Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 11-2-2020 Salt Dependence of Thermodynamic Stability of a Cold-Active DNA Polymerase I Fragment Xinji Zhu Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Zhu, Xinji, "Salt Dependence of Thermodynamic Stability of a Cold-Active DNA Polymerase I Fragment" (2020). LSU Doctoral Dissertations. 5389. https://digitalcommons.lsu.edu/gradschool_dissertations/5389 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. SALT DEPENDENCE OF THERMODYNAMIC STABILITY OF A COLD-ACTIVE DNA POLYMERASE I FRAGMENT A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Xinji Zhu B.S., East China University of Science and Technology, 2012 December 2020 ACKNOWLEDGMENTS As many long-term research projects, my dissertation demanded a lot of energy and time. During this process, many people have contributed to my dissertation and I am grateful for their support and time. I would like to express my great appreciation to Dr. Vince LiCata, my major supervisor, for his patient guidance, enthusiastic encouragement, and valuable critiques of my research work. Without his support, this dissertation would not have been possible. I would also like to thank my committee members, Dr. Naohiro Kato and Dr. John Battista, who give me a lot of advice and support in my research. I would also extend my thanks to the members of the LiCata Lab: Dr. Chin-Chi Liu, Dr. Hiromi Brown, Dr. Jaycob Warfel, Tod Baker, Wendy Hansing, Erin LeBoeuf, and Katelyn Jackson for their technical and emotional support. I would like to thank the financial, academic and educational support of the Louisiana State University Department of Biological Sciences and its staff; Dr. Ted Gauthier and Dr. Rafael Cueto for their technical support in data collection; Dr. Bill Wischusen, Dr. Jane Reiland and Dr. Christopher Gregg for their teaching guidance. Finally, I would especially thank my family. My wife, Yuli, is extremely supportive of me and has made extraordinary sacrifices to help me get his point. Also, I would like to thank my mother and father for their continued support and encouragement. ii TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………………....ii LIST OF TABLES………………………………………………………………....v LIST OF FIGURES………………………………………………………………vii LIST OF ABBREVIATIONS……………………………………………………...x ABSTRACT……………………………………………………………………….xi CHAPTER 1. GENERAL INTRODUCTION……………………………………………1 1.1 Life Adaptation in Extreme Environments ............................................................................ 1 1.2 Protein Folding in Psychrophiles and Halophiles .................................................................. 4 1.3 Thermodynamics and Kinetics in Protein Folding ................................................................ 6 1.4 DNA Polymerase I ............................................................................................................... 13 1.5 Intrinsically Disordered Protein (IDPs) ............................................................................... 17 CHAPTER 2. MATERIALS AND METHODS………………………………………...19 2.1 Materials .............................................................................................................................. 19 2.2 Methods…………………………………………………………………………………….20 CHAPTER 3. SALT DEPENDENCE OF THE THERMAL STABILITY OF KLENPIN POLYMERASE………………………………………………..39 3.1 General Characterization of Klenpin Polymerase ................................................................ 39 3.2 Effect of Salt on the Thermal Stability of Klenpin and Klenow .......................................... 43 3.3 PH Effects on Polymerase Secondary Structures ................................................................ 53 3.4 Summary .............................................................................................................................. 57 CHAPTER 4. EFFECTS OF SALT ON THE FOLDING FREE ENERGY AND STRUCTURE OF KLENPIN DNA POLYMERASE………………...59 4.1 Thermodynamic Stability of Klenpin by Chemical Denaturation ....................................... 59 4.2 Chemical Denaturation of Klenpin: Reversibility and Kinetics .......................................... 64 4.3 Effect of Salt on Chemical Denaturation of Klenpin and Klenow ...................................... 69 4.4 Structural Effect of Salt on Klenpin .................................................................................... 75 CHAPTER 5. COMPUTATIONAL BASED INVESTIGATION OF THE STABILITY OF KLENPIN POLYMERASE………………………….84 5.1 Comparison of the Electrostatic Potential of Klenpin, Klenow, and Klentaq ..................... 85 5.2 Amino Acid Composition Comparison among Klenpin, Klenow, and Klentaq.................. 87 5.3 Acidic Signature in DNA Polymerase I of P. ingrahamii ................................................... 89 iii 5.4 Comparison of Helix Content through Computational Method .......................................... 91 5.5 Amino Acid Composition Preferences from P. ingrahamii, E. coli, and T. aquaticus ....... 93 5.6 Intrinsic Disordered Regions (IDRs) ................................................................................... 96 5.7 Summary .............................................................................................................................. 97 CHAPTER 6. DISCUSSION AND CONCLUSION…………………………………..99 6.1 Discussion ............................................................................................................................ 99 6.2 Conclusion ......................................................................................................................... 105 APPENDIX A. SUMMARY OF MELTING TEMPERATURES(TMS) OF KLENPIN AND KLENOW…………………………………………...107 APPENDIX B. RESEARCH SUMMARY OF GREEN FLUORESCENT PROTEIN…………………………………………………………………109 REFERENCES……………..………………………………………………………………..121 VITA…………………………………………………………………………………………..148 iv LIST OF TABLES 1.1. Pairwise sequence alignment among Klenpin, Klenow, and Klentaq……………….17 2.1. Composition of bacterial growth media…………………………………….............. 20 2.2. Components for resolving gel .................................................................................... 21 2.3. Components for stacking gel. .................................................................................... 22 3.1. Summary of binding affinity (K50) and maximal Tm shift of Klenpin…….............49 4.1. Summary of 훥퐺퐻2푂 at different temperatures…………………………….............60 4.2. Comparison of Klenpin, Klenow, and Klentaq .......................................................... 60 4.3. Thermodynamic Stability comparison ....................................................................... 62 4.4. The relaxation rate constants (푙표푔Krel) of Klenpin folding/unfolding. .................... 68 4.5. Thermodynamic Stability comparison in GdnHCl and urea denaturation…………..70 4.6. Thermodynamic parameters of Klenow in urea denaturation at. ............................... 73 4.7. Thermodynamic parameters of Klenpin in urea denaturation. .................................. 73 4.8. Thermodynamic parameters of Klenpin in GdnHCl denaturation…………..............73 4.9. Summary of Z-average of Klenpin, Klenow and Klentaq ......................................... 79 4.10. Summary of Ksv values for Klenpin and Klenow. ................................................ 80 4.11. Solvent accessibility of Tryptophan in Klenpin and Klenow by STRIDE. ............. 82 5.1. Amino acid composition of Klenpin, Klenow and Klentaq………………………....87 5.2. Comparison of charged and hydrophobic residues .................................................... 89 5.3. Comparison of the acidic features.............................................................................. 90 5.4. Comparison of helical content. .................................................................................. 92 5.5. The Difference in individual amino acids of 100 proteins. ....................................... 94 5.6. Distribution of S, D, T, A, E, L in coli regions and helical regions. ......................... 95 5.7. Comparison of the intrinsic disorder among Klenpin, Klenow, and Klentaq. ........... 97 v 5.8. Intrinsically disordered region comparison. .............................................................. 97 vi LIST OF FIGURES 1.1. Gibbs free energy of homologous extremophilic proteins……………………….….7 1.2. Gibbs energy diagram……………………………………………………………...11 1.3. Single-mixing refolding kinetics of Klenpin by circular dichroism………………..13 1.4. 3D structure of DNA polymerase I from Thermus aquaticus………………………14 1.5. An overview of DNA polymerization reaction……………………………………..15 2.1. An idealized denaturation curve of the three-state model…………………..............28 2.2. An idealized chevron plot showing the relaxation rate constant. .............................. 31 2.3. The intensity distribution of Klenpin in the absence of salt by DLS. ........................ 35 2.4. The electrostatic surface potential