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Comparative Thermodynamics of Partial Agonist Binding to An COMPARATIVE THERMODYNAMICS OF PARTIAL AGONIST BINDING TO AN AMPA RECEPTOR BINDING DOMAIN A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Madeline Martínez May 2015 © 2015 Madeline Martínez ALL RIGHTS RESERVED Comparative Thermodynamics of Partial Agonist Binding to an AMPA Receptor Binding Domain Madeline Martínez, Ph.D. Cornell University 2015 AMPA receptors, ligand-gated cation channels endogenously activated by glutamate, mediate the majority of rapid excitatory synaptic transmission in the vertebrate central nervous system and are crucial for information processing within neural networks. Their involvement in a variety of neuropathologies and injured states makes them important therapeutic targets, and understanding the forces that drive the interactions between the ligands and protein binding sites is a key element in the development and optimization of potential drug candidates. Calorimetric studies yield quantitative data on the thermodynamic forces that drive binding reactions. This information can help elucidate mechanisms of binding and characterize ligand- induced conformational changes, as well as reduce the potential unwanted effects in drug candidates. The studies presented here characterize the thermodynamics of binding of a series of 5’substituted willardiine analogues, partial agonists to the GluA2 LBD under several conditions. The moiety substitutions (F, Cl, I, H, and NO2) explore a range of electronegativity, pKa, radii, and h-bonding capability. Competitive binding of these ligands to glutamate-bound GluA2 LBD at different pH conditions demonstrates the effects of charge in the enthalpic and entropic components of the Gibb’s free energy of binding. They suggest that the structure of agonists can be changed to improve the enthalpic energetic component to binding by building additional charge interactions in the portion of the molecule interacting with lobe 2. The differences in heat capacity change between the analogue-bound and glutamate-bound states highlights the effect of charge and additional hydrogen bond formation on the temperature dependence of thermodynamic parameters, and how this dependence affects the driving forces of the competitive binding reactions at physiological temperatures. Competitive binding experiments in multiple buffers yield the contributions of buffer ionization to the measured enthalpy of binding, as well as the differences in protonation states between the protein bound to glutamate, willardiine, and 5-substituted analogues. The exchange of glutamate for willardiine entails additional proton uptake into the LBD-ligand complex. The addition of more electronegative substituents into the 5-position eliminates the protonation differences between the glutamate and analogue-bound complexes. Bulkier substituents result in a ligand exchange where more protons are released into the buffer. Excision of buffer ionization contribution to the observed enthalpic parameters revealed that there is no statistical difference between the enthalpic components of binding to glutamate and fluorowillardiine. While there is no buffer-independent significant differences between the enthalpic parameters of binding of willardiine and the remaining halogenated analogues, their differences in protonation events suggests their intrinsic enthalpies of binding might be different from each other. This information allowed the estimation of thermodynamic parameters of binding at physiological temperature, 37oC. At this temperature the competitive binding of willardiine and the halogenated analogues is driven by favorable entropic change. The additional hydrogen bonding and electrostatic interactions possible with the nitro 5-substituent result in a competitive binding driven by favorable enthalpic change that occurs at significant entropic penalty. Biographical Sketch Madeline Martínez was born in San Juan, Puerto Rico in 1981. She obtained a Bachelor of Science from the University of Puerto Rico, Río Piedras, in 2005 where she majored in chemistry. As an undergraduate she acquired research experience in biochemistry and electrophysiology. Her research projects in the University of Puerto Rico, under the supervision of Prof. Kai Griebenow focused on the stabilization and preservation of protein structure and activity with the goal of increasing the range of available protein pharmaceuticals. She studied the effects of covalent modifications and exposure to solvents and excipients used in drug encapsulation on the enzymatic and structural properties of model enzymes through spectrophotometric techniques such as CD and FTIR spectroscopy. As part of the Leadership Alliance Summer Research-Early Identification Program (SR-EIP) she conducted research under the supervision of Prof. George Hess at Cornell University. She measured the effects of cocaine exposure on glutamate-elicited whole cell currents in kainate receptors expressed in HEK 293 cells. During this time she became fascinated with neurotransmitter receptors and signaling. In 2005 Madeline Martínez was admitted to Cornell University to pursue her PhD in Pharmacology, where she joined the laboratory of Prof. Robert Oswald, and has focused on understanding the dynamics and thermodynamics of glutamate receptors. v Dedication This work is dedicated to my spouse Jaime J. Benítez, my parents Arnaldo and Madeline, my sisters Ileana and Tania. Everything is possible with your support and love. vi Acknowledgements First of all I thank my, complex, extended, and wonderful family. My parents, grandparents, stepmother Gladys, aunts and uncles who, along with my parents, ensured I had everything I needed to develop and learn. I thank Grandmother Elba who tutored and guided my studies and upbringing through all my school years, and Grandmother Olga who showed me firsthand how scientific knowledge can save lives and small kindnesses can transform communities. I thank my advisor Dr. Robert Oswald for giving me the opportunity to perform my doctoral research in his laboratory. I am grateful for his mentorship, advice, and for encouraging me to persevere when the systems under study seemed incompatible with the techniques used to study them. I would also like to acknowledge all the members of the Oswald group, past and present, for all our conversations, help, and camaraderie. I’m especially thankful for the help and collaboration of Dr. Ahmed H. Ahmed, whose crystallographic and NMR studies of willardiine protonation and deprotonation allowed a greater understanding of their binding thermodynamics. I would also like to express thanks to the members of my committee, Dr. Linda Nicholson, Dr. Manfred Lindau, and Dr. Gregory Weiland, who provided me with advice, ideas, and guidance throughout this endeavor. I am very thankful for the help, advice, and encouragement I have received from Dr. Linda Nowak and Susan Coombs. I am also grateful of Dr. Adrienne Loh for allowing me to use her calorimeter for such a long period of time, as well as for all her help and ideas. Few things are as pretty as the thermogram baselines in her calorimeter! vii Table of Contents Biographical Sketch ...................................................................................................................................... v Dedication .................................................................................................................................................... vi Acknowledgements ..................................................................................................................................... vii Table of Contents ....................................................................................................................................... viii List of Figures .............................................................................................................................................. xi List of Tables ............................................................................................................................................. xiii List of Abbreviations ................................................................................................................................. xiv Chapter 1 Glutamate Receptors and Calorimetry ......................................................................................... 1 L-Glutamate .............................................................................................................................................. 4 Glutamate Receptor Classification ............................................................................................................ 5 Ionotropic Glutamate Receptor Subtypes ................................................................................................. 6 AMPA Receptors .................................................................................................................................. 6 Kainate Receptors ............................................................................................................................... 11 NMDA Receptors ............................................................................................................................... 14 Delta Receptors ................................................................................................................................... 18 AMPA Receptor Structure and Dynamics .............................................................................................
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