University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Protein Dynamics and Entropy: Implications for Protein-Ligand Binding Kyle William Harpole University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Harpole, Kyle William, "Protein Dynamics and Entropy: Implications for Protein-Ligand Binding" (2015). Publicly Accessible Penn Dissertations. 1756. https://repository.upenn.edu/edissertations/1756 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1756 For more information, please contact [email protected]. Protein Dynamics and Entropy: Implications for Protein-Ligand Binding Abstract The nature of macromolecular interactions has been an area of deep interest for understanding many facets of biology. While a great deal of insight has been gained from structural knowledge, the contribution of protein dynamics to macromolecular interactions is not fully appreciated. This plays out from a thermodynamic perspective as the conformational entropy. The role of conformational entropy in macromolecular interactions has been difficulto t address experimentally. Recently, an empirical calibration has been developed to quantify the conformational entropy of proteins using solution NMR relaxation methods. This method has been demonstrated in two distinct protein systems. The goal of this work is to expand this calibration to assess whether conformational entropy can be effectively quantified from NMR-derived protein dynamics. First, we demonstrate that NMR dynamics do not correlate well between the solid and solution states, suggesting that the relationship between the conformational entropy of proteins is limited to solution state-derived NMR dynamics. We hypothesize that this may be partially due to the role of hydration of the protein in its dynamics. Next, we expand our empirical calibration to over 30 distinct protein systems and demonstrated that the relationship between NMR dynamics and conformational entropy is both robust and general. Furthermore, we demonstrate that conformational entropy plays a significant role in macromolecular interactions. Using our empirical calibration, we then look to address if conformational entropy could be an important contribution to drug design. The latter process is often a brute force approach, and subsequent optimization of initial drug candidates is often a guess and check process. In silico drug design was thought to offer a more efficient and rational approach, but often relies on static structures. This minimizes or completely neglects the role that conformational entropy may play in binding. Here we experimentally determine the role of conformational entropy in the drug target p38a MAPK in binding to two potent inhibitors. We demonstrate evidence that conformational entropy may represent a tunable parameter in affinity optimization of lead compounds. This has important implications for lead optimization and strongly suggests that the role of conformational entropy be considered in drug design efforts. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor A. Joshua Wand Keywords Drug Design, Entropy, Protein Dynamics, Protein-Ligand Binding Subject Categories Biochemistry | Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1756 PROTEIN DYNAMICS AND ENTROPY: IMPLICATIONS FOR PROTEIN-LIGAND BINDING Kyle William Harpole A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2015 Supervisor of Dissertation ________________ A. Joshua Wand, Ph.D., Benjamin Rush Professor of Biochemistry and Biophysics Graduate Group Chairperson ________________ Kim A. Sharp, Ph.D., Associate Professor Dissertation Committee Ronen Marmorstein, Ph.D., Professor of Biochemistry and Biophysics Yale E. Goldman MD, Ph.D., Professor of Physiology Jeffery G. Saven, Professor of Chemistry Kim A. Sharp, Ph.D., Associate Professor of Biochemistry and Biophysics Gregory D. Van Duyne, Ph.D., Jacob Gershon-Cohen Professor of Medical Science Andrew L. Lee, Ph.D., Professor, Division of Chemical Biology and Medicinal Chemistry PROTEIN DYNAMICS AND ENTROPY: IMPLICATIONS FOR PROTEIN-LIGAND BINDING COPYRIGHT 2015 Kyle William Harpole This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ To my wife, Grace, for her unyielding love and support. iii ACKNOWLEDGMENT First, I must acknowledge my thesis mentor, Josh Wand, for all of his guidance and support over the course of my graduate career. I imagine that being a successful mentor takes a number of essential skills. The mentor must be patient. The mentor must also challenge. Often the mentor must also do both simultaneously. However, I feel that one of the marks of a great mentor is to believe in their students and the work they do, even when the student may not believe in themselves at times. This is something that Josh has done for me on various occasions, and I will always owe a great deal of success to him in this regard. I also wish to thank Kathy Valentine for teaching me the basics of NMR as a technique. I must also thank her for her support and assistance, both scientific and otherwise. Like Josh, she has dedicated a good deal of her career to foster both the scientific and personal development of all members of the lab. I thank my committee – Ronen Marmorstein, Yale Goldman, Jeff Saven, and Kim Sharp, for challenging my ideas and pushing me in the direction of good science. Also thanks to Greg Van Duyne for supplementing my committee for the final defense and Andrew Lee for serving as an external examiner. I thank the members of the Wand Laboratory, both past and present. I must especially thank a few members of the lab who played a variety of important roles: Jakob Dogan and Vonni Moorman for assistance during the early days of my graduate career, Christine Jorge for her insightful discussion but also for her knowledge of the table of contents function in Microsoft Word. I thank Bryan Marques, Jack Wee Lim, Nathaniel Nucci, Evan O’Brien, Alfredo Caro, and fellow Oklahoma expat Brian Fuglestad for helpful scientific discussions. I thank Matt Stetz for his deep knowledge of NMR relaxation. Special thanks to him for the development of pulse sequences and software that had a significant positive impact on the lab as a whole, including many crucial aspects of this dissertation. I thank Vignesh Kasinath, fellow architect of the entropy meter, for discussion of conformational entropy and all aspects of life, several of which were, like both of us, a bit quirky. I thank Sabrina Bédard for all-around support in all aspects of my graduate career. iv Further thanks to BMB administrators extraordinaire, Ruth Keris and Angie Young. You made me blissfully unaware of all of the paperwork that I must have required. I must thank several friends, especially Sandya Ajith, Helen Chen, Rob Culik, Sam Getchell , and Bridgin Lee for their support during the mutual endeavor of graduate school. I also thank a number of folks that served in various capacities toward my scientific development. Old mentors and colleagues from summers doing research at ConocoPhillips: Bernie Baldwin, James Howard, Jim Stevens , Amy Briggs, and Rachel Stryffeler. Coming from those 2 MHz magnets, I can truly say that I have come from humble magnetic beginnings. Mentors and colleagues from my undergraduate research lab at the University of Iowa: Heejin Lee, Sean Gu, and Lawrence Gray, and my undergraduate advisor Shahram Khademi. Of course, I would not be where I am without the unconditional love and support of my parents. My father, always the inquisitive engineer, sought to understand the details of my research and even playfully challenge it at times. I like to think that he only saw a prototypical version of the ‘entropy meter’ (Chapter 3) and, upon reviewing this work, is now more or less convinced that it is real. My mother’s support was also always important, even when she didn’t understand precisely what it was I was doing, she always encouraged me to discover great things. I believe that the work enclosed provides a great deal of support for these wishes. Finally, I must thank my wife Grace. She has seen me through the latter half of my graduate career and all that it has entailed. She played a crucial role in this work, though no scientific contributions were offered. Her role in my personal development is easily of comparable magnitude to the role that Josh played in my professional development. My life would be far more entropic without her. v ABSTRACT PROTEIN DYNAMICS AND ENTROPY: IMPLICATIONS FOR PROTEIN-LIGAND BINDING Kyle William Harpole A. Joshua Wand, Ph.D. The nature of macromolecular interactions has been an area of deep interest for understanding many facets of biology. While a great deal of insight has been gained from structural knowledge, the contribution of protein dynamics to macromolecular interactions is not fully appreciated. This plays out from a thermodynamic perspective as the conformational entropy. The role of conformational entropy in macromolecular