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Small and Large Cosolutes Modulate Enzyme Activity and Protein Folding Kinetics

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Small and Large Cosolutes Modulate Enzyme Activity and Protein Folding Kinetics SMALL AND LARGE COSOLUTES MODULATE ENZYME ACTIVITY AND PROTEIN FOLDING KINETICS Annelise Hocevar Gorensek A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry. Chapel Hill 2017 Approved by: Gary J. Pielak Eric M. Brustad Sharon L. Campbell Bo Li Marcey L. Waters ©2017 Annelise H. Gorensek ALL RIGHTS RESERVED ii ABSTRACT Annelise H. Gorensek: Small and Large Cosolutes Modulate Enzyme Activity and Protein Folding Kinetics (Under the direction of Gary J. Pielak) Proteins are the powerhouse of the cell, catalyzing chemical reactions, serving as signaling hubs, and maintaining cellular structure. Recently, several investigators have advanced our understanding of how the crowded cellular interior, and common cytoplasm mimetics such as synthetic polymers and commercially available proteins, affect protein equilibrium stability. These efforts show that attractive chemical interactions between proteins and macromolecular crowding agents, can destabilize proteins, contradicting the long-held idea that steric repulsions under crowded conditions increase protein stability. My work expands on these studies by examining the effect of crowding on enzyme activity and protein folding kinetics. In the first part of my dissertation, I examine the effects of synthetic polymers on the activity of the monomeric enzyme E. coli dihydrofolate reductase as a function of polymer concentration, fractional volume occupancy and viscosity. I compare my results to those in the literature, and find that crowding effects are small, difficult to predict, and depend on the crowder, protein, and even the substrate. To investigate the influence of crowding on folding kinetics, I examined the temperature-dependent folding and unfolding of the small, metastable, N-terminal SH3 domain of the drosophila signaling protein drk. The results contradict classical crowding theory in that osmolytes and polymers alter folding barriers via both enthalpic and entropic contributions. Additionally, the entropic components suggest that solvent and cosolute entropy, rather than configurational entropy, are the dominating effect. Comparing activation parameters from synthetic polymer cosolutes to iii those from the biologically relevant crowder, lysozyme, indicate that synthetic polymers are poor mimics of the cellular interior. However, my results build a foundation for studies to interpret folding landscapes in biologically-relevant crowded conditions. iv To my parents, Maximilian Boris Gorensek and Annmarie Hocevar Gorensek. v ACKNOWLEDGEMENTS I would not be where I am today without the blessing of a lineage of strong mentors who helped shaped me. Thank you to my first mentors: my father, who answered my endless questions about how the world worked, and my mother who encouraged me to read as much as I possibly could. In high school, my chemistry teacher Shannon Williams captured my interest by describing the elements as people (“Francium, he can’t keep his electrons together…”) while Dottie Andreassen showed me how to communicate my thoughts through writing, which has served me well. As a nervous senior visiting Furman University on accepted students’ day I met Dr. Karen Buchmueller, who became my academic, research, and essentially life, advisor. Karen gave me the skills I needed to become a scientist, but the dedication she showed to me and her other students inspired me to become an educator. Thank you, Karen for investing in me even after I graduated, and for talking me through some of my most difficult times both at Furman and at UNC. It was in Karen’s lab at Furman that I met Austin Smith, who at first terrified me (I ‘gave him up for Lent,’ which is still part of departmental lore) but after working together in the Pielak lab at UNC, he became my mentor and personal NMR 911 hotline. Thank you, Austin for teaching me to purify protein, how to not break the NMR, for getting me started on the SH3 project, and finally for challenging me to think deeply about my work. vi I joined Gary Pielak’s lab because I wanted to study proteins and thermodynamics was my second-favorite class after biological chemistry, so it seemed like a natural fit. I chose well, even if I ended up studying kinetics instead. Gary taught me the importance of reading the manual first, of thinking before speaking, and in the process constantly kept me on my toes. He has challenged me more than anyone else ever has and helped me realize my own potential as a scientist, teacher, and mentor. But most importantly, he helped me take the first step towards my dream of being a professor by allowing me to mentor two dedicated, hardworking undergraduates. Thank you, Gary for challenging me and supporting me as I prepare for my career after graduation. Thank you to the Pielak lab members, old and new, for your help and encouragement along the way. Thank you to Will Monteith, Mohona Sarkar and Jillian Tyrrell for teaching me the ways of the lab. Thank you to Larry Zhou for showing me the ropes of SH3 purification and Michael Senske, for suffering through it along with me. Rachel Cohen, thank you for encouraging me to stay active, to take my time, to take a chance and apply for an internship and for the countless encouraging conversations we had over coffee, lunch, or at Weaver Street. I would not have made it through Year 4 without you. Thank you to Sam Stadmiller, Pixie Piskiewicsz, Thomas Boothby, Alex Guseman, Candice Crilley and Shannon Speer for encouragement, for thoughtful conversations about science, and for helping me unwind during this last semester. Finally, I need to recognized my undergraduate mentees, Luis Acosta and Gerardo Perez Goncalves, who worked tirelessly and gifted me with my first opportunity to be a research mentor. Thank you to the other people who have kept me going outside of the lab: to Greg Young and Karl Koshlap for keeping me company in the basement and answering my NMR questions and to Reggie Singleton and Karen Gilliam for always being happy to chat and for having a full candy jar. vii Thank you to my sister, Natalie Gorensek, for loving science along with me and for being my first student (she can still recite the Mercury 7 in alphabetical order). Finally, thank you to my husband, Ryan Benitez for being the ultimate partner and support system during my 5 years here, all while living nearly 150 miles away. Thank you for staying up past your bedtime to talk me through science drama, for helping me figure out my statistical analysis and for working alongside me on weekends when you’d rather play Ultimate. But most of all thank you for your inexhaustible belief in me, it has made all the difference. viii TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................ xii LIST OF TABLES ................................................................................................................. xiii LIST OF ABBREVIATIONS AND SYMBOLS ....................................................................... xiv CHAPTER 1: A HISTORY OF PROTEIN FOLDING KINETICS IN BUFFER AND IN COSOLUTES .......................................................................................................................... 1 Introduction .......................................................................................................................... 1 Transition state theory ......................................................................................................... 2 Activation parameters and protein folding ........................................................................... 5 Activation parameters in buffer ............................................................................................ 9 Activation parameters in denaturants ................................................................................ 12 Activation parameters in stabilizing osmolytes .................................................................. 16 Activation parameters in synthetic polymers ..................................................................... 17 Activation parameters in live cells ..................................................................................... 19 Conclusions ....................................................................................................................... 20 Tables ................................................................................................................................ 22 Figures ............................................................................................................................... 23 CHAPTER 2: LARGE COSOLUTES, SMALL COSOLUTES AND ENZYME ACTIVITY ...... 27 Introduction ........................................................................................................................ 27 Results ............................................................................................................................... 29 Polymer and monomer properties ................................................................................. 29 DHFR activity ................................................................................................................. 29 Discussion ......................................................................................................................... 30 Crowder identity ............................................................................................................
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