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The Complex Role of Sequence and Structure in the Stability and Function of the TIM Barrel Proteins

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The Complex Role of Sequence and Structure in the Stability and Function of the TIM Barrel Proteins University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2017-11-03 The Complex Role of Sequence and Structure in the Stability and Function of the TIM Barrel Proteins Yvonne H. Chan University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Biochemistry Commons, Biophysics Commons, and the Molecular Biology Commons Repository Citation Chan YH. (2017). The Complex Role of Sequence and Structure in the Stability and Function of the TIM Barrel Proteins. GSBS Dissertations and Theses. https://doi.org/10.13028/M2J09H. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/934 Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. THE COMPLEX ROLE OF SEQUENCE AND STRUCTURE IN THE STABILITY AND FUNCTION OF TIM BARREL PROTEINS A Dissertation Presented By YVONNE HOI YAN CHAN Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester In partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY November 3, 2017 Biochemistry and Molecular Pharmacology THE COMPLEX ROLE OF SEQUENCE AND STRUCTURE IN THE STABILITY AND FUNCTION OF TIM BARREL PROTEINS A Dissertation Presented By YVONNE HOI YAN CHAN This work was undertaken in the Graduate School of Biomedical Sciences Biochemistry and Molecular Pharmacology Under the mentorship of C. Robert Matthews, Ph.D., Co-Thesis Advisor Konstantin B. Zeldovich, Ph.D., Co-Thesis Advisor Brian A. Kelch, Ph.D., Member of Committee David G. Lambright, Ph.D., Member of Committee William E. Royer, Ph.D., Member of Committee Eugene I. Shakhnovich, Ph.D., External Member of Committee Daniel N. Bolon, Ph.D., Chair of Committee Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences November 3, 2017 III Dedication To my parents, for your unconditional support. To my sisters, for letting me shop at your houses. To my brother-in-laws, for reading all my applications. To my boyfriend, for being there when I need you the most. To my friends, for letting me be myself. IV Acknowledgments This thesis is the product of the guidance from my two co-advisors: Dr. C. Robert Matthews and Dr. Konstantin B. Zeldovich. They trained me with their expertise; they inspired me with their passion for science. Dr. Daniel N. Bolon served as my unofficial third advisor. He welcomed me into his lab and helped me bring my experiments to life. My committee members, Dr. Brian A. Kelch, Dr. David G. Lambright, and Dr. William E. Royer, always asked thought-provoking questions. They pushed me to think critically and refined my research in countless ways. My external committee member, Dr. Eugene I. Shakhnovich, set the bar for seamlessly integrating multiple fields of science to which I aspire. My friend and collaborator, Dr. Sergey V. Venev, added new dimensions to our paper and greatly improved my bioinformatics skills along the way. Dr. Troy Whitfield provide numerous useful discussions. The past and current members of the Matthews Lab have contributed immensely to my research. Dr. Sagar Kathuria always made time for me and others in the lab. Dr. R. Paul Nobrega led the graduate students with his dedication and intelligence. Kevin Halloran provided invaluable discussion, suggestions, and reminders. Dr. Jill Zitzewitz always knew what to say. Dr. Osman Bilsel had the calmest demeanor and thoughtful suggestions. My interaction with each lab member has trained me to be a better scientist: Noah Cohen, Meme Tran, Dr. Brian Mackness, Dr. Sujit Basak, Dr. Rohit Jain, and Dr. Nidhi Joshi. V Members of the Bolon lab, Dr. Ryan Hietpas, Dr. Ben Roscoe, Dr. Parul Mishra, Dr. Li Jiang, and Dr. Julia Flynn, provided incredibly helpful insights to my project. Adam Choi, a Ph. D. candidate working under the guidance of Dr. Stephen Miller, synthesized a key compound for my enzyme kinetic assay and provided valuable discussions throughout my training career. VI Abstract Sequence divergence of orthologous proteins enables adaptation to a plethora of environmental stresses and promotes evolution of novel functions. As one of the most common motifs in biology capable of diverse enzymatic functions, the TIM barrel represents an ideal model system for mapping the phenotypic manifestations of protein sequence. Limits on evolution imposed by constraints on sequence and structure were investigated using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Exploration of fitness landscapes of phylogenetically distant orthologs provides a strategy for elucidating the complex interrelationship in the context of a protein fold. Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape. These results suggest that fitness landscapes of point mutants can be successfully translocated in sequence space, where knowledge of one landscape may be predictive for the landscape of another ortholog. VII Analysis of a surprising class of beneficial mutations in all three IGPS orthologs pointed to a long-range allosteric pathway towards the active site of the protein. Biophysical and biochemical analyses provided insights into the molecular mechanism of these beneficial fitness effects. Epistatic interactions suggest that the helical shell may be involved in the observed allostery. Taken together, knowledge of the fundamental properties of the TIM protein architecture will provide new strategies for de novo protein design of a highly targeted protein fold. VIII Table of Contents Contents 1. DEDICATION ........................................................................................... III 2. ACKNOWLEDGMENTS ........................................................................... IV 3. ABSTRACT .............................................................................................. VI 4. TABLE OF CONTENTS ......................................................................... VIII 5. LIST OF FIGURES ................................................................................... XI 6. LIST OF TABLES ................................................................................... XIII 7. LIST OF ABBREVIATIONS .................................................................. XIV 8. PREFACE .............................................................................................. XVI 9. CHAPTER I – INTRODUCTION .............................................................. 17 General overview: Intersecting protein biophysics and molecular evolution ....................... 17 Historical highlights and general principles of protein folding .............................................. 18 Protein sequence and stability ................................................................................................... 19 Types of protein interactions ...................................................................................................... 20 BASiC hypothesis ........................................................................................................................ 21 Protein evolution and physical constraints .............................................................................. 22 Inverse protein folding problem ................................................................................................. 24 TIM barrels as a model system for intersecting protein biophysics and protein evolution 24 Structure and stability of IGPS ................................................................................................... 27 IX IGPS enzyme chemistry .............................................................................................................. 27 Tryptophan biosynthetic pathway and regulation .................................................................... 28 Fitness landscapes ...................................................................................................................... 29 EMPIRIC fitness screen ............................................................................................................... 29 Scope of project ........................................................................................................................... 30 10. CHAPTER II – CORRELATION OF FITNESS LANDSCAPES FROM THREE ORTHOLOGOUS TIM BARRELS ORIGINATES FROM SEQUENCE AND STRUCTURE CONSTRAINTS .................................. 33 Introduction .................................................................................................................................. 34 Results .......................................................................................................................................... 36 αβ-loops ....................................................................................................................................
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