University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2009 Thermodynamics and Kinetics of Iso-1-cytochrome c Denatured State Franco Ollan Tzul The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Tzul, Franco Ollan, "Thermodynamics and Kinetics of Iso-1-cytochrome c Denatured State" (2009). Graduate Student Theses, Dissertations, & Professional Papers. 1112. https://scholarworks.umt.edu/etd/1112 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. THERMODYNAMICS AND KINETICS OF ISO-1-CYTOCHROME C DENATURED STATE By FRANCO OLLAN TZUL B. Sc. Chemistry & Biology, Regis University, Denver, CO, USA, 1997 Dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry & Biochemistry The University of Montana Missoula, MT March 2009 Approved by: Perry Brown, Associate Provost for Graduate Education Graduate School Dr. J.B. Alexander Ross, Committee Chairperson Department of Chemistry & Biochemistry Dr. Bruce E. Bowler, Committee Member Department of Chemistry & Biochemistry Dr. Klára Briknarová, Committee Member Department of Chemistry & Biochemistry Dr. Michael DeGrandpre, Committee Member Department of Chemistry & Biochemistry Dr. Michele A. McGuirl, Committee Member Division of Biological Sciences Tzul, Franco O., Ph. D., Spring 2009 Chemistry & Biochemistry Thermodynamics and Kinetics of Iso-1-cytochrome c Denatured State Chairperson: J. B. Alexander Ross Various diseases result from protein misfolding. Curing these conditions requires understanding the principles governing folding. Efforts toward understanding how proteins fold have focused on the transition state rather than the earliest folding events. We study these initial events using the assumption that protein folding must involve the formation of the most primitive structure possible – a simple loop. Our laboratory has developed a system of studying simple loops in the denatured state using c-type cytochromes. New insights into how the properties of these loops impact the denatured state are outlined in this thesis. First, studies on a 22-residue loop revealed a previously unreported finding that equilibrium loop formation was not strongly affected by sequence composition. While loop formation rates depended only on sequence composition, loop breakage rates also depended on sequence order. Second, thermodynamic and kinetic studies on homopolymeric inserts in “poor” and “good” solvents revealed that homopolymeric non- foldable protein sequences behave like a random coil. However, heteropolymeric foldable sequences have scaling factors higher than those of a random coil, suggesting the presence of residual structure in denatured proteins. Thus, peptide models with homopolymeric sequences do not adequately describe the nature of foldable sequences. Third, we investigated the kinetics of reversible oligomerization in the denatured state using a P25A yeast iso-1-cytochrome c variant. The findings indicated that intermolecular aggregation in a denatured protein is extremely fast – 107-108 M-1s-1 and that the P25A mutation strongly affects intermolecular aggregation. This work suggests that equilibrium control of folding versus aggregation is advantageous for productive protein folding in vivo. Fourth, we use time-resolved FRET to follow compact and extended distributions of a protein under denaturing conditions. Our findings revealed three major populations in the unfolded state when no loop is present whereas only two populations remain when the loop forms. The most extended population is lost upon loop formation showing that simple loop formation dramatically constrains the denatured state. Thus, thermodyamic and kinetic studies on simple loops using a variety of spectroscopic techniques have enhanced understanding of the initial events of protein folding and the role of the denatured state in modulating protein aggregation. ii Acknowledgements I would first like to thank my advisor Dr. Bruce Bowler for all his patience and guidance throughout my graduate educational development. He has been an inspiration of the type of scientist I would like to mature into and has shown me the dedication and hard work required to be successful in academia. I will forever cherish the kindness he has shown me, both in and out of the lab environment. I would also like to thank past and present members of the Bowler group, especially Jasmina, Saritha and Eydiejo for their mentoring; as well as Swati and Michael for their added friendship – making it a joy to work in the Bowler group. I would also like to thank Natasa Mateljevic and Nicole Branan for assistance in the preparation of the AcA25H26I52 variant; Sudhindra, Tanveer and Melisa for valuable discussions and insights into life beyond graduate school; Ayesha Sharmin, Reuben Darlington and Dr. Ross for data analysis and guidance with FRET experiments and software training. I must thank my dear friends, Edmir, Nelda, Michael, Behrang, Joe, Tom, Ricardas, Ryan, Jessica, Kurtis, Becky and Jeff for making my educational experience a wonderful one even during star-crossed situations. I especially would like to thank my parents, Martha and Franco Sr., for all they have sacrificed of themselves so that I may be able to pursue a tertiary education; my sisters Marsha and Jasmine for their continued support and encouragement; my wife, Gianna, for her selflessness, support, encouragement, sacrifice and patience during my studies. Finally, to my committee members – Drs. Michele McGuirl, Klára Briknarová, Michael DeGrandpre and J. B. Alexander Ross – thank you for your guidance and time. iii Table of Contents Abstract ii Acknowledgements iii Table of Contents iv List of Figures vii List of Tables x Chapter 1 The Protein Denatured State 1.1 Proteins and the Polypepide Chain 1 1.2 Protein Folding Models 3 1.3 Difficulty Studying the Denatured State 8 1.4 Random or Non-Random Denatured State? 10 1.5 Experimental Evidence Supporting Non-random Coil 11 1.6 Denatured State Loop Formation 17 1.7 Inspiration for Dissertation 21 1.8 Goals of Dissertation 22 iv Chapter 2 Local Sequence and Composition Effects on a 22-residue Loop with a Single Gly_Ala Insert at the N-terminus of Iso-1-cytochrome c 2.1 Introduction 26 2.2 Materials and Methods 33 2.3 Results 46 2.4 Discussion 62 2.5 Conclusion 75 Chapter 3 Thermodynamics and Kinetics of Loop Formation with Homopolymeric Polyalanine Sequences from the N-terminus of Iso-1-cytochrome c in Poor and Good Solvent Conditions 3.1 Introduction 76 3.2 Materials and Methods 82 3.3 Results 90 3.4 Discussion 106 3.5 Conclusion 115 Chapter 4 Competition between Reversible Aggregation and Loop Formation in Denatured Iso-1-cytochrome c 4.1 Introduction 117 4.2 Materials and Methods 119 4.3 Results 126 4.4 Discussion 151 4.5 Conclusion 162 v Chapter 5 Probing Denatured State Distributions Relevant to the Initial Events of Protein Folding using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) 5.1 Introduction 164 5.2 Materials and Methods 168 5.3 Results 176 5.4 Discussion 188 5.5 Conclusion 196 References 199 Appendices 216 Appendix A 216 Appendix B 217 Appendix C 219 vi List of Figures FIGURE PAGE 1.1 Predetermined Pathway-Optional Error Folding Model 5 1.2 Folding Funnel Energy Landscape 6 1.3 Folding Funnel with Intermolecular Aggregation 8 Schematic Representation of m-values and Denatured State 1.4 12 Compaction 1.5 Hydrogen Exchange Experimental Scheme 14 Schematic Representation of Loop Formation in the Denatured 1.6 18 State – Heme Edge versus Wrap Around 2.1 Schematic Representation of Denatured State His-heme Ligation 29 2.2 B-factor Putty Representation of Disordered Region of Insertions 34 2.3 MALDI-TOF Spectrum of Fragmented Protein 40 MALDI-TOF Spectrum of EDTA Minimized Protein 2.4 41 Fragmentation 2.5 MALDI-TOF Spectrum of Optimized Purification with EDTA 42 2.6 Typical CD Denaturation Curve Obtained with AP PiStar 180 46 2.7 Typical CD Denaturation Curve Obtained with AP Chirascan 47 Representative Equilibrium Loop Formation Titration with DU- 2.8 51 640 2.9 pH Titration Reproducibility 54 pK Plotted as a Function of Glycine Percentage Segregated by 2.10 a 55 Local Sequence Loop Breakage and Formation Rate Constants as a Function of 2.11 61 Glycine Percentage Segregated by Local Sequence Typical CD Denaturation Curve of Poly(Ala) Variants Obtained 3.1 90 with AP Chirascan Typical CD Denaturation Curve of Cytochrome c’ Variants 3.2 91 Obtained with AP Chirascan Stability Plots of Cytochrome c’ Global Stability and m-values as 3.3 93 a Function of Loop Size Representative Equilibrium Loop Formation Titration of 3.4 94 Poly(Ala) Variants with Isoplot Equilibrium Loop Formation Titration with DU-800 in 3 M and 6 3.5 95 M gdnHCl Equilibrium obtained Scaling Factors of Poly(Ala) Variants in 3.6 97 “Poor” and “Good” Solvents Representative Equilibrium Loop Formation Titration of Biphasic 3.7 98 Cytochrome c’ Variants vii Representative Equilibrium Loop Formation Titration of 3.8 99 Monophasic Cytochrome c’ Variants Comparison of Scaling Factors for