numerous biophysical techniques. Although each of the techniques were able to characterize certain aspects of the protein structural transitions, it is apparent that despite the be taken to obtain such information, and even then all information was not always able to be attained. This signifies the necessity for utilizing multiple biochemical and biophysical techniques, especially on more complex IDP systems. By optimizing SDSL EPR for investigating IDPs, it will provide another means with which to study these proteins, and hopefully assist in providing information on IDPs that was otherwise difficult or even unattainable with other techniques.

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44 Figure 1 1. The 20 standard amino acids that comprise proteins.

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45 Figure 1 2 Levels of structure in proteins.

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46 Figure 1 3. Depiction of a peptide bond. The planar nature of the peptide bond allows for only two degrees of freedom per residue for the peptide chain. Rotation is allowed about the bond linking the carbon and the carbon of the peptide bond ( ) and the bond linking the nitrogen of the peptide bond and the adjacent carbon ( ). Figure modified from Garrett et al. ( 10 )

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4 7 Figure 1 4. Forbidden peptide bond configurations. Due to steric crowding some angles are unfavorable and are typically prohibited. Figure modified from Garrett et al. ( 10 ) Figure 1 5. Ramachandran plot fo r L Ala residues. Plot shows favorable conformational parameters for different / values. Figure modified from Lehninger reference ( 3 )

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48 Figure 1 6. The alternative hypothesis to the class ical structure function paradigm. This model indicates that function can arise from any of the three structural states, not just the ordered state. Figure modified from Dunker et a l. 2001 ( 18 ) Figure 1 7. Schematic of protein structure and functions (modified from Uversky and Dunker 2010) ( 15 ) This schematic shows that the classical structure function paradigm cannot describe many function s that proteins perform and that the protein trinity model is a more sufficient way to describe protein structure and function relationships.

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49 Figure 1 8 X ray crystal structure of IA 3 (dark grey) bound to YPRA (light grey) that shows residues 2 34 of IA 3 helical conformation. The remaining residues (35 68) are not resolved in the X ray c rystal structure (PDB ID 1DPJ) and are drawn in graphically with their single letter amino acid codes to signify an unstructured sequence. Figure made in Chimera.

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50 Figure 1 9 Depiction of the amphipathic nature t he helical conformation of the N termi nal residues of IA 3 upon interaction with YPRA. Figure made in Chimera.

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51 Table 1 1 Common NMR Techniques for Characterizing Protein Structure and Flexibility NMR Experiment Information Determined Comments Nuclear Overhauser Effect (NOE) Secondary And Tertiary Structure Determination Primary NMR method for generating long range distance constraints Relaxation Measurements Flexibility and Motion Measurement of global and local correlation times, order parameters, and chemical exchange Chemical Shifts Secondary Structure Characterization Backbone chemical shifts are often correlated to protein secondary structure Hydrogen Exchange Protein Stability and Hydrogen Bonding Measurement of protection rates in protein folding Dipolar Couplings Structural Cha racterization Orientation Information and local order parameters Diffusion Measurements Hydrodynamic Characterization Global shape information can distinguish folded and unfolded structures Paramagnetic Probes Solvent Binding and Protein Accessibility Di stance constraints in proteins by paramagnetic broadening Scalar Couplings Secondary Structure Information about bond torsion angles Table recreated from Bracken reference ( 36 )

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52 T able 1 2 Order/Disordered c omposition p rofile Order Promoting Residues a Disorder Promoting Residues a Residues Listed From Order Promoting to Disorder Promoting b Number of Each Residue in IA 3 Asn Ala Trp 0 Cys Arg Phe 2 Ile Gln Tyr 2 Leu Glu Ile 1 Phe Gly Met 2 Trp Lys Leu 2 Tyr Pro Val 3 Val Ser Asn 3 Cys 0 Thr 3 Ala 5 Gly 4 Arg 0 Asp 7 His 2 Gln 7 Lys 13 Ser 6 Glu 6 Pro 0 (a) Data from Vihinen et al. ( 62 ) (b) Data from Campen et al. ( 63 )

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53 CHAPTER 2 BACKGROUND AND THEOR Y OF MULTI FREQUENCY CONTINUOUS WAVE ELECTRON PARAMAGNETI C RESONANCE SPECTROS COPY Introduction to Electron Paramagnetic Resonance Spectroscopy The first microwave spectrometer was constructed in 1934 by Cleeton and Williams thus mar king the beginning of microwave spectroscopy. Although the concepts of this technology were being investigated in the early part of the 1930s, it paramagnetic resonan ce would fully take shape ( 64 ) In 1944, E. Zavoisky published the first article on paramagnetic resonance ( 65 ) the field of paramagnetic resonance excelled. By the end of the WWII, advancements in microwave and electronic technologies allowed paramagnetic resonance experim ents to be conducted with the better sensitivity and resolution ( 64, 66 ) Since that time, there has continued to be vast improvements in spectrometer development with concomitant emergence of new applications Paramagnetic resonance is now referred to as e lectron paramagnetic resonance (EPR) or electron spin resonance (ESR), and is used as a spectroscopic technique that detects the transitions of unpaired electrons in an applied magnetic field (B 0 ). The unpaired electrons resonate between energy states when irradiated with microwaves at a given frequency that equals the energy difference between the states at a specific applied magnetic field ( 64 ) One main area of interest for EPR studies is in biological processes. Biological samples that contain naturally occurring unpaired electrons are applicable for investigation by EPR ( 66 ) Examples include the following: free radical intermediates in metabolic reactions, many transition metal ions, and free radicals occurring from

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54 external radiation just to name a few ( 66 ) Although, many biological samples do not contain unpaired electrons rendering them EPR inactive, the lack of unpaired electrons can be advantageous. In the case where unpaired electrons are absent, an external spin probe, which supplies an unpaired electron, can be incorporated at a desired location within the molecule allowing the system to become EPR active and amen able for stu dy by this technique. EPR can provide insightful information about a system of interest. This chapter will discuss the theory of EPR, provide information on the background of site direct spin labeling (SDSL), discuss multi frequency EPR, and give insight into kinds of information attainable by various simulation methods of EPR spectral line shapes. Zeeman Effect The Zeeman Effect was first described in 1896, when Zeeman observed that spectral lines split in the presence of a magnetic field ( 66 ) This effect would not be fully understood until the discovery of quantum mechanics in the early 20th century. Quantu m mechanics revealed that e lectron s have a magnetic moment and a spin quantum number of 1/2 As shown in Figure 2 1, for the simplest case of a f ree electron in the absence of a magnetic field the electron magnetic moment is degenerate. Upon applying an external magnetic field, the magnetic moment of the electron will diverge and align either parallel or anti parallel to the external applied magnetic field, splitting the energy into two state s; the energy difference is proportional to the magnitude of th e external applied magnetic field ( B 0 ) the spectroscopic g factor ( g ), and the Bohr magneton ( e ), as given in Equation 2 1 ( 67 ) (2 1)

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55 The Bohr magneton (9.274 10 24 J T 1 ), is a proportionality constant defined in Equa tion 2 2 where e is the electric charge carried by a proton (1.6021 10 19 C), constant divided by 2 (1.054 10 34 Js), and m e is the mass of the electron (9.109 10 31 kg) (2 2) W hen the applied energy ( ) is equal to the splitting betwe en levels, the free electrons will resonate between the two energy levels by either absorbing or emitting electromagnetic r adiation. Hyperfine Interaction In many cases the system being studied is more complex than a free electron in solution. Often a ma gnetic moment of nuclear spin from nearby nuclei will couple with the electron magnetic spin moment. This interaction is known as h yperfine coupling ( 68 ) The magnetic moment of t he nucleus creates an additional magnetic field interacting with the electron either opposing or adding to the applied external magnetic field. The interactions with the nuclear magnetic field will further split the energy levels usually by the 2 nI +1 rule where n in the number of equivalent nuclei and I is the nuclear spin ( 69 ) Hyperfine splitting can be used to identify radical species, and coupling nuclei. However, as the number of equivalent nuclei increase, the number of energy level splitting will also increase, which can cause some systems to be extremely complex, and can even cause the signals to overlap and create what appear to be fewer, more broad looking signals ( 70 ) In ordered to make samples which do not contain naturally occurring unpaired electrons EPR active, an external spin probe can be attached to samples. Commonly

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56 this probe is a nitroxide probe, often referred to as a s pin label, and has an unpaired electron that couples with the neighboring 14 N, with a nuclear spin moment of I =1. The 2 nI +1 rule indicates that the hyperfine interaction will further split the Zeeman splitting into three hyperfine splitting, shown in Figu re 2 2. The ability to attach spin labels to biological samples making them EPR active radically changed the biological application of EPR. Spin Labeling In 1964, Burr and Koshland introduced the concept of labeling a biological sample with an external pr obe that could be used as an EPR reporter group ( 71 ) In order to be deemed an adequate probe, they stated three main requirements that a reporter group must meet. The requirements were: first, that the repor sensitive moiety that can be introduced into specific centers of the system of interest and it must subsequently report changes in its environment to an appropriate detector. rter group must be either unique or perturbation(s) in its structure and function as ( 67 ) A year later, in 1965, nearly twenty years after the first published work on p aramagnetic resonance, McConnell and colleagues incorporated a paramagnetic nitroxide spin label into a biomolecule and for the first time a diamagnetic biological sample was investigate by EPR spectroscopy ( 66 ) This technique was first applied to the study of cooperative interactions in hemoglobin ( 72 ) However, one of the early successful application s of spin labeling was investigating the structure and dynamics of

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57 lipid membranes ( 66 ) The use of spin lab els to study diamagnetic systems was directed mutagenesis that incorporation of spin labels at desired positions within protein systems would become possible. Site Direct ed Spin Labeling (SDSL) First investigated in the lab of Wayne Hubbell, the development of site directed mutagenesis provided the ability to substitute one amino acid residue for another. Using this method a reactive cysteine residue could be incorporated at any location with the amino acid sequence. This substitution is achieved by manipulating DNA such that a codon for a cysteine residue is mutated into the DNA sequence at a desired position T he modified DNA sequence results in a cysteine substituted protein construct upon protein expression The newly added cysteine residue can then be reacted with a nitroxide spin label, resulting in a modified side chain. Spin labels are most commonly incorporated into a protein via a thiol based chemistry reactio n as shown in the reaction scheme of the spin label (1 oxyl 2,2,5,5 tetramethyl pyrroline 3 methyl)methanethiosulfonate (MTSL) in Figure 2 3. The development of SDSL allowed for spin labels to be incorporated anywhere within most proteins permitting them to be investigated by EPR; however, the sensitivity of EPR spectrometers at the time were not adequate for studying small aqueous samples. The sensitivity issue would be rectified in 1982 when Froncisz and Hyde introduced the loop gap resonator ( 73 ) This resonator allowed for limited quantities of aqueous samples to be studied by EPR, which enabled SDSL to be used for many various applications.

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58 In the late 1980s the Hubbell laboratory showed that SDSL in conjunction with EPR was successful in extracting global structural information by investigation bee venom protein ( 74 ) and colici n E1 ( 75 ) By the early 1990s, many papers were published using SDSL EPR to study biological systems. Analysis of the EPR spectral line shapes reported in those studies revealed information such as: topology determination in membrane proteins ( 76 ) orientation and location of helical regions within proteins ( 45 ) immersion depths of transmembrane helix residues ( 77 ) and protein conformational changes ( 78 ) In 1994 protein structure at the level of the backbone fold, study equilibrium dynamics of the ( 46, 79 ) Since the mid 1990s numerous papers have been published utilizing the SDSL technique. SDSL EPR has been shown to provide key structural information on protein systems, and one could say has exceeded the original goals of the technique. T his technique is currently assisting in the elucidation of structural, dynamic, and conformational changes in systems that can otherwise be difficult to study by other biophysical techniques. Spin Label Structures Most spin labels used for SDSL studies ar e nitroxide radicals which are well suited for examining the structure and fl exibility of biomolecules due to their simple line shape and its high sensitivity to molecular motion The n itroxide radical is protected by bulky methyl groups that are p reserve d in a n enclosed five o r six member ring structure which makes the radical extremely persistent and very exploitable in SDSL experiments The MTSL spin label, shown in Figure 2 3(a), is the most commonly used in SDSL studies; however, occasionally other s pin labels are used in various studies due to advantages

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59 the structural differences can provide. Examples of three other spin labels used in SDSL experiments are shown in Figure 2 3(b d). Numerous spin labels are currently commercially available that have various ring structure s, reaction chemistri es and flexible linker regions. Spin Label Motion One of the goals of SDSL is to investigate protein structure at the backbone level. In order to obtain such information it is important to first understand that the nitroxide EPR line shape is modulated by motion of the spin label which in turn is affe cted by various modes of motion. E ach mode of motion that the spin label undergoes provides information about the system being studied. These are discussed in mor e detail below. Most SDSL EPR studies are conventionally collected at X band (9.5 GHz) frequency. At this frequency the EPR spectral line shape from the nitroxide spin label changes considerably as the rotational correlation time, termed varies in the 0.1 50 ns time scale. The effect of the correlation time can be seen in Figure 2 4 The correlation time is affected by three main modes of motion as depicted in Figure 2 5. The first mode is the overall tumbling of the molecule in solution, called rot ational correlation time ( R ) R relies heavily on the size of the molecule as well as environmental factors such as temperature, viscosity, and the presence of solutes. The second mode is loc al structural and dynamic backbone fluctuation s of the macromolecule ( B ). B is affec ted by changes in secon dary and tertiary structure, as well as conformational changes occurring within the macromolecule. The third mode is torsional oscillations about the connecting bonds of the spin label to the macromolecule ( i ) i varies depending on the number of rotatable bonds in the linker region of the spin label, and is also dictated by the steric restrictions of the local environment All three

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60 modes of motion provide information about the macromolecule; however, only i and B are affected by the local environment and therefore provide site specific information about the system ( 80 ) EPR at Variable Frequencies Approximately 30 years after the first EPR exper iment was conducted by Zavoisky, the laboratory of Yakov Lebedev developed high frequency EPR spectroscopy. The development of a 140 GHz spectrometer, proved to exhibit enhanced sensitivity to fast molecular motion, provided a means to observe the effects of local polarity on nitroxide g factor, and allowed for other applications ( 81 ) It became readily apparent that EPR spectral line shapes collected at differen t microwave frequencies are sensitive to motions at different time scales. The early high frequency EPR experiments were ground breaking; however, at the time the instrumentation was not sensitive enough for lossy liquid aqueous spin labeled samples. Ove r time and with vast improvements in instrumentation, high frequency EPR has been employed in numerous spin labeled experiments ( 82 95 ) To date, high frequency spectrometers have been constructed in various labor atories ( 96 ) and a W band (95 GHz) spectrometer is now commercially available from Bruker The availability of EPR spectrometers at various frequencies has permitted a multi frequency EPR approach to be utilized to study protein dynamics ( 82, 86, 95 ) This technique allows for data collection of spectral line shapes that are sensitive to different time scales; therefore provi des a means to de convolute the three main motional contributions of the spin label. As stated previously, EPR experiments are commonly collected at X band frequencies. At X band, the EPR spectrum can provide information about the local structure and dyn amics of the particular site at which the

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61 label has been attached within the protein. However, the spectrum also reports on the overall tumbling rate of the protein in solution, which at times is the most dominate mode of motion, making site specific data difficult to obtain ( 81 ) Data collected at frequencies in the W band range or higher are more sensitivity to faster dynamics and frequently the slow overall tu This characteristic of high frequency EPR provides a means to observe more site specific motional modes within the protein ( 85 ) A list of frequencies that are currently used in EPR studies are given in Table 2 1 By exploiting a multi frequency approach, additional quantitative information can also be obtained. In an EPR experiment, as the magnetic field increases the EPR spectrum changes dramatically At X band, the EPR spectrum is dominated by the axial hyperfine term dictated by the spin label at that particular applied magnetic field. At higher fields, and therefore higher frequencies, the nitrogen hyperfine interactions start to be dominated by the anisotropy of the Zeeman term in the spin H amiltonian. The more dominate rhombic Zeeman term at frequencies in the W band range and higher provides new and additional information on protein structure and dynamics that are inaccessible when X band is used solely ( 81 ) Another useful feature of using a multi frequency EPR approach is that the magnetic hyperfine A tensor and g tensor values can be accurately determined from rigid limit spectra ( 95 ) The hyperfine interactions between the nitrogen nucleus and the free electron within the spin label are intrinsic to the molecular environment of the spin label, causing the A tensor values to be independent of the applied magnetic field; therefore, the hyperfine splittings are equivalent at all frequencies. In contrast, the

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62 Zeeman interaction is a field dependent parameter at higher frequencies the g tensor values have a more significant effect on the EPR spectrum and therefore are more resolved ( 97 ) Simulations of EPR Spectral Line Shapes A variety of simulation programs have been written to extract spectral parameters from experimental EPR line shapes. Each simulation program is generally written using different models and algorithms based on the motional time range that is being observed. Spectral p arameters such as rotational motion and molecular orientation of the spin label can be determined by dynamic and static effects on the magnetic hyperfine A tensor and g tensor anisotropies, respectively ( 98 ) The Zeeman and hyperfine interactions of the EPR spectrum provide information on the molecular orientation by monitoring angular anisotropy with respect to the applied magnetic field orientation. Molecular motion of the spin label will cause an averaging of the anisotropy, which is determined by the angular ampl itude of the rotation ( 98 ) The EPR spectral line shapes are determined by the molecular reorientational dynamics of the spin label and its constraints over correlation times. The rotational correlation time of the spin labeled molecule in the fast motionally narrow ed regime can be determined from the Lorenzian line widths of the EPR spectrum provided by Redfield theory ( 98 ) These line widths are determined by the transverse relaxation time, which is dependent on the rate of modulation of the anisotropies by the molecular moti on. Analyses of spectra that are in the slow motional regime (10 9 s t R 10 6 s) are more difficult to simulate and require that the full stochastic Liouville equation be used ( 99 ) In 1996, Budil et al from the laboratory of Jack Freed at Cornell University, published a paper describin g a non linear least squares analysis of slow motion EPR spectra using a

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63 modified Levenberg Marquardt algorithm ( 100 ) Most simulation software to date are based on the theory described within the Budil reference, and pro vide information on EPR spectral line shapes in the slow motional regime.

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64 Figure 2 1. Energy diagram for a single free electron in an applied magnetic field.

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65 Figure 2 2 Energy diagram depicting the hyperfine splitting of an electron coupled to a nuclear spin of I=1 and the corresponding EPR line shape with three transitions. ( A) Hyperfine interaction diagram for a system with m s =1/2 and m i =1. ( B) EPR spectral line shape corresponding to a system with a hyperfine interaction of a single electron with a nuclear spin of I=1.

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66 Figure 2 3. Commonly used spin labels for EPR studies. ( A) (1 oxyl 2,2,5,5 t etramethyl pyrroline 3 methyl) m ethanethiosulfonate (MTSL); ( B) 3 (2 i odoacetamido) PROXYL (IAP); ( C) 4 m aleimido TEMPO (MSL) and ( D) 4 (2 i odoacetamido) TEMPO (IASL).

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67 Figure 2 4. Dependence of EPR spectral line shape s on motion. Line shapes simu lated assuming isotropic motion.

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68 Figure 2 5. Depiction of the three modes of motion affecting a spin label. R is the overall tumbling of the protein, B is the movement of the protein backbone including local oscillations and conformational change s, and i is the movement of the label about the flexible linker

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69 Figure 2 6 Illustration of differences in EPR spectral line shapes fro m nitroxide spin labels change with frequency ( Left ) Simulations illustrating increased orientational sensitivit y of EPR spectral line shapes from low to high frequencies. The derivative spectra are characteristic of a spin label in a frozen amorphous matrix (i.e., the rigid limit). ( Right ) EPR spectral line shape simulations for characteristic frequencies utilized in EPR studies demonstrating the snapshot property of EPR when studying tumbling spin labeled molecules. The large variations that the spectrum undergoes as the frequency is changed are very sensitive to the character of the motion. From Borbat et.al ref erence ( 101 ) Reprinted with permission from AAAS.

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70 Table 2 1. Frequency b ands in EPR s pectroscopy Band Name Typical Frequency (GHz) Wavelength (mm) Energy (cm 1 ) Resonance Field (Gauss) L band 1 300 0.033 360 S band 3 100 0.10 1100 X band 10 30 0.33 3600 Q band 35 8.60 1.2 12500 W band 90 3.30 3.0 32200 D band 130 2.30 4.3 46400 G band 180 1.67 6.0 64300 J band 270 1.11 9.0 96400 Table modified from Hagen reference ( 102 )

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71 CHAPTER 3 REDUCING THE EFFECTS OF THE OVERALL ROTATIONAL CORRELATION TIME OF PROTEINS SDSL EPR is a spectroscopic technique that can assist in the characterization of structural, dynamic, and confo rmational changes of proteins ( 46, 49, 103, 104 ) SDSL EPR involves the introduction of a paramagnetic spin label at a selected site within the protein. The motional dynamics of the spin label are correlated with the general features of the protein fold and are determined from the EPR spectral line shape. Based on the local environment in which the spin label is located within the protein, the EPR spectral line shapes will differ dramatically. These differences result from variations in the three main modes of motion that affect the spin labeled moiety. As discussed extensively in chapter 2, the first mode of motion is the overall tumbling of the protein ( R backbone ( B ), and torsional oscillations about the connecting bonds of the spin label to the protein ( i ). The latter two modes of motion ( B and i ) are those that provide s ite specific information about the environment in which the spin label is located. These modes of motion also provide information for the determination of protein structure at the level of the backbone fold, for the study of equilibrium dynamics of the ba ckbone, and for resolving conformational changes in the protein fold, which according to Hubbell were the original goals of SDSL ( 103 ) In comparison to NMR or X ray studies, early SDSL EPR studies provided more qualitative and less resolved structural information. It was not until the mid 1990s that analysis of SDSL EPR provided more quantifiable parameters and therefore enhanced

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72 the resolution of structural data obtained from EPR spectral line shapes would start to take place ( 49 ) The interpretation of EPR spectral line shapes and the understanding of the time dependent changes in terms of local structure based on the mobility of the spin label at a given site within the protein are not trivial EPR spec tral line shapes are inherently complex since they report on all three modes of motion simultaneously Furthermore, the choice of spin label employed also has an effect on EPR spectral line shapes given that i reports on the tortional oscillations about the bond that connects the label to the protein This fact only further complicates analysis of the line shapes since there is a depend ence on the structure of the spin label itself. Most SDSL EPR studies em ploy the use of the MTSL spin label; however, in the studies reported within the scope of this dissertation, MSL and IAP spin labels were also used and differences in EPR spectral line shapes based on differences in the spin label had to be taken into acco unt. B oth qualitative and quant it ative information are attained from EPR spectral line shapes utilizing both experimental and theoretical data analysis ( 49 ) Qualitatively, the intensity and breadth of the EPR spectral line shape provide information about the environment in which the spin label is located. When the line shape has sharp narrow peaks where in intensities of each of th e peaks are relatively equal, then the label is experiencing nearly completely isotropic motion. As the spin label is located into sites within the protein where it is more motionally constrained it will experience more anisotropic motion, and the line sh ape will be broad and the intensities of the peaks will no longer be equivalent. Clearly, information can be obtained by monitoring the EPR spectral line shapes, it has been shown that investigating these features using semi

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73 empirical parameters can provi de more detailed information than qualitative methods alone ( 46, 49, 105 ) Often, semi empirical parameters report on the mobility of the spin label. The inverse line width of the central resonance peak ( H 0 1 ) and the inverse of the spectral second moment ( 1 ), which is a representation of the EPR spectral breadth, are two such semi empirical parameters (depictions of each parameter are shown in Figure 3 1) ( 46 106 ) are primarily determined by the amount of averaging of the anisotropic hyperfine A tensors as well as the averaging of the anisotropic g tensors As such 1 and H 0 1 are affected by both the rate and order of the spin label motion and are useful means to quantify the mobility of nitroxide spin label moieties ( 46 ) In order to determine more quantitative information, a more theoretical approach has been employed. EPR s pectral line shape simulation software has been developed in order to fit experimental data and extract quantitative spectral parameters. Various models have been employed based on the motional regime of the spin label. When the rotational correlation ti me of the spin labeled molecule is in the fast motionally narrowed regime, Lorentzian line widths of the EPR spectrum can be determined from Redfield theory ( 98 ) Analyses of spectra in the slow motional regime (10 9 s t R 10 6 s) are more difficult to simulate and require that the full stochastic Liouville equation be employed. Typically a model, such as microscopic order macroscopic disorder (MOMD), or slowly relaxing local structure (SRLS) are used in simulations of spectra in the slow motional regime ( 99 ) The simulations from these models pro vide information on the motion of the spin label in terms of two parameters: an order parameter (S), which is

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74 related to the amplitude of motion, and an overall rotational correlation time ( ), which is related to the rate of movement ( 100 ) MOMD simulations can establish whether the experimental EPR spectral line shape is undergoing isotropic or anisotropic motion, and can determine whether the line shape is comprised of multiple components. A line shape that is comprised of multiple components may indicate that there are either various protein conformational states that are in slow exchange on the EPR timescale or various conformations of the spin label itself. Although major advancements have been made in experimental and t heoretical data analysis of X band spectral line shapes, obtaining site specific information from the spin labeled protein can still present a challenge. For small proteins, which tumble quickly in solution, the overall rotational correlation time will do minate the EPR spectrum and mask any site specific information that is being reported from B and i For these proteins, it is often necessary to reduce the contribution of the overall correlation time so that the motion of the spin label relative to the protein backbone can be revealed in the EPR line shape. To accomplish this, it has become common for viscosgens, such as sucrose, glycerol, ficol, or PEG, to be added to the solution, which in turn slows the overall correlation time of the protein ( 46, 107 112 ) This chapter reports experimental data analysis of IA 3 by the semi empirical parameters, 1 and H 0 1 Also provided are data on the optimization of slowing the rotational correlation time of IA 3 in solution with different techniques. Because SDSL EPR has not been extensively used to investigate IDPs it was important to optimize experimental param eters and data analysis methods in order to extract useful

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75 information from the EPR line shapes on the chemically induced conformational changes occurring in the presence of TFE. Materials and Methods Materials 3 (2 Iodoacetamido) PROXYL (IAP) spin label was purchased from Sigma Aldrich (St. Louis, MO). His Bind resin was purchased from Novagen (Gibbstown, NJ). The QuikChange site directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). BL21(DE3) cells were purchased from Invitrogen (Carlsba d, CA). Unless otherwise stated, all other reagents were purchased from Fisher Scientific (Pittsburg, PA) and used as received. Protein Expression and Purification of IA 3 mutants E. Coli codon optimized DNA encoding for the IA 3 gene was purchased from DN A2.0. The gene was cloned into the pET 22b vector (Novagen). Cysteine substitutions for spin labeling were introduced using the QuikChange site directed mutagenesis kit, and the sequence was confirmed by DNA seq uencing. C ysteine variants were expressed via an E. Coli system modified from the original protocol ( 113 ) The bacterial host strain for expression was BL21(DE3) cells. Cells were grown at 37 C in LB medium to an OD 600 of ~0.6 before induction with 1 mM IPTG. Aft er approximately two hours of expression, the cells were pelleted, resuspended, and lysed by sonication and three passes through a 35 mL French pressure cell (Thermo Scientific, Waltham, MA). After lysis, the supernatant was boiled for 5 minutes to precip itate proteins and assist in purification and the supernatant was centrifuged to remove insoluble material (18500 g, 20 minutes, 4 C). The soluble recombinant protein was purified utilizing a C terminal His tag encoded by the pET 22b vector by affinity

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76 ch romatography as described previously ( 113 ) Protein was estimated to be pure by a 16.5% Tris Tricine SDS PAGE gel. Protein gel images of purified protein are shown in Figure 4 2. 1 mM EDTA was added to purified IA 3 to rem ove any residual nickel that may have been leeched off the affinity column Spin Labeling Proteins containing the cysteine substitutions were buffer exchanged into 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.4 using a HiPrep 26/10 desalting colu mn (Amersham, Pittsburg, PA). 100:1 molar excess of dithiothreitol (DTT) was using a desalting column (as described above) equilibrated and eluted with 50 mM sodium phos phate, 300 mM sodium chloride, pH 7.4 for both IAP or MSL, or pH 6.9 for MTSL. Protei n was then spin labeled with molar excess of IAP, MSL, or MTSL dissolved in ethanol, in the dark at room temp erature (22 C) for 4 hours. Excess spin label was removed via the desalting procedure described above. Protein was eluted in 50 mM sodium phosphate, 3 00 mM sodium chloride, pH 7.4. EPR Sample Preparation Samples were prepared by adding 0% to 40% TFE (v/v) in 5% increments to approximately 100 M spin labeled protein. Samples were prepared with IA 3 either free in solution or bound to His Bind resin charged with 0.1 mM nickel sulfate and equilibrated in 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.4 buffer containing 0% to 40% TFE. Exces s protein was rinsed away from the resin with the aforementioned buffer. Spin labeled IA 3 samples were also prepared in solutions containing 30% sucrose.

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77 Continuous Wave (CW) X Band EPR Spectra CW X band EPR spectra were collected on a modified Bruker ER2 00 spectrometer with an ER023 M signal channel, an ER032 M field control unit, and a loop gap resonator (Medical Advances, Milwaukee, WI). Samples of ~10 L were loaded into 0.60 i.d. X 0.84 o.d. capillary tubes (Fiber Optic Center, New Bedford, MA). All CW EPR experiments were performed at 27 C unless otherwise stated as some spectra were collected at 5 C. All spectra were collected as 100 Gauss (G) sca ns with 2 mW incident microwave power. The 100 kHz field modulation amplitude and time constant of the detector were optimized to provide maximum signal to noise ratio with no line broadening. All spectra are reported as the average of 10 scans. Spectra are plotted with intensities scaled to normalized absorption area. LabVIEW software was used for baseline correction and double integral area normalization, which was generously provided by Drs. Christian Altenbach and Wayne Hubbell ( University of Califor nia, Los Angeles ). Results and Discussion Qualitative Assessment of the EPR Spectral Line Shapes CW X ban d EPR spectra were collected for IA 3 samples labeled at sites S14C and N58C with IAP spin label in concentrations of 0% and 30% TFE Spectra were coll ected for protein in a buffer solution at both 27 C and 5 C and in a 30% sucrose solution at 27 C. The EPR spectral line shapes are shown in Figure 3 2. As expected, the EPR spectral line shapes collected for both S14C IAP and N58C IAP in 0% TFE buffer solution at 27 C have very sharp narrow peaks, indicative of isotropic motion. In the 30% TFE buffer solution at 27 variants were reduced and the line shapes were slightly broadened in comparison t o the

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78 0% TFE samples, which indicates some sort of environmental change had occurred at the labeled site. It was well know n from literature that IA 3 adopts an helical conformation at TFE percentages above 23%, so the change in the line shape is likely reporting on the conformational change from an unstructured state to an helical state. Although there were differences in the line shapes collected at 0% and 30% TFE, the 30% TFE data does not resemble a common line shape of a spin label located within an helical segment of a structured protein. This is likely due to the small dynamic nature of IA 3 in solution, where the spectral line shapes are dominated by the fast overall correlation time of the entire protein. In an attempt to slow the overall correlation time of the protein, data were collected in 30% sucrose solutions at 27 C, and also in buffer solution at 5 C and the data are shown in the lower po rtion of Figure 3 2. EPR spectra were collected for both variants in 0% and 30% TFE solutions with the addition of 30% sucrose (w/v) at 27 C. The peak intensities of the 0% TFE line shapes for both variants in the presence of sucrose were reduced in com parison to the spectra collected in buffer solution alone. This is a direct result from the viscous sucrose which prevents the protein from tumbling as quickly in solution ( 46 ) Despite the decrease in the spe ctral intensity, it is postulated that the line shape is still reporting on the unstructured state of the protein at both sites and is indicative of isotropic motion. The peak intensities of the spectral line shapes collected in the 30% TFE buffer solutio n with sucrose for both sites also decreased and the line shapes were further broadened. Once again, the addition of sucrose slowed the overall correlation time. The additional decrease in peak intensities and the broadening of the line shape is attribut ed to the conformational change that occurred to

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79 an helical state. The sucrose did appear to slow the tumbling of the entire protein; however, the line shapes collected in 30% TFE with sucrose still do not appear to have the shape of data collected at helical sites within structured proteins. This could indicate that the overall correlation time is still having a dominating effect on the spectral line shape. In another effort to slow the overall correlation time of the protein, data was collected for both variants in buffer solution at 5 C, and is shown in the bottom panel of Figure 3 2. For both the 0% and 30% TFE spectra at both sites, the line shapes have decreased peak intensities and are broader than the data collected at 27 C. Similar to the sucrose data, the overall correlation time is reduced when the data is collected at 5 C. The 0% TFE data still show relatively sharp, narrow peaks, which indicate isotropic motion associated with the unstructured state of the protein. The 30% TFE data c ollected at 5 C more clearly reveal line shape changes, in comparison to both the buffer samples and sucrose sample that were collected at 27 C. Data collected at colder temperatures may slow the overall correlation time of the protein where more site s pecific information on the environment of the spin label at a particular site within the protein can be obtained. Semi Empirical Parameters The EPR spectral line shapes, shown in Figure 3 2 were evaluated in terms of the semi empirical parameters, H 0 1 and 1 ,and were compared to similar data reported in literature. The results are presented in Figure 3 3. The plot of the reciprocal of the central line width versus the reciprocal of the second moment is a data analysis tool utilized in studies repo rted in literature which can reveal environmental conditions of the spin label ( 46, 105 ) The grey scaled data take from literature shown in Figure 3 3,

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80 illustrate how values of H 0 1 versus 1 for particular sites within proteins fall into one of the following categories: buried, contact, loop/contact, loop/surface, or unstructured. The H 0 1 and 1 values obtained for the IA 3 spectral line shapes are plotted along with the data obtained from literature in Figure 3 3. It is apparent that the IA 3 data points lay outside of the range of values reported in literature for both the unstructured data collected at 0% TFE and the helical data collected at 30% TFE. Even the da ta collected in the presence of sucrose or at 5 C do not fall within the appropriate categories. From Figure 3 3, it can be concluded that small, dynamic, IDP systems, such as IA 3 cannot simply be analyzed in the same terms as larger structured protein systems and requires further data analysis to obtain the desired site specific environmental conditions at the labeled site. As will be discussed extensively in chapters 4 and 5, alternative data analyses have been investigated to obtain information on IA 3 in the presence of TFE. These analyses provide additional information than the use of the semi empirical parameters. Addition Method to Slow the Overall Correlation Time An alternative way to slow the global protein tu mbling is to tether the spin labeled molecule to a much larger molecule. A noted example from literature is a study on a small 7.6 kDa RNA duplex, the RNA duplex was tethered a large 60 kDa avidin molecule via a biotin moiety present at the 5 terminus of RNA strand ( 80 ) The study revealed that tethering the RNA duplex to an avidin molecule slowed the overall correlation time allowing site specific features to be observed without perturbing the labeling site by adding viscogens.

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81 A similar experiment was performed in attempt to slow the overall correlation time of IA 3 in solution at room temperature without adding a viscogen like sucrose. Utilizing the 6x His tag already lo cated at the C terminal end of the protein, IA 3 was bound to loose Ni IDA resin, as illustrated in Figure 3 4. Spectra were collected for both the S14C IAP and N58C IAP variant of IA 3 bound to Ni IDA resin and compared to data collected for protein free i n solution (unbound) and are shown in Figure 3 5. For both variants, collected in increasing TFE concentrations, the unbound and bound data show peak intensity decreases and the breadth of the line shape increases, which was expected. It is apparent t hat the line shapes of the bound IA 3 data are different from the unbound data, which was anticipated because tethering the protein was expected to reduce the overall correlation time. Despite the fact that the tethering alters the global molecular motion from an isotropic tumbling to a tethered cone on the surface, the conformational change can still be inferred from an increased line shape broadening that occurs upon increasing concentration of TFE. Upon further analysis of the bound data, it is clear t hat the spectral line shapes from the C terminal N58C IAP site were dramatically more reduced than the line shapes from the N terminal S14C IAP site. This data indicated that since the protein was tethered at only the C terminal end, the differences in th e environment of the two sites in respect to the Ni IDA resin was causing an additional effect on the EPR spectral line shapes. Therefore, it was concluded that the closer the spin labeled site is to the surface, the more motionally restricted that site b ecomes due to the tethering process, terminal N58C IAP site, the

PAGE 82

82 effects of TFE upon the spectral line shape parameters are reduced which interfered with the spectroscopic monitoring of the conform ational change. Summary EPR spectral line shapes can provide information on protein structure, dynamics, and conformational changes. Both qualitative and quantitative analysis of the line shapes can offer details on such information about the spin labeled protein. Qualitatively, the peak intensities and breadths of the spectral line shapes give some indication on the isotropic or anisotropic movements of the spin label at a particular site within the protein. Based on the data presented here, the semi em pirical parameters, H 0 1 and 1 have been shown to supply more detailed information on the environment of the spin label for structured proteins. However, these parameters do not appear to provide the same detailed information for the small dynamic IDP, IA 3 even when the effect of the overall correlation time of the protein is minimized in the EPR spectral line shape via a viscogen or colder temperatures. Additionally, the reduction of the overall correlation time by tethering the protein to large Ni IDA resin was inv estigated. Although the line shapes from bound data did seem to indicate that there was a conformational change that occurred upon increased TFE concentrations, the altered global molecular motion to a tethered cone on the broa terminal N58C IAP. As expressed within this chapter, experimental and theoretical data can be utilized to extract spectral parameters from the experimentally collected line shapes. The following chapters will show both experimental and th eoretical EPR analyses that provide structural information on the IA 3 and help monitor the chemically induced conformational change.

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83 Figure 3 1. Semi empirical parameters illustrated on IA 3 S14C IAP data. (A) Depiction of the measurement of the centr al line width ( H 0 ), where as mobility decreases H 0 will increase. (B) Depiction of the components related to the second moment () which is related to the breadth of the spectrum.

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84 Figure 3 2. 100G X band EPR spectra for S14C IAP (red) and N58C IAP (blue) in 0% TFE and 30% TFE collected in buffer and 30% sucrose solutions at 27 C, and in buffer at 5 C. Figure 3 3. Plot of the inverse second moment ( 1 ) versus inverse central line width ( H 0 1 ). Grey scaled data was taken from Kim et al ( 105 ) to compar e IA 3 data for the S14C IAP site (red) and N58C IAP (blue)

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85 Figure 3 4. Depiction of the tethering scheme. IA 3 was bound to Ni IDA resin via the C terminal 6x his tag. Figure 3 5. 100G X band EPR spectra for S14C IAP (red) and N58C IAP (blue) in 0%, 15% and 30% TFE collected in buffer (unbound) and tethered to Ni IDA resin (bound).

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86 CHAPTER 4 CHARACTERIZATION OF THE DISORDERED TO ALPHA HELICAL TRANSITION O F IA 3 BY SDSL EPR SPECTROSCOPY I ntrinsically disordered proteins (IDPs) are proteins or pr otein segments (> 50 residues) that lack uniform secondary and tertiary structure under physiological conditions ( 18, 31, 50, 114 117 ) Many proteins from higher eukaryotic systems contain regions of disorder in t heir structure ( 118 ) which often correlate to vital functional roles in biology such as transcriptional and translational regulation, signal transduction, and protein phosphorylation ( 19, 115 ) In other instances, IDPs undergo coupled folding and binding, where conformational changes in segments of the IDP are induced upon binding to their target protein ( 50, 119, 12 0 ) IA 3 is a 68 amino acid protein found in the cytoplasm of Saccharomyces cerevisiae that acts as a potent inhibitor of yeast proteinase A (YPRA). X ray crystallography studies reveal that the first 34 amino acids of IA 3 helical conformation upon binding in the active site of YPRA, whereas the 34 C terminal amino acids have not been resolved in the X ray data as shown in Figure 1 8 ( 53, 113 ) Far UV circular dichroism (CD) studies show that IA 3 is unstructured in solution. In the presence of the secondary structural stabilizer 2,2,2 trifluoroethanol (TFE) a two state transition from an unstructured to helix conformation is induced ( 53 ) The CD analysis however, cannot establish site specific information regarding the extent to which a specific residue within the IA 3 helical structure or whether the TFE induced two state transition was uniform throughout the protein. 2D 15 N 1 H NMR spectroscopy coupled with singular valued decomposition (SVD) analysis shows that the N terminal residues adopt more pronounced helical structure with TFE than the C terminal residues ( 60 )

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87 As extensively discussed in chapter 2, SDSL EPR has become a powerful technique for investigating macromolecular structure, conformational changes, and protein dynamics ( 49, 50, 104, 117, 121 123 ) In this method, an EPR active reporter group, such as a nitroxide spin label, is introduced into the biological system to obtain site specific information about structure and flexibility. Typically, a non native cysteine (CYS) residue is incorporated into a desired location within the protein via site direct ed mutagenesis, which is then chemically modified with a spin label. The EPR spectral line shape generated from the spin label provides valuable structural information ( 49, 50, 104, 117, 121, 122 ) When the overa ll motion of the spin label varies in the 0.150 ns regime, the motion has a dramatic effect on the observed EPR line shape ( 77 ) The following three primary modes of motion contribute to the EPR spectrum: the over all tumbling of the molecule ( R ), the motion of the spin label about the bonds that connect it to the protein ( i ), and the motion of the protein backbone to which the spin label is attached ( B ) The backbone motion and the motion of the spin label abou t the tethering bonds provide the most structural information on biomolecules in SDSL studies ( 80 ) A more thorough description is given in chapter 2, and an illustration is shown in Figure 2 5 Although SDSL EP R has been shown to be a useful technique to study ordered to disordered transitions in numerous proteins ( 105, 123 126 ) it is not widely recognized as a tool to study IDP systems ( 19 ) One notable example found in the literature is the characterization of an induced folding event of the intrinsically disordered C terminal domain of the measles virus nucleoprotein ( 50, 117, 127 ) Detailed information on this system is discussed in chapter 1.

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88 The work presented here extends the applicability of SDSL EPR to study conformational changes in IDPs. Here we use SDSL EPR spectroscopy to characterize t he TFE induced helical conformational change in IA 3 Because of the relative simplicity of this protein and detailed characterization previously performed by CD and NMR spectroscopy, IA 3 serves as an excellent model system for further development of the SDSL EPR methodology for studying IDPs. Two cysteine substitutions were generated in IA 3 ; one, in the N terminus which has been shown by X ray crystallography to undergo an unstructured to helical transition upon binding to YPRA, and one in the C termin us that remained unresolved ( 113 ) in the crystal structure but has been shown by NMR to adopt helical structure in the presence of TFE ( 60 ) The amino acid sequence is given in Figure 4 1 and highlights the positions of the two single cysteine substitutions. Materials and Methods Materials (1 Oxyl 2,2,5,5 tetramethyl 3 pyrroline 3 methyl) methanethiosulfonate spin label (MTSL) was purchased from Toronto Research Chemicals, Inc (North York, ON, Canada). 3 (2 Iodoacetamido) PROXYL (IAP) and 4 Maleimido 2,2,6,6 tetramethyl 1 piperidinyloxy (4 Maleimido TEMPO, MSL) spin labels were purchased from Sigma Aldrich (St. Louis, MO). His Bind resin was purchased from Novagen (Gibbstown, NJ). The QuikChange site directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). BL21(DE3) cells were purchased from Invitrogen (Carlsbad, CA). Unless otherwise stated, all other reagents were purchased from Fisher Scientific (Pittsburg, PA) and used as received.

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89 Protein Expression and Purification of IA 3 mutants E. Coli codon optimized DNA encoding for the IA 3 gene was purchase d from DNA2.0. The gene was cloned into the pET 22b vector (Novagen). Cysteine substitutions for spin labeling were introduced using the QuikChange site directed mutagenesis kit, and the sequence was confirmed by DNA sequencing. Wild type and cysteine v ariants were expressed via an E. Coli system modified from the original protocol ( 113 ) The bacterial host strain for expression was BL21(DE3) cells. Cells were grown at 37 C in LB medium to an OD 600 of ~0.6 before inducti on with 1 mM IPTG. After approximately two hours of expression, the cells were pelleted, resuspended, and lysed by sonication and three passes through a 35 mL French pressure cell (Thermo Scientific, Waltham, MA). After lysis, the supernatant was boiled for 5 minutes to precipitate proteins and assist in purification and the supernatant was centrifuged to remove insoluble material (18500 g, 20 minutes, 4 C). The soluble recombinant protein was purified utilizing a C terminal His tag encoded by the pET 22 b vector by affinity chromatography as described previously ( 113 ) Protein was estimated to be pure by a 16.5% Tris Tricine SDS PAGE gel. Protein gel images of purified protein are shown in Figure 4 2. 1 mM EDTA was added to purified IA 3 to remove any residual nickel that may have been leeched off the affinity column Spin Labeling Proteins containing the cysteine substitutions were buffer exchanged into 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.4 using a HiPr ep 26/10 desalting column (Amersham, Pittsburg, PA). 100:1 molar excess of dithiothreitol (DTT) was using a desalting column (as described above) equilibrated and eluted with 50 mM

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90 sodium phosphate, 300 mM sodium chloride, pH 7.4 for both IAP and MSL, or pH 6.9 for MTSL. Protein was then spin labeled with 10x molar excess of IAP, MSL, or MTSL dissolved in ethanol, in the dark at room temp erature (22 C) for 4 hours. Exc ess spin label was removed via the desalting procedure described above. Protein was eluted in 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.4. Spin labeling efficiency was evaluated by double integration of the EPR signal and comparison to a TEMPO standard curve. The labeling yields of both the S14C and N58C variants were estimated to be approximately 30% for both MTSL and MSL, and 50% for IAP. EPR Sample Preparation Samples were prepared by adding 0% to 40% TFE (v/v) in 5% increments to approximat ely 100 M spin labeled protein. Continuous Wave (CW) X Band EPR Spectra CW X band EPR spectra were collected on a modified Bruker ER200 spectrometer with an ER023 M signal channel, an ER032 M field control unit, and a loop gap resonator (Medical Advances, Milwau kee, WI). Samples of ~10 L were loaded into 0.60 i.d. X 0.84 o.d. capillary tubes (Fiber Optic Center, New Bedford, MA). All CW EPR experiments were performed at 27 C unless otherwise stated as some spectra were collected at 5 C. All spectra were coll ected as 100 Gauss (G) scans with 2 mW incident microwave power. The 100 kHz field modulation amplitude and time constant of the detector were optimized to provide maximum signal to noise ratio with no line broadening. All spectra are reported as the ave rage of 10 scans. Spectra are plotted with intensities scaled to normalized absorption area. LabVIEW software was used for baseline correction and double integral area normalization, which was

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91 generously provided by Drs. Christian Altenbach and Wayne Hubb ell (U niversity of C alifornia, L os A ngeles ). Data Analysis The EPR line shapes were analyzed using various analysis techniques. The first technique used monitors h (+1) / h (0) as a function of TFE percentage. All collected intensity values are located in Ap pendix C. The data can be fit to a 2 state Boltzmann function, and mid points of transition are determined. The Boltzmann function is shown in Equation 4 1, were A 1 is the initial value of the curve, A 2 is the final value of the curve, and the mid point is given by the x o term. (4 1 ) Monitoring the local tumbling volume (V L ) of the spin label is another analysis technique that can be used to investigate conformational changes in biomolecules ( 128 ) It has been shown that this parameter reports on structural information of the protein system with external environment dependent quantities removed ( 128 ) The expression for V L is given by Equation 4 2, (3 2) where k is the Boltzmann constant, T is the absolute temperature, R is the rotational correlation time, and R were determined from line shape simulations using the EWVoight program generously provided by Alex Smirnov (NCSU). S imulation s provide values for the Lorentzian line widths of the three transitions denoted as Lor (+1) Lor (0) and Lor ( 1) for the low field, center field, and high

PAGE 92

92 field line widths; respectively. S ubsequently these values are used to obtain the l ine shape parameters A, B, and C in Equation 4 3, (4 3 ) where M is the nuclear spin quantum number of the M th hyperfine line, T 2 ( M ) is the spin spin relaxation time (which is inversely proportional to the homog eneous line width) of that line In the f ast motional regime, assumptions can be made and B and C alone can be used to determine rotational correlation time ( 98 ) R were determined using Equation 4 4, (4 4 ) where the value of B is obtained from Equation 4 3. The calculated values used to determine V L are provided in Appendix C. Viscosity Measurements Viscosity measurements were performed using Cannon Fenske Viscometer (size 50) suspended in a water bath at 6 C and 27 C. TFE solutions of 0% 40% (v/v) in increments of 5% were made by combining the appropriate amount of TFE, and the pH 7.4 buffer previously mentioned. Measurements were repeated four times to ensure reproducibility. The values collected are expressed as kinematic viscosity in units of centistokes (cS) and needed to be converted to 1 1 ) utilizing the densities of each of the solutions. All pertinent values fo r determining the viscosities used in this study are located in Appendix C.

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93 Results Effects of Spin Label Moiety on Monitoring the Helical Conformational Change The following three different spin labels were used in this study: methanethiosulfonate (MTSL) 4 maleimido TEMPO (MSL), and 3 (2 iodoacetamido) Proxyl (IAP). The structures of each label and the resultant modified C YS residue are given in Figure 4 3. The effects of the CYS substitution and incorporation of the spin label on the secondary structu re of IA 3 were investigated with far UV CD. Spectra were collected for 0% and 30% TFE for wild type (WT) IA 3 and for each CYS variant labeled with MTSL, MSL, or IAP and are shown in Figures 4 4 through 4 7 CD spectra of the modified IA 3 constructs are nearly identical to the results obtained for the WT protein. The structure of the modified IA 3 constructs are determined to be predominantly random coil in solution, indicated by a negative band near 200 nm and some weak bands between 220 nm and 230 nm. F or 30% TFE, the conformation of the modified proteins becomes helical, as indicated by double minima at 222 nm and approximately 210 nm and a maximum at 190 nm ( 53 ) The CD spectra of the spin labeled constructs indicate that the amino acid substitution and spin labeling do not alter the disorder to helical transition in IA 3 upon induction by TFE Continuous wave (CW) X band EPR spectra were collected for IA 3 samples labeled at sites S14C and N58C with MTSL, MS L, or IAP in increasing concentrations of TFE that ranged from 0% to 40% in 5% increments. Figures 4 8 through 4 10 show stack plots of double integral (area) normalized 100 G X band EPR spectra for samples

PAGE 94

94 Two general conclusions can be drawn from the EPR spectra line shapes First, the spectral line shapes for the three spin labels follow the expected trend of mobility given their individual structures and tethering geometries. Mobility of the spin label is defined as both the rate of motion and the restricted/unrestricted conformations, which it samples, often described as order of the spin label ( 104, 121 ) The spectra are plotted with normalized areas; hence, the overall intensity of the signal is proporti onal to the mobility. The higher the intensity, the more motional averaging the spin probe undergoes. As mobility decreases, the normalized intensity will decrease and the spectra will have an overall broadening. For both sites investigated, the line sh apes have the following expected mobility trend: IAP > MTSL > MSL. A second general observation is that upon addition of TFE, the overall intensity of all of the spectra decrease and broaden; indicative of a conformational change. To ensure the reduced m obility seen in the spectral line shapes did not arise from changes in solution viscosity, control experiments shown in Figure 4 11 and 4 12, were performed for IA 3 in solutions of 6% sucrose, which is iso viscous to the 30% TFE at 27 C. Negligible chan ges were observed in comparison of the 0% TFE EPR spectra to those obtained with 6% sucrose; thus, indicating that the changes seen upon addition of TFE in the X band spectra arise from conformational changes of the protein backbone, which lowers the overa ll mobility of the spin label. Quantitative Analysis of the TFE Induced Change Observed via SDSL The previous section provides a qualitative approach in discussing the spectral line shapes. However, a more quantitative approach can be taken by analyzing va rious EPR spectral line shape parameters. Figure 4 13 shows a representative nitroxide EPR spectrum. The three observed transitions arise from hyperfine coupling of the electron

PAGE 95

95 with the magnetic moment of the nitrogen nucleus ( 67 ) As shown, the low field transition is designated as h (+1) the center field transition is designated as h (0) and the high field transition is designat ed as h ( 1) When spectra are plotted with normalized area, the intensities of the three transitions provide information about the spin label mobility ( 50 ) Although the high field transition of the X band CW EPR line shape is often correlated to the most sensitive molecular motio n ( 129 ) values of the h (+1) / h (0) ratio were utilized i n a previous SDSL study of a highly dynamic protein S pecifically, values of this parameter were used to monitor conformational changes in the measles virus nucleoprotein upon binding to its target ( 50 ) We also utilized this parameter, Figure 4 14 plots values of h (+1) / h (0) as a function of %TFE for IA 3 sites S14C and N58C labeled with MTSL, MSL, at 27 C and with IAP at 27 C and 5 C. All of the data sets have a sigmoid al shape that could readily be fit to the 2 state Boltzmann function (Eq 4 1), where the results from the best fits are given with solid lines through the data points. From fitting the data to Eq. 4 1, values of the %TFE transition mid points were determined to be 17 2%, 20 1%, 20% 1%, 14% 1%, 19% 1%, and 18% 2% for S14C MTSL, S14C MSL, S14C IAP, N58C MTSL, N58C MSL, and N58C IAP; respectively. The average mid point value of 18 1% determined from the EPR data collected at 27 C, agrees well with the value of 18.3% obtained from previously reported NMR data analysis ( 60 ) The effects of temperature were also investigated. Spectra were collected at 5 C for S14C IAP and N58C IAP, where the global tumbling of the protein should be reduced Data for samples collected at 5 C are shown in the bottom panels of Figure 4

PAGE 96

96 14 (only IAP labels were collected at the colder temperatures). The magnitude of the change in the values of this parameter are greater at the lower temperature, reflecting a helical character, which is consistent with degree of helicity seen in CD spectra of IA 3 with TFE at lower temperatures ( 61 ) As stated above the solid lines represent the best fits with Eq. 4 1, and the mid points were found to be 12% 2% and 9% 2% for S14C IAP and N58C IAP; respectively, which are lower than the values obtained at 27 C. These r esults agree well with data obtained from previously reported CD analysis, which showed the transition occurs at a lower TFE percentage at colder temperatures, and those thermodynamic parameters predict a mid point %TFE of 12% at 5 C ( 61 ) An additional way to analyze the EPR line shapes is by analyzing how the local tumbling volume, V L of the spin label changes upon addition of TFE. Plots of V L as a func tion of %TFE for both S14C and N58C labeled with IAP at both 27 C and 5 C are shown in Figure 4 15 In the unstructured state, the local tumbling volume is expected helical character is induced, the Stokes Einstein radius of a rigid helix should increase, thus increasing the local tumbling volume of the spin label. For both S14C and N58C at 27 C, as expected, V L is seen to increase linearly upo n addition of TFE to percentages below 25%. Above 25%, the slope flattens, indicating a change in the dependence of V L with %TFE. For the data collected at 5 C, a transition where the slopes of the linear regions differ can also be observed. The greater negative slope for 25% to 40% TFE at 5 C is believed to arise from inaccuracy of the simulation method for these more anisotropic line shapes (discussed below) and errors in viscosity measurements arising from salt precipitation at the lower temperature t hat occur for

PAGE 97

97 high %TFE. Note, no protein precipitation was observed in the EPR samples, but salt precipitation was observed over time in viscosity measurements. The intersection points for each site are found to be roughly 20% and 24% TFE for 27 C and 5 C, respectively. These points of intersection represent the concentration of TFE for where the conformational change has completed and the points agree well with previous results from CD and NMR analysis. An interesting observation is that at both temp eratures, the magnitude of the change of V L for site S14C IAP is greater than that of site N58C IAP, indicating that the N terminal site is undergoing a larger induced conformational change than the N58C site. This finding is also in agreement with previou sly published NMR data ( 60 ) A third line shape parameter used to monitor the TFE induced conformational change of IA 3 is the percentage change in the normalized intensity of the high field transition, h ( 1) as a function of % TFE. Figure 4 16 plot s the absolute value of this parameter upon increasing % TFE determined from spectra of both S14C and N58C IA 3 in solution for the various spin labels used here. Analyzing the data in this fashion provides a similar trend as the results obtained from the local volume a nalysis given in Figure 4 15 For each sample, the dependence of the value of the percentage change in the normalized intensity of the high field transition, h ( 1) on %TFE can easily be seen to fall into two linear regions described as foll ows: for TFE < 20%, the value of this parameter is highly dependent upon %TFE, and for TFE > 20%, this line shape parameter is nearly independent of TFE concentration. Again, the significance of 20% TFE is indicative of the concentration for which the co nformational change is complete. At concentrations TFE > 20%, the values of h ( 1) do not significantly change upon further

PAGE 98

98 increases in TFE concentration, indicating that the spin label mobility and, hence, protein structure, remain constant above this con centration. For concentrations < 20% TFE, the EPR spectra broaden progressively as TFE is added, which is consistent with an increase in the relative percentage of helical conformation that would change the relative mobility of the spin label in a dose d ependent manner. The value of 20% TFE identified from this analysis is again consistent with prior CD analyses of IA 3 which found that complete helical structure is obtained at 23% TFE ( 53 ) In addition, as note d above, the magnitude of the overall change in this line shape parameter, regardless of spin label used, is less for the C terminal site than for the N terminal site, reflecting a greater degree of conformational change in the N terminus of IA 3 Discussi on Effects of Spin Label Choice on Data Analysis In an effort to discern if various spin label structures report more readily on the induced unstructured to helical conformational change, three separate spin labeled samples were prepared for each site in IA 3 MTSL (Fig. 4 3a ) modifies cysteine sites by forming a disulfide bond which is easily reduced in the presence of other reactive thiol groups or reduci ng agents, which may be a drawback of utilizing this spin label in highly dynamic unstructured proteins, especially if the protein under investigation is not completely labeled. The MTSL probe when attached to a protein, is often considered to have restri cted mobility around only two rotatable bonds described as the 4/ 5 model ( 113 ) and may report most effectively on backbone conformational changes. MSL (Fig. 4 3b ) forms a non reducible thioet h er bond with the cysteine side chain and is considered to be rigid with limited rotations ar ising from the bulkiness of the 4

PAGE 99

99 maleimido TEMPO group. IAP (Fig. 4 3c ) a lso forms a non reducible thioe t h er bond but has an extended and flexible linker region in comparison to the other two labels; thus, having the greatest degree of motionally averaged line shape. Line shape analyses shown here reveal that all three spin labels readily report on the global conformational change of IA 3 upon undergoing an unstructured to helical conformational change induced with TFE. However, we did find that IAP was due to difficulties in labeling efficiencies with MTSL due to the high propensity of this peptide to disulfide bond with itself during protein purification and subsequent labeling. The unrestricted mobility of the IAP lab el was quite useful for analysis of changes in local tumbling volume and reporting on the global conformational change from an unstructured random coil to a more rigid helical rod that had an altered Stokes Einstein radius and slower global tumbling correl ation time. Data A nalysis via h (+1) /h (0) In a previous SDSL study of a highly dynamic IDP, the h (+1) / h (0) parameter was used to monitor conformational changes occurring in the measles virus nucleoprotein ( 50, 117 ) Here, we also utilized this parameter to characterize the conformational changes in IA 3 upon the addition of TFE. Our results show that both the N and C termini of IA 3 are undergoing a conformational change as the percentage of TFE increases. Control experiments in both urea and various concentrations of sucrose indicate that the changes in the spectral line shapes seen upon addition of TFE do not simply arise from changes in solution viscosity, but are indeed indicative of the induced conformational c hange in the protein as illustrated in Figures 4 11 and 4 12 Regardless of the choice of spin label used, all plots of h (+1) / h (0) give a sigmoid al shape

PAGE 100

100 that could be readily fit to a 2 state Boltzmann function Ganesh et al. observed by SVD analysis of their NMR data that the overall effect of TFE on the coil to helix transition of IA 3 can be evaluated as a two components of the TFE dependence of both the 1 H N and 15 N chemical shifts exhibit sigmoidal trends with increasing amounts of TFE. These movements are strongly indicative of a two state transition, where one state is progressively depopulated in favor of a second state as the TFE concentration rises ( 60 ) The h (+1) / h (0) data in this study al so seem to be indicative of a conformational change from the unstructured state to the helical conformation. The h (+1) / h (0) method gave mid point values that were in agreement with those previously reported in a detailed NMR study, where NMR coupled w ith singular value decomposition was used to determine the degree of helical conformation and the mid point of the conformational transition for IA 3 ( 60 ) The h (+1) / h (0) analysis provides information regarding the global conformational change from a hig hly dynamic unstructured peptide segment to a more structured rigid protein that has changes in both local spin label correlation times and global protein tumbling, R where R is likely dominating the motional averaging of the nitroxide hyperfine tensor. Comparison of V L to the P ercent C hange of h ( 1) An additional parameter that we used to characterize the unstructured to helical conformation of IA 3 was the lo cal tumbling volume parameter, V L introduced by Freed and co workers. Previously, this line shape parameter was also extensively utilized by Millhauser and co workers to characterize cold temperature induced helical conformational changes in short alani ne synthetic peptides, where as the temperature was decreased a more pronounced helix was formed ( 128 ) Values for V L represent the

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101 volume of the spin labeled protein as the nitroxide reorients on the time scale of R ( 128 ) The V L parameter provid es structural information without the affect of external environmental factors such as viscosity and temperature, therefore providing information solely on the change in protein structure ( 128 ) Values of V L for IA 3 increase as a function of %TFE indicating a conformational change from d isordered to ordered system. When IA 3 is unstructured the nitroxide s ability to reorient back to its original starting position is fast and therefore gives a smaller value of V L helical structure is induced upon addition of TFE, the amount of tim e it takes for the nitroxide to reorient is slowed and results in a larger tumbling volume. The data shown in Figure 4 15 indicate that V L is providing site specific information, where the volume in the S14C site undergoes a much greater change than the v olume in the N58C site. V L does not report on the overall global change like the h (+1) / h (0) parameter, and therefore plots as a function of %TFE do not have a sigmoid al shape, but instead change linearly until the conformational change is completed. One difficulty we faced, however, in utilizing V L is the need to determine the rotational correlation time, which may not always be straightforward. As shown, the EPR data collected at 5 C have significant broadening of the high field transition upon additi on of TFE, indicating more anisotropic motion become concurrent wi th a correlation time that can no longer be accurately modeled by the simpler isotropic motion limit and likely would more accurately be described by simulations in the intermediate motion r egime ( 100, 121 ) This fact may contribute to the negative slopes of V L in Figure 4 15 Even the data obtained at higher temperatures have line shapes indicative of slower motion, outside the regime of the simple isotropic model used here.

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102 Another parameter we found to report on site specific changes in conformation in IA 3 is the percentage change of the intensity of the h ( 1) resonance. Both h ( 1) and V L parameter profiles give similar results, where two distinct regions are identified (Fig ures 4 15 & 4 16) and the point where the transition was complete can readily be determined from the intersection of the slopes of the two regions. Both parameters provided information on the degree of change in each of the spin labeled sites, and indicate that the N terminal site is undergoing a larger conformational change than the C terminal site. This finding is in agreement with the NMR data previously reported ( 60 ) The percent change in h ( 1) has not been previously us ed as a parameter to gain structural information for IDPs. This study shows that the h ( 1) parameter provides similar information as does calculating V L and does not require line shape simulations. All three parameters used in this study provide valuabl e information on the induced conformational change occurring as a function of TFE. From this study it appears that for small dynamic IDP systems that data analysis parameters that mainly rely on changes occurring in the high field transition seem to be re porting on site specific conformational changes, whereas parameters that rely on changes in the low and center field transitions provide information on the global conformational change. The local tumbling volume and the percent change of the h ( 1) paramete rs appear to yield the TFE percentage needed to compl ete the conformational change. These parameters also indicate that the N terminus is undergoing a larger conformation al change than the C terminus. The h (+1) / h (0) parameter appears to be reporting on t he global conformational change, where it provides information on the overall global unstructured to helical transition that both the N and C terminal regions are

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103 experiencing. Although the h (+1) / h (0) parameter has been shown to be site specific in a s tudy on the C terminal domain of the measles virus nucleoprotein ( 50, 117, 127 ) it does not appear to report the same information on IA 3 which is significantly smaller, therefore the EPR spectral lin e shapes are b eing dominated by R However, this parameter is still useful in investigating small IDP systems as i t provides the transition mid point of the conformational change Summary SDSL EPR spectroscopy was used to monitor the disordered to helical transition of IA 3 Three diffe rent nitroxide probes were used, and it was shown that due to the tendency of MTSL to dissociate from the protein, it is not the best spin label choice for studying highly dynamic IDP systems. Although qualitative information can be obtained by studying th e EPR line shapes, more information about the transition comes from analysis of various line shape parameters. To gain more quantitative information from the resultant line shapes, three techniques were employed. The percent change in the h ( 1) intensity and values of V L provide similar information, indicating at what point the conformational transition are complete and provide site specific information which indicate that N58C, in the C terminus, does not undergo as large of a change as does site S14C in the N terminus. The other parameter employed was analysis of h (+1) / h (0) which has previously been used to monitor an induced conformational change in an IDP system ( 50 ) Here, we find upon titration of TFE, this parameter gave a sigmoidal trend that could readily be fit with a 2 s tate Boltzmann function providing information about the mid point of the conformational change that agrees well with previous NMR and CD investigations ( 53, 60, 61 ) The results from this model system show that th e SDSL EPR data can be interpreted in either global or site specific conformational changes and

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104 imply that SDSL EPR methods should be applicable to other IDP systems. EPR methods may provide an advantage over NMR methods given that the SDSL EPR has no uppe r size limitations and the h (+1) / h (0) parameter, as well as the percentage change in the h ( 1) line shape analyses are straightforward.

PAGE 105

105 Figure 4 1. Sequence of IA 3 The bold and underlined residues represent the sites substituted with cysteine (S14 C and N58C) that were then spin labeled for EPR investigations.

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106 Figure 4 2. Protein gel of S14C (A) and N58C (B) purification. Lane 1 is the polypeptide SDS standard. Lane 2 is the total cell lysate. Lane 3 is the S/N from the first centrifugation Lane 4 is the pellet from the first centrifugation. Lane 5 is the S/N from the second centrifugation after boiling. Lane 6 is the pellet from the second centrifugation after boiling. Lane 7 is the flow through of the pH 8.0 wash. Lane 8 is the flow th rough of the pH 6.3 wash. Lane 9 is protein labeled with IAP. Lane 10 is protein labeled with MSL. Lane 11 is protein labeled with MTSL.

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107 Figure 4 3 Structures of spin labels used in this study and the resulting chemical modification of the cysteine side chain. (A) MTSL, (1 oxyl 2,2,5,5 tetramethyl D3 pyrroline 3 methyl) methanethiosulfonate; (B) MSL, 4 maleimido TEMPO; (C) IAP, 3 (2 iodoacetamido) Proxyl.

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108 Figure 4 4. CD spectra of wild type IA 3 and IA 3 variant S14C collected at 27 C CD spect ra were recorded as a function of TFE percentage from 0% 40% TFE for both sites labeled with each spin label MTSL (red), MSL ( green ), IAP (blue) and compared to Wild type (black).

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109 Figure 4 5. CD spectra of wild type IA 3 and IA 3 variant S14C collected at 5 C CD spectra were recorded as a function of TFE percentage from 0% 40% TFE for both sites labeled with each spin label MTSL (red), MSL ( green ), IAP (blue) and compared to Wild type (black).

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110 Figure 4 6. CD spectra of wild type IA 3 and IA 3 vari ant N58C collected at 27 C CD spectra were recorded as a function of TFE percentage from 0% 40% TFE for both sites labeled with each spin label MTSL (red), MSL ( green ), IAP (blue) and compared to Wild type (black).

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111 Figure 4 7. CD spectra of wild t ype IA 3 and IA 3 variant N58C collected at 5 C CD spectra were recorded as a function of TFE percentage from 0% 40% TFE for both sites labeled with each spin label MTSL (red), MSL ( green ), IAP (blue) and compared to Wild type (black).

PAGE 112

112 Figure 4 8. A rea normalized 100G X band EPR spectra of IA 3 variant S14C labeled with MTSL, MSL, and IAP in increasing TFE concentration. Spectra were collected at 27 C.

PAGE 113

113 Figure 4 9. Area normalized 100G X band EPR spectra of IA 3 variant N58C labeled with MTSL, MS L, and IAP in increasing TFE concentration. Spectra were collected at 27 C.

PAGE 114

114 Figure 4 10. Area normalized 100G X band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in increasing TFE concentration. Spectra were collected at 5 C.

PAGE 115

115 Fig ure 4 11. Area normalized 100G X band EPR spectra of IA 3 variants S14C and N58C labeled with MTSL, MSL, and IAP in 0% TFE and 8M urea. Spectra were collected at 27 C.

PAGE 116

116 Figure 4 12. Area normalized 100G X band EPR spectra of IA 3 variants S14C and N58 C labeled with MTSL, MSL, and IAP in 0% TFE 30% TFE, and 6% Sucrose Spectra were collected at 27 C.

PAGE 117

117 Figure 4 13. 100G X band CW EPR spectrum of IA 3 S14C IAP with labeled transitions indicating the peak to peak intensities of the low field, h (+1) center field, h (0) and high field, h ( 1) resonances.

PAGE 118

118 Figure 4 14. Plots of h (+1) / h (0) as a function of TFE for S14C ( red, left) and N58C ( blue, C C. The sizes of the data points are larger than errors in the measurements.

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119 Figure 4 15. The local tumbling volume ( V L ) of the protein as a function of increasing percentage TFE for S14C IAP ( red, left) and N58C IAP ( blue, right) collected at 27 C ( ) and 5 C ( ). Lines represent linear regression fits to the data. The sizes of the data points are larger than errors in the measurements.

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120 Figure 4 16. The absolute value of the percent change of the intensity of the high field resonance, h ( 1) a s a function of increasing percentage TFE for S14C ( red, left) and N58C ( blue, right) labeled with IAP collected at 27 C and 5 C The percent change was calculated by subtracting the h ( 1) intensity for spectra collected in the presence of TFE f rom the intensity of the spectrum in the absence of TFE ( I 0 ), dividing that quantity by I 0 and multiplying by 100%. The sizes of the data points are larger than errors in the measurements.

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121 CHAPTER 5 MULTI FREQUENCY EPR ANALYS IS OF THE C TERMINUS OF IA 3 As discussed in more detail in the preceding chapters, IA 3 is a 68 residue IDP that undergoes a disorder ed to helical conformational change in the presence of its target protein, YPRA, or in the presence of the secondary structural stabilizer, TFE. Because this structural transition has been well characterized by X ray crystallograp hy, CD, and NMR analysis, the chemically induced conformation al change of IA 3 by TFE serves as an excellent model system to investigate by EPR spectroscopy. Previous X band SDSL EPR studies of the N terminal position S14C and the C terminal position N58C of IA 3 revealed both global and possibl e site specific conformational change s occur in the presence of TFE which were determined from various line shape analysis A sigmoidal trend in the ratios of h (+1) / h (0) as a function of TFE percentage for both sites was observed. This behavi or is characteristic of a two state transition from the unstructured state to the helical state. The data were fit with a 2 state Boltzmann function, providing information about the mid point of the conformational change These X band EPR r esults analy sis agree wel l with previous ly published NMR and CD investigations ( 53, 60 ) The data however, appear to only provide information on global conformational change s occurring in the presence of TFE and distinctions between the two sites could not be clearly determined ( 53, 60, 61 ) Monitoring two other line shape parameters, the percent change in the h ( 1) intensity and V L as a function of TFE revealed the percentage of TF E required to fully i nduce the helical transition for S14C and N58C differed This suggests the C terminal N58C residue, undergoes less of a structural change than N terminal S14C residue Analysis of V L also appeared to provide site specific informati on of the conformational change. Previously reported

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122 studies indicated that the N terminus is more helical, than the C terminus, which is in agreement with our V L results ( 53, 60, 61 ) The previously described EPR data analysis provided an excellent basis for further investigation and collection of site specific information on addition al residues within the IA 3 sequence. X band EPR studies w ere conducted on five sequential residues, Y57C, N58C, K59C, L60C, and K61C, located in the C t erminal region of the protein. Changes in V L as a function of TFE percentage w ere monitored to investigate the residue specific conformational changes induced by increasing amounts of TFE. Additionally, high frequency W band spec tra were collected on all the C terminal residues mentioned above at 0%, 15%, and 30% TFE A multi frequency fitting of both X and W band EPR line shapes collected at 0%, 15%, and 30% TFE were performed to more accurately determine the overall rotational correlation time at each residue and extract EPR spectral parameters As discussed in chapter 2, multi frequency EPR allows for data collection of spectral line shapes that are sensitive to different time scales; therefore providing a means to de convolut e the three main motional contributions of the spin label. H igh frequency EPR (above 95 GHz) provides a means to capture faster motional dynamics where X band fails to resolve these faster motions, resulting in motionally narrowed spectra. Monitoring fa ster motion dynamics with frequencies >95 GHz reveals slow er motion al features at the same motional rate A m ulti frequency EPR approach may also provide information from spin labeled systems by providing a means to more accurately acquire dynamic and or dering parameters ( 86 ) To acquire such parameters, various s imulation software packages

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123 ha ve been developed to fit the EPR spectral line shapes based on particular motional models ( 100 ) T he slowly relaxing local structure (SRLS) model is commonly used to de convolute the motion of the overall tumbling of the protein from the internal motions of the label. Unlike other models such as the m icroscopically ordered macroscopically disordered (MOMD) model t he SRLS model accounts for the overall rotational correlatio n diffusion of the protein allowing data to be collected in solution without the need to slow the rotational correlation time of th e protein ( 86, 95 ) Although the SRLS model can provide information on a protein system in solution, a large number of fitting parameters are often needed to accurately simulate the EPR spectral line shapes. Mult i frequency EPR spectra can be fit simultaneously with a common set of magnetic and dynamic parameters in order to reduce the number of variable parameters associated with the SRLS model. Various multi frequency EPR studies have shown this technique to be amenable for studying complex modes of motion of structure proteins ( 82, 85, 86, 89, 90, 93, 95, 130 ) DNA ( 85 ) and lipid membranes ( 87 ) Although multi frequency EPR has been used to study a variety of systems, there are no known reports in the literature of multi frequency studies on IDP systems. With advantages of high frequency EPR such as enhanced spectral resolu tion and sensitivity for probing fast motional dynamics ( 96 ) utilizing a multi frequency EPR approach to investigate IDPs will further assist in elucidat ing site sp ecific information on these hard to characterize proteins Materials and methods Materials 3 (2 Iodoacetamido) PROXYL (IAP) spin label was purchased from Sigma Aldrich (St. Louis, MO). The QuikChange site directed mutagenesis kit was purchased from

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124 Str atagene (La Jolla, CA). BL21(DE3) cells were purchased from Invitrogen (Carlsbad, CA). Unless otherwise stated, all other reagents were purchased from Fisher Scientific (Pittsburg, PA) and used as received. Protein Expression and Purification of IA 3 mut ants E. Coli codon optimized DNA encoding for the IA 3 gene was purchased from DNA2.0 ( Menlo, CA ) The gene was cloned into the pET 22b vector (Novagen ) Cysteine substitutions for spin labeling were introduced using the QuikChange site directed mutagene sis kit, and the sequence was confirmed by DNA sequencing. Wild type and cysteine variants were expressed via an E. Coli system modified from the original protocol ( 54 ) The bacteria l host strain for expression was BL21(DE3) cells. Cells were grown at 37 C in LB medium to an OD 600 of ~0.6 before induction with 1mM IPTG. After approximately two hours of expression, the cells were pelleted, resuspended, and lysed by sonication and th ree passes through a 35 mL French pressure cell (Thermo Scientific, Waltham, MA). After lysis, the supernatant was boiled for 5 minutes to precipitate structured proteins and assist in purification and the supernatant was centrifuged to remove insoluble m aterial (18500 g, 20 minutes, 4 C). Boiling the lysate versus boiling the purified protein was a modification from the original protocol. It has been shown that boiling the lysate assists in protein purification of IDPs ( 131 ) The soluble recombi nant protein was purified utilizing a C terminal His tag encoded by the pET 22b vector by affinity chromatography as described previously ( 54 ) Protein was estimated to be pure by a 16.5% Tris Tricine SDS PAGE gel. 1mM EDTA was added to purified IA 3 to remove any residual nickel that may have been leeched off the affinity column.

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125 Spin Labeling Proteins containing the cysteine substitutions were buffer exchanged into 50 mM sodium pho sphate, 300 mM sodium chloride, pH 7.4 using a HiPrep 26/10 desalting column (Amersham, Pittsburg, PA). 100:1 molar excess of dithiothreitol (DTT) was using a desalting column (as described above) equilibrated and eluted with 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.4. Protein was then spin labeled with 10x molar excess of IAP, dissolved in ethanol, in the dark at room temp erature (22 C) for 4 hours. Excess spin label was removed via the desalting procedure described above. Protein was eluted in 50 mM sodium phosphate, 300 mM sodium chloride, at pH 7.4. Continuous Wave (CW) X Band EPR Spectra CW X band EPR spectra were collected on a modified Bruker ER200 spectrometer with an ER023 M signal channel, an ER032 M field control unit, and a loop gap resonator (Medical Advances, Milwaukee, WI). A quartz Dewar (Wilmad Labglass, Buena, NJ) surrounded the loop gap resonator where nitrogen gas was passed through a copper coil submerged in a recirculating bath (Thermo Scientific San Jose, CA ) in order to control the temperature. Samples were prepared by adding 0% to 40% TFE (v/v) in 5% increments to a n approximate concentration of 2 tubes (Fiber Optic Center, New Bedford, MA) for X band data collection. All reported CW EPR experiments were performed at 27 C unless otherwise stated. All s pectra were collected as 100 Gauss (G) scans with 2 mW incident microwave power. The 100 kHz field modulation amplitude and time constant of the detector were optimized to

PAGE 126

126 provide maximum signal to noise ratio with no line broadening. All spectra are rep orted as the average of 10 scans. Spectra are plotted with intensities scaled to normalized absorption area. LabVIEW software was used for baseline correction and double integral area normalization, which was generously provided by Drs. Christian Altenbac h and Wayne Hubbell ( University of California, Los Angeles ). Continuous Wave (CW) W Band EPR Spectra CW W band EPR spectra were collected on a Bruker Elexsys 680 spectrometer equipped with a W band ENDOR resonator at the N ational H igh M agnetic F ield L ab (N HMFL) in Tallahassee, FL Temperature was regulated by use of a CF935 Cyrostat (Oxford Instruments). Samples were prepared by adding 0% to 40% TFE (v/v) in 5% increments to an approximate concentration of 2 mM spin labeled protein. Samples were loaded i nto 0.15 i.d. X 0.25 o.d. suprasil quartz capillary tubes (Vitrocom, Mountain Lakes, NJ) and sealed with X Sealant. The capillary tube with the sample was placed inside a quartz tube with a 0.50 i.d.X 0.90 o.d. that was sealed at one end. All experiment s reported were performed at 27 C unless otherwise stated. All spectra were collected as 180 Gauss (G) scans with 0.0042 mW incident microwave power. The 100 kHz field modulation amplitude and time constant of the detector were optimized to provide maxi mum signal to noise ratio with no line broadening. All spectra are reported as the average of ~15 scans. Spectra are plotted with intensities scaled to normalized absorption area. X band Data Analysis The X band EPR line shapes were analyzed by monitori ng the local tumbling volume (V L ) of the spin label. This analysis technique can be used to investigate conformational changes in biomolecules ( 128, 132 ) T his parameter reports on

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127 structural information of the pr otein system with external environment dependent quantities removed ( 128, 132 ) The expression for V L is given by Equation 5 1, (Eq. 5 1) where k is the Boltzmann constant, T is the abs olute temperature, R is the rotational correlation time, and is the solvent viscosity. Values of R were determined from line shape simulations using the EWVoight program generously provided by Alex Smirnov ( North Carolina State University ). The simulation provides value s for the Lorentzian line widths of the three transitions and are subsequently used in Equation 5 2, (Eq. 5 2) where M is the nuclear spin quantum number of the M th hyperfine line, T 2 ( M ) is the spin spin relaxation time (which is i nversely proportional to the Lorentzian line width s of the three transitions ) of that line, and A B and C are the line shape parameters. In the fast motional regime, assumptions can be made and B and C alone can be used to determine rotational correlatio n time ( 98 ) V alues of R were determined using Equation 5 3, (Eq. 5 3) where the value of B is obtained from Equation 5 2. Viscosity M easurements Viscosity measurements were performed using Cannon Fenske Viscometer (size 50) suspended in a water bat h Experiments were performed at 6 C and 27 C. TFE solutions of 0 40% (v/v) in increments of 5% were made by combining the appropriate amount of TFE, and the 50 mM sodium phosphate, 300 mM sodium

PAGE 128

128 chloride pH 7.4 buffer The efflux time is converted to kinematic viscosity with units of centistokes ( m m 2 1 ) by multiplying the efflux time in seconds by the viscometer constant 0.004. The kinematic viscosity is then converted to 1 1 ) utilizing the densities of each of the solutio ns. X and W band EPR Spectral Line Shapes Fitting The EPR spectral line shapes of the C terminal sites, Y57C, N58C K59C, L60C, and K61C labeled with the IAP spin label at both X and W band were simulated using t he Labview program for fitting multi compon ent CW EPR spectra, written by Dr. Christian Altenbach from the laboratory of Dr. Wayne Hubbell at U niversity of C alifornia L os A ngeles The experimental line shapes were n ormalized and phased before being loaded into the program. All the EPR spectral line shapes could be fit with one component assuming isotropic motion. The phase and the shift parameters were allowed to vary to optimize the fit. The g tensor values for gxx, gyy, and gzz were determined experimentally from the rigid limit spectrum of free IAP spin label in varying TFE solution s collected at W band at 150 K and are : 2. 00821, 2. 00641, and 2.00205 ; respectively T he g tensor values were held constant for each fit. The A tensors determined from the fits for the X band data are listed in T able 5 1, and for the W band data are listed in T able 5 2 The isotropic A tensor (A1) the isotropic rotational diffusion tensor (R bar ), and the isotropic Lorentzian linewidth tensor (W1) were all allowed to vary. The definitions of the parameters were described by Budil et al. ( 100 ) The values of R reported below were determined from the rotational diffusion tensor based on the relationship described in Equation 5 4. (Eq. 5 4)

PAGE 129

129 Results and Discussion Qua litative Analysis of the TFE Induced Change in the Spectral Line Shapes Continuous wave (CW) X band EPR spectra were collected for IA 3 samples labeled at sites Y57C, N58C, K59C, L60C, and K61C with IAP in increasing concentrations of TFE ranging from 0% to 40% in 5% increments. Figure 5 1 shows stack plots of double integral (area) normalized 75 G X band EPR spectra for samples. A general trend is observed from the EPR spectral line shapes plotted in Figure 5 1, where for each residue the overall intensity of the spectra decrease s and broaden s as a function of TFE percentage Because t he spectra ar e plotted with normalized areas, the peak intensities are proportional to the mobility. The larger the peak intensity, the more motional averaging the spin probe undergoes. As mobility decreases, the spectra will broaden and the n ormalized intensity will decrease The plots in Figure 5 1 also show that comparatively the spectra of each residue at a given TFE percentage have slight differences in peak intensities and overall breadth. The observed variations between the spectra of each residue at a given TFE percentage indicate that the spin label experience d minor difference s in mobility; therefore, indicat ing site specific differences from residue to residue. To further examine these differences, a quantitative analysis approach was performed in an attempt to verify differences between the N terminus and C terminus. Quantitative Analysis of the TFE Induced Change in the Spectral Line Shapes As shown in C hapter 4 a quantitative approach may also be used by analyzing the local tumb ling volume (V L ) of the spin label as a function of TFE percentage. Plots of V L as a function of increasing TFE percentage for the C terminal sites Y57C, N58C K59C, L60C, and K61C labeled with IAP at 27 C are shown in Figure 5 2 T he value of

PAGE 130

130 V L increa ses as the spin For the C terminal sites V L increase d linearly for TFE percentages below 25%. Above 25%, the slope changes and becom ing less steep indicating a change in the dependence of V L with TFE, similar to the pr eviously reported data. Figure 5 2 also show the magnitude of the change of V L for each site varied indicating site specific differences For easier comparison, Figure 5 3 is a histogram of the values of V L at 0%, 15%, and 30 % TFE From these plots, it is apparent that there are considerable variations in the values of V L as a function of TFE among the C terminal sites. It is also noteworthy that the change in magnitude of all of the studied C terminal residues are less than those observed for the N term inal site S14C reported in C hapter 4. Previously published NMR data indicated that even in the absence of TFE the C terminus of IA 3 was a moderately populated helix ( 53, 60 ) NMR data also revealed that the C t erminus does not undergo as much of a pronounced conformational changes as the N terminus in increasing concentrations of TFE. Similarly, the values of V L for the C terminal residues at 0% TFE are significantly larger than the values for the N terminal re sidue S14C, which indicates that the EPR data is in good agreement with the data reported in literature Multi Frequency EPR A nalysis In an effort t o extract accurate values for the overall rotational correlation times from EPR spectral line shapes on the C terminal residues, high frequency (W band) SDSL EPR studies were also performed Initial W band experiments were collected on a home built W band spectrometer in the laboratory of Ale x Smirnov and Tatyana Smirnova at North Carolina State University. Th e W band spectra of the C terminal variants labeled with IAP collected in 0%, 15%, and 30% TFE solutions are displayed in Figure 5 4. P ronounced differences from residue to residue were observed in the

PAGE 131

131 spectra particular ly in the high field peak of the 3 0% TFE samples From this data, it appeared the W band spectra revealed more apparent site specific differences from residue to residue tha n the corresponding X band data T he se initial studies were not however, collected under rigorous temperature contr ol. Because temperature variations alter molecular motions, there was concern that the differences observed may have originated from temperature differences during data collection In an effort to discern whether the observed differences in the initial W band data were artifacts of variations in temperature during data collection, temperature effect experiments were performed. Temperature control studies W band spectra for IA 3 variants S14C and N58C labeled with IAP were collected on a Bruker W band spe ctrometer at the NHMFL under temperature control at 19 C, 22 C, 25 C, and 27 C in 0% and 30% TFE solutions. For comparison, X band data was also collected under the same conditions. The area normalized X and W band spectra of S14C and N58C variants collected in 0% TFE solution as a function of tem perature are shown in Figure 5 5 At X band as temperature was increased, differences in both S14C and N58C line shapes were observed where the peak intensities increased, and the overall breadth s decrease d resulting from the faster motion of the protein at higher temperatures Conversely, at 0% TFE the W band line shapes were not as dramatically a ffected by changes in temperature and only showed slight variations as temperature was increased The qualit ative e ffects in the line shapes as a function of temperature for both X and W band data can be more readily observed in the overlaid s tack plots shown in Figure 5 6 The insets within the figure provide a magnified view of the high field peak where the change in the spectral intensity and breadth are best illustrated

PAGE 132

132 The area normalized X and W band spectra of S14C and N58C variants were also collected in 30% TFE as a function of temperature and are shown in Figure 5 7. Similar to the 0% TFE data at X band, the line shapes for both variants showed increased peak intensity and decreased spectral breadths as temperature was increased. The W band spectra collected at 30% TFE showed the same trend as the X band data w h ere peak intensities increased and spectral breadths decreased as temperature was increased. The variations as a function of temperature for the spectra collected in 30% TFE solutions are most apparent in the high field peak at X band, and the center field and high field peaks at W band The overlaid spectra and magnified views of the center and high field peaks are shown in Figure 5 8. From this data, it is clear that EPR spectral line shapes can be effected by temperature variations. This indicates that precise temperature control is necessary to exclude temperature as a variable in line shape analysis. Experimental m ulti frequency EPR spectral line shapes The previous studies indicated that temperature control was necessary to rule out affects contributed from variations in temperatur e Because the initial W band studies were not collected under temperature control the data was recollected on the Bruker W band spectrometer at the NHMFL under regulated temperature at 27 C Both X and W band data collected at 27 C for the C terminal sites in 0%, 15%, and 30% TFE are displayed in Figure 5 9. As shown in the initial W band studies the W band spe ctra particularly at 30% TFE have distinct differences from residue to residue particularly in the central and high field peaks The Y57C, N 58C, and K61C spectra appeared to have more restricted motion than K59C, and especially L60C. This qualitative assessment of the W band data follows the same general trend seen in the V L data determined from

PAGE 133

133 the X band data. Qualitatively, the variations in the EPR spectral line shapes are clearly more evident at W band than at X band, indicating that monitoring conformational changes in small dynamic IDPS by EPR may be more straightforward at higher frequencies. Multi frequency fitting of the EPR spectr al line shapes Spectral simulations of the X and W band data described in the previous section are overlaid on the experimental spectra shown in Figure 5 9. The g tensor values used in the simulations were determined from the spectrum shown in Figure 5 10 and the values are given in the material and methods sections above The A tensor values used in the simulations for the X and W band data are provided in Tables 5 1 and 5 2 respectively. X band data were fit using two different simulation programs, the EWVoight program based on Redfield theory and the multi component fitting program based on the SRLS model, both described previously. W band data could only be fit using the multi component fitting program. All data was fit with a single component a ssuming isotropic Brownian motion. The spectral fitting parameters the overall correlation times ( R ), the rotational diffusion tensors (R bar ), and the Lorentzian line width tensors (W), were extracted from the simulations for each variant as a function of TFE percentage and the values are giv en in Tables 5 3, 5 4, and 5 5. The Lorentzian line width t ensor, which reflects orientation dependent inhomogeneous broadening caused by magnetic interactions of the el ectronic and nuclear spins, was constant for all collected X band spectra. In contrast the Lorentzian broadening parameter determined at W band had to be varied to obtain good fits. The values for the Lorentzian broadening were shown to vary from residue to residue and

PAGE 134

134 were also shown to broaden as a function of TFE percentage. Based on these observations the Lorentzian line width broadening ten sor appears to have a greater effect on the line shapes collected at hig her frequencies The values of the overall correlation times at X band determined from both simulation programs are in good agreement with one another. As expected, the overall correl ation time slowed as the TFE percentage was increased for all the C terminal variants. Slight differences were detected in the correlation times from residue to resi due at each TFE percentage and t he differences among residue s increased as the percent TFE increased The trends observed for the differences in R among the C terminal residues follow the same trends observed in the V L data. From this comparison, s imulation s of the EPR spectral line shapes provided a n additional quantitative analysis tool to measure the site specific differences observed among the C terminal residues further elucidating information on the induced conformational change occurring in IA 3 as a function of TFE. The overall correlation times determined from the W band spectra at e ach TFE percentage followed the same trend as the X band values whe re the correlation times slowed as a function of TFE Similar values for the overall correlation times of the 0% TFE samples were obtained at X and W band However, as the TFE percentag e was increased, t he values of the overall correlation times determined at X and W band began to deviate from one another. Several justifications for the deviations at higher TFE percentages have been considered. The divergence in R between the X and W band data may be attributed to variations in sample preparation and c oncentration. T he same protein samples were

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135 not used in the X and W band data collections and the protein concentrations of the W band samples were 10 fold higher than the X band samp les Due to the extremely high protein concentrations used at W band, the local environment of the spin label may have be en affected as a result of differences in solution viscosity Additionally at such high concentrations of protein the EPR spectral line shape may be affected by dipolar broadening and could be a factor in the differences seen in overall rotational correlation time reported at each frequency To eliminate the effects caused form differences in sample preparation and concentration X and W band data should be recollected on the same protein samples at a given concentration Summary Multi frequency EPR has been shown to be a useful tool for investigating protein dynamics in a variety of biological systems; however, it has never been u tilized to study IDPs. In this study, X and W band EPR spect roscopy were performed to monitor the chemically induced disordered to helical transition in the intrinsically disordered protein, IA 3 The X band spectra were analyzed by monitoring changes i n V L as a f unction of TFE. It was shown that analyzing data in this fashion provided a means to distinguish variations occurring from residue to residue. In an effort to extract spectral parameters to further characterize the induced conformational chan ge, simulations were performed on the multi frequency spectra. The overall correlation times were acquired for each residue as a function of TFE T he general trends of the overall correlation times followed the trends seen in the local tumbling volume an alysis for each residue, which indicates that fitting the data can provide site specific information However, the overall rotational correlation time determined at X and W band differed slightly from one another at higher TFE

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136 percentages which may be a ttributed to differences in sample preparation and concentrations. Recollecting the X and W band data using the same protein sample may provide a means to obtain comparable values of R at the two frequencies A m ulti frequency EPR approach appears to be a valuable tool for investigating of IDPs. Using a variety of data analysis parameters, we have shown IDPs which undergo unstructured to structured transitions can be characterized w ith EPR. With further optimization of the technique, these methods can provide a way to characterize IDPs which have proven difficult to study using other biophysical techniques.

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137 Figure 5 1 Area normalized 75G X band EPR spectra of IA 3 variants Y5 7C, N58C, K59C, L60C, and K61C labeled with IAP in increasing TFE concentration. Spectra were collected at 27 C. Experimental dat a (solid black). Simulated data (dashed red).

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138 Figure 5 2 The local tumbling volume ( V L ) of the protein as a function of increasing percentage TFE for Y57C, N58C, K59C, L60C, and K61C labeled with IAP collected at 27 C Lines represent linear regression fits to the data. The sizes of the data points are larger than errors in the measurements. Figure 5 3 Bar graph plots of the local tumbling volume (V L ) of the protein as a function of 0%, 15%, and 30% TFE for Y57C, N58C, K59C, L60C, and K61C labeled with IAP collected at 27 C.

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139 Figure 5 4. 180G area normalized W band data ofY57C, N58C, K59C, L60C, and K61C lab eled with IAP in 0%, 15%, and 30% TFE solutions. The intensities of the 15% and 30% TFE spectra were magnified 2X and 3X, respectively. Data collected on a home built W band spectrometer at NCSU.

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140 Figure 5 5 Plots of area normalized X band and W b and EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 0% TFE monitoring changes in spectral line shapes based on variations in temperature. Spectra were collected at 19 C (black), 22 C (red), 25 C (blue) and 27 C (green). X band data colle cted on a Bruker ER200. W band data collected on a Bruker Elexsys E580 at the NHMFL.

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141 Figure 5 6 Overlaid plots of area normalized X band and W band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 0% TFE monitoring changes in spectral l ine shapes based on variations in temperature. Spectra were collected at 19 C (black), 22 C (red), 25 C (blue) and 27 C (green). Inset shows magnified high field peak to accentuate differences in peak intensity as a function of temperature. X band da ta collected on a Bruker ER200. W band data collected on a Bruker Elexsys E580 at the NHMFL.

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142 Figure 5 7 Plots of area normalized X band and W band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 30% TFE monitoring changes in spectra l line shapes based on variations in temperature. Spectra were collected at 19 C (black), 22 C (red), 25 C (blue) and 27 C (green). X band data collected on a Bruker ER200. W band data collected on a Bruker Elexsys E580 at the NHMFL.

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143 Figure 5 8 Overlaid plots of area normalized X band and W band EPR spectra of IA 3 variants S14C and N58C labeled with IAP in 30% TFE monitoring changes in spectral line shapes based on variations in temperature. Spectra were collected at 19 C (black), 22 C (red), 25 C (blue) and 27 C (green). Inset shows a magnified portion of the spectra to accentuate differences in peak intensity as a function of temperature. X band data collected on a Bruker ER200. W band data collected on a Bruker Elexsys E580 at the NHMF L.

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144 Figure 5 9 Experimental (solid black) and simulated (dashed red) EPR spectra collected at 27 C. (A) 100G area normalized X band data of Y57C, N58C, K59C, L60C, and K61C labeled with IAP in 0%, 15%, and 30% TFE solutions. (B) 180G area normaliz ed W band data ofY57C, N58C, K59C, L60C, and K61C labeled with IAP in 0%, 15%, and 30% TFE solutions. The intensities of the 15% and 30% TFE spectra were magnified 2X and 3X, respectively. X band data collected on a Bruker ER200. W band data collected on a Bruker Elexsys E580 at the NHMFL.

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145 Figure 5 10. W band spectrum of free IAP spin label in aqeous solution collected at 150 K. Spectrum used to measure g tenor values.

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146 Table 5 1. X b and m agnetic A t ensor c omponents d etermined f rom c alculations % TFE A xx A yy A zz 0 6.52 5.6 34.48 5 6.5 5.58 34.46 10 6.46 5.54 34.42 15 6.41 5.49 34.37 20 6.35 5.43 34.31 25 6.32 5.4 34.28 30 6.28 5.36 34.24 35 6.26 5.34 34.22 40 6.24 5.32 34.2 Th e estimated error in the A tensor component is 0.4G Table 5 2. W b and m agnetic A t ensor c omponents d etermined f rom c alculations % TFE A xx A yy A zz 0 6.93 5.98 34.86 15 6.81 5.89 34.77 30 6.61 5.69 34.57 The estimated er ror in the A tensor component is 0.4G

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147 Table 5 3. Best f it p arameters of IAP l abeled v ariants in 0% TFE b uffer s olution X band W band Variant R (1) R (2) R bar W 9.5GHZ R (2) R bar W 95GHZ ( 10 9 s 1 ) ( 10 9 s 1 ) ( 10 8 s 1 ) (Gauss) ( 10 9 s 1 ) ( 10 8 s 1 ) (Gauss) Y57C IAP 0.25 0.27 6.17 0.25 0.27 6.16 0.5 0 N58C IAP 0.28 0.31 5.37 0.25 0.33 5.01 0.83 K59C IAP 0.27 0.27 6.17 0.25 0.27 6.17 0.51 L60C IA P 0.24 0.25 6.76 0.25 0.27 6.46 0.43 K61C IAP 0.24 0.24 6.76 0.25 0.28 6.03 0.43 (1) Determined using EWVoight software from Smirnov (2) Determined using Multi component fit software from Altenbach Table 5 4. Best f it p arameters of IAP l abeled v ariants in 15% TFE b uffer s olution X band W band Variant R (1) R (2) R bar W 9.5GHZ R (2) R bar W 95GHZ ( 10 9 s 1 ) ( 10 9 s 1 ) ( 10 8 s 1 ) (Gauss) ( 10 9 s 1 ) ( 10 8 s 1 ) (Gauss) Y57C IAP 0.45 0.48 3.47 0.25 0.39 4.27 1.25 N58C IAP 0.47 0.54 3.09 0.25 0.42 3.98 1.04 K59C IAP 0.47 0.47 3.55 0.25 0.38 4.37 1.00 L60C IAP 0.41 0.40 4.17 0.25 0.36 4.57 0.85 K61C IAP 0.47 0.45 3.72 0.25 0.36 4.68 0.98 (1 ) Determined using EWVoight software from Smirnov (2) Determined using Multi component fit software from Altenbach

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148 Table 5 5. Best f it p arameters of IAP l abeled v ari ants in 30% TFE b uffer s olution X band W band Variant R (1) R (2) R bar W 9.5GHZ R (2) R bar W 95GHZ ( 10 9 s 1 ) ( 10 9 s 1 ) ( 10 8 s 1 ) (Gauss) ( 10 9 s 1 ) ( 10 8 s 1 ) (Gauss) Y57C IAP 0.66 0.67 2.45 0.25 0.59 2.95 1.76 N58C IAP 0.62 0.65 2.57 0.25 0.57 2.82 1.82 K59C IAP 0.62 0.64 2.57 0.25 0.48 3.47 1.79 L60C IAP 0.54 0.55 3.02 0.25 0.45 3.72 1.53 K61C IAP 0.62 0.64 2.57 0.25 0.57 2.95 2.09 (1 ) Determined using EWVoight software from Smirnov (2) Determined using Multi component fit software from Altenbach

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149 CHAPTER 6 FUTURE WORK Full Cysteine Scanning of IA 3 f or Further X and W B and EPR Studies SDSL EPR has been utilized to characterize structur al dynamic, and conformational changes in a variety of biological systems; however, it has not been used extensively to investigate IDPs Furthermore, there are no kno wn reports of a multi frequency EPR approach to study IDPs. This work explored the use of multi frequency EPR to investigate structural changes in IDPs by monitoring the induced conformational changes in IA 3 at both X and W band frequencies. The data pr esented here showed that multi frequency SDSL EPR was able to elucidate site specific information on the chemically induced conformational change using both experimental and theoretical data analysis The multi frequency EPR approach compared well to pre viously reported NMR studies which showed that the N terminal region of IA 3 undergoes a more pronounced conformational change than the C terminal region of the protein ( 60 ) Although only a few sites in the N and C terminal regions were investigated the experiments show the applicability of a multi frequency EPR approach to characterizing IPDs. Indeed, b y investigating each residue within the IA 3 sequence with multi frequency EPR, the site specific differences as well as each region of the protein we re observable. Moreover, a full cysteine scanning of the protein should further demonstrate the use of multi frequency EPR for investigating IDPs and provide additional insight into the types of data analysis necessary to characterize structur al dynamic, and conformational changes in IDPs.

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1 50 Additional High Frequency EPR Studies As discussed within this work, EPR spectra l line shapes collected at various frequencies are sensitive to motions on different time scales and allow spectral parameters to be mo re accu rately determined Chapter 5 detailed the X and W band EPR studies performed to extract motional spectral parameters from the line shapes of the C terminal residues of IA 3 by simulating the data using the SRLS model in the multi component fitting program. A recent multi frequency EPR study revealed that investigating the protein, T4 lysozyme, with a more extensive multi frequency EPR approach at 9 GHz, 95 GHz, 140 GHz, and 250 GHz allowed better fits of the data that to be obtained than when data were collected at only 9 GHz and 250 GHz frequencies ( 95 ) This study indicates that the different perspectives of the molecular motions revealed at various frequencies may be necessary to obtain truer fits of the data. The data reported in the T4 lysozyme study signifies that a more extensive multi frequency EPR approach may provide a means to acquire better fit s for IA 3 spectral line shapes and therefore extract more accurate spectra parameters from the data. SDSL EPR Study of IA 3 Variants B ound to YPRA T he SDSL EPR studies presented in this work monitor ed the chemically induced conformational change in IA 3 by the secondary structural stabilizer, TFE. Although these studies showed the inherent propensity of IA 3 to form an helix it does no t necessarily show how the protein would act upon interaction with its specific target protein, YPRA. Future EPR studies should be conducted for IA 3 bound to YPRA for comparison to the spectral line shapes obtained in high TFE percentages. These

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151 studies would give a good indication if the conformational change observed in TFE, also occur in biologically relevant samples.

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152 APPENDIX A : IA 3 WILD TYPE AND VARIENT PRO TEIN AND AMINO ACID SEQUENCE S Table A 1. IA 3 Wild Type s equence M N T D Q Q K V S E I F Q S S K ATG AAT ACA GAC CAA CAA AAA GTG AGC GAA ATA TTT CAG AGC TCA AAG E K L Q G D A K V V S D A F K K GAA AAG TTG CAG GGC GAT GCA AAG GTA GTG AGT GAC GCT TTT AAG AAA M A S Q D K D G K T T D A D E S ATG GCT AGT CAA GAC AAA GAC GGC AAG ACT ACC GAT GCT GAT GAA AGT E K H N Y Q E Q Y N K L K G A G GAA AAA CAC AAC TAT CAG GAG CAA TAC AAC AAG CTG AAA GGG GCG GGG H K K E CAT AAG AAG GAG Table A 2. IA 3 GC o ptimized w ild t ype s equence M N T D Q Q K V S E I F Q S S K ATG AAC ACG GAT CAG CAG AAG GTT AGC GAG ATT TTC CAG TCC AGC AAG E K L Q G D A K V V S D A F K K GAG AAA CTG CAA GGC GAT GCG AAG GTT GTG AGC GAC GCG TTT AAG AAG M A S Q D K D G K T T D A D E S ATG GCT AGC CAG GAC AAA GAT GGT AAA ACG ACC GAC GCA GAC GAA AGC E K H N Y Q E Q Y N K L K G A G GAG AAG CAC AAC TAT CAG GAG CAG TAT AAC AAG CTG AAG GGC GCA GGT H K K E CAC AAG AAG GAG Table A 3. IA 3 GC o ptimized S14C s equence M N T D Q Q K V S E I F Q C S K ATG AAC ACG GAT CAG CAG AAG GTT AGC GAG ATT TTC CAG TGC AGC AAG E K L Q G D A K V V S D A F K K GAG AAA CTG CAA GGC GAT GCG AAG GTT GTG AGC GAC GCG TTT AAG AAG M A S Q D K D G K T T D A D E S ATG GCT AGC CAG GAC AAA GAT GGT AAA ACG ACC GAC GCA GAC GAA AGC E K H N Y Q E Q Y N K L K G A G GAG AAG CAC AAC TAT CAG GAG CAG TAT AAC AAG CTG AAG GGC GCA GGT H K K E CAC AAG AAG GAG

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153 Table A 4. IA 3 Y57C s equence M N T D Q Q K V S E I F Q C S K ATG AAT ACA GAC CAA CAA AAA GTG AGC GAA ATA TTT CAG AGC TCA AAG E K L Q G D A K V V S D A F K K GAA AAG TTG CAG GGC GA T GCA AAG GTA GTG AGT GAC GCT TTT AAG AAA M A S Q D K D G K T T D A D E S ATG GCT AGT CAA GAC AAA GAC GGC AAG ACT ACC GAT GCT GAT GAA AGT E K H N Y Q E Q C N K L K G A G GAA AAA CA C AAC TAT CAG GAG CAA TGC AAC AAG CTG AAA GGG GCG GGG H K K E CAT AAG AAG GAG Table A 5. IA 3 N58C s equence M N T D Q Q K V S E I F Q C S K ATG AAT ACA GAC CAA CAA AAA GTG AGC GAA ATA TTT CA G AGC TCA AAG E K L Q G D A K V V S D A F K K GAA AAG TTG CAG GGC GAT GCA AAG GTA GTG AGT GAC GCT TTT AAG AAA M A S Q D K D G K T T D A D E S ATG GCT AGT CAA GAC AAA GAC GGC AAG ACT ACC G AT GCT GAT GAA AGT E K H N Y Q E Q Y C K L K G A G GAA AAA CAC AAC TAT CAG GAG CAA TAC TGC AAG CTG AAA GGG GCG GGG H K K E CAT AAG AAG GAG Table A 6. IA 3 K59C s equence M N T D Q Q K V S E I F Q C S K ATG AAT ACA GAC CAA CAA AAA GTG AGC GAA ATA TTT CAG AGC TCA AAG E K L Q G D A K V V S D A F K K GAA AAG TTG CAG GGC GAT GCA AAG GTA GTG AGT GAC GCT TTT AAG AAA M A S Q D K D G K T T D A D E S ATG GCT AGT CAA GAC AAA GAC GGC AAG ACT ACC GAT GCT GAT GAA AGT E K H N Y Q E Q Y N C L K G A G GAA AAA CAC AAC TAT CAG GAG CAA TAC AAC TGC CTG AAA GGG GCG GGG H K K E CAT AAG AAG GAG

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154 Table A 7. IA 3 L60C s equence M N T D Q Q K V S E I F Q C S K ATG AAT ACA GAC CAA CAA AAA GTG AGC GAA ATA TTT CAG AGC TCA AAG E K L Q G D A K V V S D A F K K GAA AAG TTG CAG GGC GAT GCA AAG GTA GTG AGT GAC GCT TTT AAG AAA M A S Q D K D G K T T D A D E S ATG GCT AGT CAA GAC AAA GAC GGC AAG ACT ACC GAT GCT GAT GAA AGT E K H N Y Q E Q Y N K C K G A G GAA AAA CAC AAC TAT CAG GAG CAA TAC AAC AAG TGC AAA GGG GCG GGG H K K E CAT AAG AAG GAG Table A 8. IA 3 K61C s equence M N T D Q Q K V S E I F Q C S K ATG AAT ACA GAC CAA CAA AAA GTG AGC GAA ATA TTT CAG AGC TCA AAG E K L Q G D A K V V S D A F K K GAA AAG TTG CAG GGC GAT GCA AAG GTA GTG AGT GAC GCT TTT AAG AAA M A S Q D K D G K T T D A D E S ATG GCT AGT CAA GAC AAA GAC GGC AAG ACT ACC GAT GCT GAT GAA AGT E K H N Y Q E Q Y N K L C G A G GAA AAA CAC AAC TAT CAG GAG CAA TAC AAC AAG CTG TGC GGG GCG GGG H K K E CAT AAG AAG GAG

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155 APPENDIX B : CIRCULAR DICROISM AN D X BAND AND W BAND CW EPR EXPERIMENTAL PARAMETERS Table B 1. Circular d ichroism e xperimental p arameters Parameter Value Experiment Type Wavelength Bandwidth 1 nm Temperature 5 C or 27 C Waveleng th Start Point 245 nm Wavelength End Point 185 nm Wavelength Step 1 nm Averaging Time 4 seconds Settling Time 1 second Multi Scan Wait Time 0 seconds Number of Scans 4 Table B 2 Typical X b and CW EPR p arameters Parameter Value Number of Points 1024 Center Field ~3250 G Number of Scans 4 32 Sweep Width 20 G/100 G Acquisition Time 40.63 Frequency ~9.450 GHz Power 20 dB 2 mW Receiver Gain 1 10 4 110 5 Modulation Amplitude ~1G (uncalibr ated) Time Constant 0.082 Receiver Phase 100 Table B 3 Typical W b and CW EPR p arameters Parameter Value Number of Points 1024 Center Field ~33500 G Number of Scans 4 32 Sweep Width 180 G Acquisition Time 40.96 Frequency ~94.50 GHz Power 30 dB Receiver Gain 42 Modulation Amplitude 2 G 5 G Time Constant 40.96

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156 APPENDIX C : LOCAL TUMBLING VOLUM E PARAMETERS AND EPR SPECTRAL INT ENSITY VALUES AND RATIOS Table C 1 Parameter v alues u sed to d ete rmine V L p arameter at 27 C % TFE Average Time (sec) Density (kgm 3 ) Kinematic Viscosity (mm 2 s 1 ) Dynamic Viscosity (kgm 1 s 1 ) 0 208.00 1019 0.8320 0.000848 5 234.75 1043 0.9390 0.000979 10 263.00 1066 1.052 0.001122 15 291.50 1092 1.166 0.001273 20 299.50 1115 1.198 0.001335 25 317.25 1134 1.269 0.001439 30 332.25 1154 1.329 0.001534 35 345.50 1172 1.382 0.001620 40 351.25 1194 1.405 0.001678 Table C 2 Parameter v alues u sed to d etermine V L p arameter at 5 C % TFE Average Time (sec) Densit y (kgm 3 ) Kinematic Viscosity (mm 2 s 1 ) Dynamic Viscosity (kgm 1 s 1 ) 0 392.5 0 1019 1.570 0.001600 5 419.25 1043 1.677 0.00174 9 10 481.75 1066 1.927 0.00205 5 15 515.75 1092 2.063 0.002252 20 600.75 1115 2.403 0.00267 9 25 629.25 1134 2.517 0.00285 5 30 656.5 0 1154 2.626 0.003031 35 700.75 1172 2.803 0.003286 40 392.5 0 1194 1.570 0.001600

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157 Table C 3 Calculated l ocal t umbling v olume r aw d ata for S14C IAP at 27 C % TFE Lor (+1) (dp) Lor (0) (dp) Lor( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 9.400 8.256 12.21 0.9184 0.807 1.193 0.8067 0.1371 0.2488 1.672E 10 816.9 5 9.260 8.247 13.32 0.9047 0.806 1.301 0.8057 0.1983 0.2973 2.419E 10 1023 10 10.51 9.410 15.70 1.0 27 0.919 1.534 0.9194 0.2533 0.3611 3.090E 10 1141 15 12.92 11.55 20.11 1.263 1.128 1.964 1.128 0.3509 0.4855 4.281E 10 1393 20 15.19 13.38 24.58 1.484 1.308 2.402 1.308 0.4591 0.6350 5.600E 10 1737 25 15.91 13.91 26.04 1.554 1.359 2.544 1.359 0.495 2 0.6906 6.042E 10 1739 30 17.79 15.35 29.53 1.738 1.500 2.885 1.500 0.5737 0.8120 6.999E 10 1890 35 18.65 15.96 31.02 1.822 1.559 3.030 1.559 0.6041 0.8668 7.372E 10 1884 40 19.49 16.64 32.39 1.904 1.626 3.165 1.626 0.6304 0.9087 7.691E 10 1899 Ta ble C 4 Calculated l ocal t umbling v olume raw data for S14C IAP at 5 C % TFE Lor (+1) (dp) Lor (0) (dp) Lor( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 12.42 10.92 21.45 1.213 1.066 2.095 1.066 0.4410 0.5879 5.380E 10 1300 5 14.99 13.02 26.00 1.464 1.272 2.540 1.272 0.5380 0.7301 6.564E 10 1447 10 19.85 16.68 34.50 1.939 1.630 3.371 1.630 0.7157 1.025 8.732E 10 1639 15 29.05 22.14 51.44 2.838 2.163 5.025 2.163 1.093 1.769 1.334E 09 2285 20 37.33 26.01 6 7.73 3.647 2.541 6.617 2.541 1.485 2.591 1.812E 09 2609 25 38.09 26.25 69.26 3.721 2.565 6.767 2.565 1.523 2.679 1.858E 09 2508 30 38.02 25.52 70.18 3.714 2.494 6.856 2.494 1.571 2.792 1.917E 09 2437 35 39.63 26.77 72.11 3.872 2.615 7.045 2.615 1.58 7 2.843 1.936E 09 2272

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158 Table C 5 Calculated l ocal t umbling v olume raw data for N58 C IAP at 27 C % TFE Lor (+1) (dp) Lor (0) (dp) Lor( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 10.38 9.198 15.13 1.014 0.898 6 1.479 0.8986 0.2322 0.3477 2.833E 10 1287 5 11.01 9.808 16.49 1.076 0.9582 1.611 0.9582 0.2677 0.3855 3.266E 10 1285 10 12.12 10.78 18.89 1.184 1.053 1.845 1.053 0.3305 0.4615 4.032E 10 1384 15 13.63 12.03 21.56 1.332 1.175 2.107 1.175 0.3872 0.54 44 4.724E 10 1430 20 14.69 12.84 23.97 1.435 1.255 2.342 1.255 0.4535 0.6341 5.533E 10 1596 25 15.04 13.06 25.03 1.470 1.276 2.445 1.276 0.4878 0.6811 5.951E 10 1592 30 15.48 13.39 25.86 1.512 1.308 2.527 1.308 0.5072 0.7119 6.188E 10 1554 35 15.78 13.52 26.53 1.542 1.321 2.592 1.321 0.5253 0.7460 6.408E 10 1523 40 15.97 13.67 26.97 1.560 1.336 2.635 1.336 0.5373 0.7618 6.556E 10 1505 Table C 6 Calculated l ocal t umbling v olume raw data for N58 C IAP at 5 C % TFE Lor (+1) (dp) Lor (0) (dp) Lor ( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 15.76 13.48 27.36 1.539 1.317 2.673 1.317 0.5669 0.7894 6.916E 10 1671 5 19.27 16.04 33.80 1.883 1.567 3.302 1.567 0.7097 1.025 8.658E 10 1908 10 23.40 18.94 41.61 2.286 1.8 50 4.065 1.850 0.8897 1.325 1.085E 09 2038 15 28.81 22.04 52.18 2.815 2.154 5.098 2.154 1.142 1.803 1.393E 09 2386 20 31.30 23.13 57.01 3.058 2.260 5.570 2.260 1.256 2.054 1.532E 09 2206 25 32.86 23.81 60.49 3.211 2.326 5.910 2.326 1.350 2.234 1.647 E 09 2222 30 33.22 23.88 60.99 3.245 2.333 5.959 2.333 1.357 2.269 1.655E 09 2105 35 33.48 23.94 62.13 3.271 2.339 6.070 2.339 1.399 2.332 1.707E 09 2003

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159 Table C 7 Calculated l ocal t umbling v olume raw data for Y57 C IAP at 27 C % TFE Lor (+1) (dp) L or (0) (dp) Lor( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 7.067 6.234 11.33 0.6904 0.6090 1.107 0.6090 0.2081 0.2895 2.539E 10 1240 5 8.040 7.100 13.15 0.7855 0.6937 1.285 0.6937 0.2498 0.3415 3.047E 10 1289 10 9.277 8.245 15.49 0.9064 0.8056 1.514 0.8056 0.3037 0.4045 3.705E 10 1368 15 10.94 9.679 18.64 1.069 0.9456 1.821 0.9456 0.3762 0.4991 4.590E 10 1493 20 12.46 10.92 21.61 1.217 1.067 2.111 1.067 0.4470 0.5970 5.453E 10 1691 25 13.55 11.79 23.71 1.324 1.152 2.317 1.152 0.4966 0.6681 6.058E 10 1743 30 14.61 12.59 25.65 1.427 1.230 2.506 1.230 0.5395 0.7366 6.582E 10 1777 35 15.45 13.22 27.07 1.509 1.291 2.645 1.291 0.5677 0.7858 6.926E 10 1771 40 16.47 14.00 28.73 1.609 1.368 2.807 1.368 0.5 988 0.8403 7.305E 10 1804 Table C 8 Calculated l ocal t umbling v olume raw data for K59 C IAP at 27 C % TFE Lor (+1) (dp) Lor (0) (dp) Lor( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 7.396 6.548 11.91 0.7226 0 .6397 1.163 0.6397 0.2203 0.3033 2.688E 10 1221 5 8.389 7.437 13.76 0.8196 0.7266 1.345 0.7266 0.2626 0.3556 3.203E 10 1260 10 9.716 8.613 16.19 0.9492 0.8415 1.582 0.8415 0.3162 0.4239 3.858E 10 1325 15 11.36 9.998 19.32 1.110 0.9768 1.888 0.9768 0 .3891 0.5220 4.747E 10 1436 20 12.57 10.98 21.65 1.228 1.073 2.116 1.073 0.4438 0.5989 5.414E 10 1562 25 13.34 11.56 23.14 1.303 1.129 2.261 1.129 0.4787 0.6525 5.840E 10 1563 30 14.04 12.11 24.38 1.371 1.183 2.382 1.183 0.5053 0.6939 6.164E 10 1548 35 14.68 12.62 25.33 1.435 1.233 2.474 1.233 0.5198 0.7211 6.342E 10 1508 40 15.31 13.13 26.30 1.496 1.283 2.570 1.283 0.5370 0.7502 6.551E 10 1504

PAGE 160

160 Table C 9 Calculated l ocal t umbling v olume raw data for L60 C IAP at 27 C % TFE Lor (+1) (dp) Lor ( 0) (dp) Lor( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 6.923 6.103 11.00 0.6764 0.5963 1.074 0.596 0.1990 0.2792 2.428E 10 1103 5 7.834 6.913 12.68 0.7654 0.6754 1.239 0.675 0.2369 0.3269 2.890E 10 1137 10 8.841 7.813 14.67 0.86 37 0.7633 1.433 0.763 0.2848 0.3852 3.474E 10 1193 15 9.938 8.766 16.73 0.9709 0.8564 1.635 0.856 0.3319 0.4464 4.050E 10 1225 20 10.92 9.554 18.74 1.067 0.9334 1.831 0.933 0.3818 0.5155 4.658E 10 1343 25 11.74 10.18 20.30 1.147 0.9948 1.984 0.995 0 .4186 0.5703 5.107E 10 1367 30 12.46 10.73 21.50 1.218 1.049 2.100 1.049 0.4412 0.6101 5.383E 10 1352 35 13.18 11.29 22.77 1.287 1.103 2.224 1.103 0.4684 0.6524 5.715E 10 1359 40 14.34 12.31 24.43 1.401 1.203 2.387 1.203 0.4931 0.6909 6.015E 10 1381 Table C 10 Calculated l ocal t umbling v olume raw data for K61 C IAP at 27 C % TFE Lor (+1) (dp) Lor (0) (dp) Lor( 1) (dp) pp (+1) (Gauss) pp (0) (Gauss) pp ( 1) (Gauss) A B C t R (sec) V L (A 3 ) 0 6.904 6.100 10.96 0.6745 0.5960 1.070 0.5960 0.197 9 0.2765 2.415E 10 1097 5 7.966 7.053 12.89 0.7782 0.6891 1.260 0.6891 0.2408 0.3299 2.938E 10 1156 10 9.180 8.129 15.24 0.8969 0.7942 1.489 0.7942 0.2960 0.3987 3.611E 10 1240 15 10.96 9.672 18.61 1.071 0.9450 1.818 0.9450 0.3737 0.4996 4.560E 10 13 80 20 12.18 10.66 21.11 1.190 1.041 2.063 1.041 0.4360 0.5853 5.320E 10 1534 25 13.06 11.31 22.87 1.276 1.105 2.234 1.105 0.4791 0.6498 5.845E 10 1564 30 13.95 12.03 24.42 1.363 1.175 2.386 1.175 0.5114 0.6995 6.239E 10 1567 35 14.71 12.64 25.64 1.4 37 1.235 2.505 1.235 0.5341 0.7363 6.516E 10 1549 40 15.66 13.40 27.12 1.530 1.309 2.650 1.309 0.5602 0.7805 6.834E 10 1569

PAGE 161

161 Table C 11 EPR s pectral l ine i ntensities, h (+1) /h (0) and, h ( 1) values for S14C MTSL c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 1.08 1.24 0.59 0.87 0.00 5 0.93 1.08 0.46 0.86 21.53 10 0.65 0.76 0.29 0.85 50.58 15 0.57 0.69 0.23 0.82 61.12 20 0.57 0.71 0.22 0.81 63.28 25 0.55 0.69 0.20 0.79 65.68 30 0.54 0.69 0.20 0.78 66.45 35 0.49 0.63 0.18 0.78 6 9.82 40 0.47 0.61 0.17 0.77 70.91 Table C 12 EPR s pectral l ine i ntensities, h (+1) /h (0) and, h ( 1) values for S14C M SL c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.69 0.74 0.37 0.93 0.00 5 0.63 0.69 0.30 0.92 17.07 10 0.47 0.51 0.20 0.91 45.4 4 15 0.35 0.40 0.12 0.87 66.37 20 0.29 0.35 0.09 0.84 74.79 25 0.30 0.36 0.09 0.84 75.01 30 0.28 0.35 0.09 0.82 76.55 35 0.32 0.39 0.09 0.82 74.63 40 0.24 0.30 0.07 0.80 80.68 Table C 13 EPR s pectral l ine i ntensities, h (+1) /h (0) an d, h ( 1) values for S14C IAP c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 1.15 1.34 0.82 0.86 0.00 5 1.15 1.32 0.70 0.87 13.83 10 1.03 1.19 0.57 0.87 29.95 15 0.83 0.98 0.40 0.85 50.76 20 0.68 0.83 0.29 0.82 64.42 25 0.63 0.79 0.26 0 .81 67.97 30 0.55 0.71 0.22 0.78 73.55 35 0.50 0.65 0.19 0.77 76.48 40 0.49 0.65 0.19 0.76 77.12

PAGE 162

162 Table C 14 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for N58 C MTSL c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0 .77 0.90 0.39 0.85 0.00 5 0.68 0.80 0.32 0.84 17.47 10 0.54 0.67 0.23 0.81 39.86 15 0.47 0.61 0.19 0.77 50.85 20 0.42 0.57 0.16 0.75 57.84 25 0.40 0.55 0.15 0.72 61.38 30 0.38 0.53 0.14 0.71 63.16 35 0.35 0.50 0.13 0.70 65.99 40 0.36 0.51 0.14 0.70 64.88 Table C 15 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for N58C M SL c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.70 0.73 0.34 0.95 0.00 5 0.62 0.66 0.28 0.94 16.84 10 0.55 0.59 0.22 0.94 33.45 15 0.47 0.51 0.17 0.92 48.87 20 0.43 0.48 0.14 0.90 58.86 25 0.39 0.45 0.12 0 .88 63 .57 30 0.38 0.43 0.11 0.87 66.16 35 0.37 0.43 0.11 0.86 67.37 40 0.36 0.42 0.11 0.86 68.11 Table C 16 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for N58C IAP c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0. 94 1.10 0.55 0.86 0.00 5 0.97 1.13 0.53 0.86 3.41 10 0.84 0.99 0.42 0.85 23.60 15 0.80 0.97 0.38 0.83 31.32 20 0.70 0.86 0.30 0.81 44.49 25 0.65 0.81 0.27 0.80 50.80 30 0.66 0.83 0.27 0.80 50.95 35 0.63 0.80 0.25 0.78 53.92 40 0.61 0.78 0.24 0.78 5 6.08

PAGE 163

163 Table C 17 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for Y57C IAP c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 1.46 1.66 0.84 0.88 0.00 5 1.32 1.51 0.70 0.88 16.56 10 1.25 1.44 0.60 0.87 27.83 15 0.99 1.17 0.43 0.85 48.47 20 0.88 1.07 0.35 0.83 58.08 25 0.78 0.97 0.30 0 .81 64.57 30 0.70 0.89 0.26 0.79 69.12 35 0.69 0.88 0.25 0.78 70.35 40 0.60 0.78 0.21 0.76 74.46 Table C 18 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for K59C IAP c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 1. 45 1.64 0.81 0.88 0.00 5 1.29 1.48 0.67 0.87 17.13 10 1.14 1.32 0.54 0.86 32.98 15 0.96 1.15 0.41 0.84 48.86 20 0.84 1.03 0.34 0.82 57.36 25 0.78 0.97 0.31 0.81 61.92 30 0.73 0.92 0.28 0.80 65.13 35 0.71 0.90 0.27 0.79 66.07 40 0.68 0.87 0.26 0.78 68.14 Table C 19 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for L60C IAP c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 1.43 1.62 0.83 0.88 0.00 5 1.43 1.63 0.77 0.88 7.59 10 1.24 1.44 0.62 0.87 25.91 15 1.07 1.25 0.50 0.85 40.52 20 1.01 1.21 0.44 0.83 47.36 25 0.93 1.14 0.38 0. 82 53.78 30 0.89 1.10 0.36 0.81 56.68 35 0.80 1.01 0.32 0.79 62.00 40 0.73 0.92 0.29 0.79 65.61

PAGE 164

164 Table C 20 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for K61C IAP c ollected at 27 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 1.5 4 1.74 0.90 0.89 0.00 5 1.36 1.54 0.73 0.88 19.41 10 1.15 1.34 0.56 0.86 37.36 15 0.95 1.13 0.42 0.84 53.58 20 0.89 1.07 0.36 0.83 60.21 25 0.82 1.01 0.32 0.81 64.76 30 0.75 0.95 0.28 0.80 68.51 35 0.70 0.89 0.26 0.78 71.03 40 0.66 0.84 0.24 0.78 7 3.03 Table C 21 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for S14C IAP c ollected at 5 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.84 1.01 0.34 0.83 0.00 5 0.69 0.86 0.26 0.80 23.65 10 0.45 0.62 0.15 0.73 55.44 15 0.24 0.41 0.07 0.58 78.19 20 0.17 0.36 0.05 0.48 84.36 25 0.16 0.34 0.05 0. 47 85. 64 30 0.15 0.33 0.05 0.46 85.78 35 0.15 0.33 0.05 0.45 86.09 40 0.14 0.32 0.05 0.45 86.61 Table C 22 EPR s pectral l ine i ntensities h (+1) /h (0) and, h ( 1) values for Y57C IAP c ollected at 5 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.69 0.85 0.28 0.82 0.00 5 0.57 0.74 0.20 0.77 28.73 10 0.43 0.60 0.14 0.72 49.63 15 0.28 0.45 0.09 0.63 69.09 20 0.20 0.36 0.06 0.54 79.47 25 0.17 0.34 0.05 0.48 82.90 30 0.16 0.35 0.05 0.46 83.40 35 0.15 0.34 0.05 0.44 83.91 40 0.14 0.33 0.04 0.43 84 .61

PAGE 165

165 Table C 23 EPR s pectral l ine i ntensities h (+1) /h (0) and h ( 1) values for N58C IAP c ollected at 5 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.65 0.84 0.24 0.78 0.00 5 0.48 0.67 0.17 0.72 31.40 10 0.36 0.54 0.12 0.66 51.54 15 0.26 0.44 0.08 0.58 68.19 20 0.23 0.41 0.07 0.55 71.36 25 0.21 0.40 0.07 0. 52 73.06 30 0.21 0.40 0.06 0.51 74.55 35 0.20 0.39 0.06 0.52 75.35 40 0.21 0.41 0.06 0.51 74.55 Table C 24 EPR s pectral l ine i ntensities h (+1) /h (0) and h ( 1) values for K59C IAP c ollected at 5 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.70 0.87 0.28 0.80 0.00 5 0.54 0.71 0.19 0.76 30.50 10 0.40 0.58 0.14 0.70 50.73 15 0.27 0.44 0.09 0.60 69.06 20 0.21 0.39 0.06 0.53 77.21 25 0.19 0.38 0.06 0.50 78.41 30 0.19 0.38 0.06 0.49 79.52 35 0.18 0.37 0.06 0.49 79.95 40 0.18 0.38 0.06 0.48 79. 53 Table C 25 EPR s pectral l ine i ntensities h (+1) /h (0) and h ( 1) values for L60C IAP c ollected at 5 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.74 0.91 0.31 0.81 0.00 5 0.61 0.80 0.22 0.77 27.82 10 0.46 0.64 0.16 0.72 48.41 15 0.35 0.53 0.11 0.66 63.52 20 0.29 0.48 0.09 0.60 71.46 25 0.24 0.44 0.07 0. 54 76.11 30 0.22 0.42 0.07 0.53 77.87 35 0.21 0.42 0.07 0.51 78.76 40 0.21 0.43 0.07 0.50 78.68

PAGE 166

166 Table C 26 EPR s pectral l ine i ntensities h (+1) /h (0) and h ( 1) values for K61C IAP c ollected at 5 C % TFE h (+1) h (0) h ( 1) h (+1) /h (0) % h ( 1) 0 0.73 0.90 0.31 0.81 0.00 5 0.59 0.79 0.22 0.76 29.29 10 0.46 0.65 0.16 0.71 49.33 15 0.30 0.48 0.09 0.62 69.43 20 0.22 0.40 0.07 0.56 78.61 25 0.19 0.37 0.06 0.51 82.04 30 0.17 0.35 0.05 0.48 84.00 35 0.17 0.36 0.05 0.46 83.96 40 0.16 0.36 0.05 0.45 84. 54

PAGE 167

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179 BIOGRAPHICAL SKETCH Natasha L. Pirman was born in 1983 in Morehead, Kentucky. She lived in West Liberty Kentucky until 1989 when she moved to Lexington, Kentucky. She graduated from Tates Creek High School in 2001, and moved to Cleveland, Tennessee where she attended Lee University. In May of 2005 sh e graduated with honors with a B achelor of S cience (B.S .) degree in chemistry, and a minor in religion. She was admitted to the Department of Chemistry graduate program at the University of Florida in 2005. She s research group In the fall of 2006, she switched research grou ps and began working under the direction of Dr. Gail E. Fanucc i. She obtained her PhD. in chemistry in August 2011.