NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY IN THE STUDY OF PROTEIN-LIGAND INTERACTIONS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Daniel Leonard Morris May 2018 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY IN THE STUDY OF PROTEIN-LIGAND INTERACTIONS Daniel Leonard Morris Dissertation Approved: Accepted: _____________________________ _____________________________ Advisor Department Chair Dr. Christopher J. Ziegler Dr. Christopher J. Ziegler _____________________________ _____________________________ Committee Member Dean of the College Dr. Leah P. Shriver Dr. John C. Green _____________________________ _____________________________ Committee Member Dean of the Graduate School Dr. Sailaja Paruchuri Dr. Chand. K. Midha _____________________________ _____________________________ Committee Member Date Dr. Aliaksei Boika _____________________________ Committee Member Dr. Abraham Joy ii ABSTRACT Numerous nuclear magnetic resonance (NMR) experiments for observing protein- ligand interactions have been proposed over the years since NMR was first used to study biomolecules. In the work that follows, a survey of the most widely used methods is presented with applications to three protein systems. First, orthologous glutaredoxins from homo sapiens and some bacterial species were compared structurally to assay their binding interactions in a fragment-based drug discovery project. This analysis yielded ortholog specific binders to be considered as antibiotic drug leads against the glutaredoxins from Brucella melitensis and Pseudomonas aeruginosa. Next, a pedagogical research initiative was conducted along with an undergraduate biochemistry laboratory course to describe the folding and iron-sulfur cluster binding properties of the mitochondrial membrane protein mitoNEET. The students created several mitoNEET mutants and used 1D and 2D NMR experiments to characterize their effect on mitoNEET’s structure and ligand binding capabilities. Finally, the lysozyme isolated from hen egg whites was observed catalyzing the polymerization of a polyacetylene. This marked the first time a hydrolase was observed catalyzing a synthetic polymer. NMR, along with X-ray crystallography, was used to describe the reaction mechanism, kinetics, and properties of the polymer product. iii ACKNOWLEDGEMENTS Foremost, I would like to appreciate my advisor, Christopher Ziegler. I cannot thank you enough for your guidance and support through my final two years of graduate school. After the tumultuous events of Dr. Leeper’s departure I was welcomed into the Ziegler group with open arms and immediately treated as an equal among the group members. If not for your faith in my ability to make that transition and pick up the lysozyme project I doubt I would have reached matriculation. Next, I would like to thank Thomas Leeper for seeing the early potential in me and being my mentor through the first three years of graduate school. Most of my science knowledge and benchtop skills come directly from you. You took me into your group as an undergrad and offered me a job washing dishes on the side, allowing me to make the lab my home away from home. During the five years I worked for you I made some of my closest friends grinding away in KNCL 208 and it’s an experience I’ll never forget. It is bittersweet finishing my graduate career alone in the lab sitting at Stephanie’s old desk. The University gave you a raw deal and tore our science family apart. It still stings. Additionally, I would like to thank my friends and fellow graduate students who were integral to helping me develop my skills including Stephanie Bilinovich, Joel Caporoso, and Laura Crandall. Your individual expertise in NMR and X-ray crystallography aided me greatly. Thank you to my other colleagues past and present for their friendship and support including Allen Osinski, Briana Schrage, Kullapa Chanawanno, Megan Cruz, Alex Taraboletti, Mangaldeep Kundu, Caroline Davis, Ram Khattri, Shaun Christie, Nita Duangjumpa, Brandon Rapier, Will Komar, Marie Southerland, Greg Buchan, Roger Shi, and Jessi Baughman, among many others. I also would like to thank the small hoard of iv undergraduates that came though the Leeper lab and worked on projects with us, especially Alex DeFabio and Jacob Sweet. Thank you to my other committee members Leah Shriver, Sailaja Paruchuri, Aliaksei Boika, and Abraham Joy. I appreciate your advice and support and am grateful for your time spent serving on my committee. I also need to thank the Magnetic Resonance Center staff, especially Venkat Dudipala for his mentoring and advice, along with Simon Stakleff and Bart Hamilton for always being there with a friendly face and a good joke. I would also like to thank Nancy Homa and Jean Garcia for being very helpful and efficient administrative staff. The chemistry department could not run without them. Finally, I would like to thank my parents, Len and Darlene, along with the rest of my family for their boundless support and enthusiasm and for the steady stream of homemade Italian food. v TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………..ix LIST OF FIGURES………………………………………...……………………………..x LIST OF SCHEMES………………………………………………………...………….xvi LIST OF ABBREVIATIONS…………………………………………………..…..…xvii CHAPTER I. INTRODUCTION AND BACKGROUND………………………………...………..…1 1.1 Evolution of Proteins and the Structure/Function Relationship……………..........1 1.2 Nuclear Magnetic Resonance for Biological Samples………………..…………19 1.3 NMR Methods for Observing Protein-Ligand Interactions……………..………37 1.4 Protein Structure Calculation by NMR……………………………..…………...46 1.5 Survey of Proteins Investigated……………………………………...…………..59 II. AN NMR-GUIDED SCREENING METHOD FOR SELECTIVE FRAGMENT DOCKING AND SYNTHESIS OF A WARHEAD INHIBITOR…………………...….66 2.1 Introduction………………………………………………………………………66 2.2 Experimental……………………………………………………………………..70 2.3 Results and Discussion………………………………………………...………...92 2.4 Conclusions……………………………………………………………………..119 III. IDENTIFYING ORTHOLOG SELECTIVE FRAGMENT MOLECULES BY NMR AND BINDING ENHANCEMENT BY MODIFICATION WITH ACRYLAMIDE WARHEADS…………………………………………………………………………...121 3.1 Introduction……………………………………………………………………..121 vi 3.2 Experimental……………………………………………………………………129 3.3 Results and Discussion……………………………………………...………….139 3.4 Conclusions……………………………………………………………………..168 IV. MULTINUCLEAR NMR AND UV-VIS SPECTROCSCOPY OF SITE DIRECTED MUTANTS OF THE DIABETES DRUG TARGET PROTEIN MITONEET SUGGEST THAT FOLDING IS INTIMATELY COUPLED TO IRON-SULFUR CLUSTER FORMATION………………………………………………………………………..…170 4.1 Introduction……………………………………………………………………..171 4.2 Experimental……………………………………………………………………177 4.3 Results and Discussion…………………………………………………...…….180 4.4 Conclusions……………………………………………………………………..186 V. LYSOZYME-CATALYZED FORMATION OF A CONJUGATED POLYACETYLENE……………………………………………………………………190 5.1 Introduction……………………………………………………………………..191 5.2 Experimental……………………………………………………………………195 5.3 Results and Discussion……………………………………………………...….198 5.4 Conclusions……………………………………………………………………..212 VI. INHIBITION OF LYSOZYME’S POLYMERIZATION ACTIVITY USING A POLYMER STRUCTURAL MIMIC…………………………………………………..214 6.1 Introduction……………………………………………………………………..214 6.2 Experimental……………………………………………………………………216 6.3 Results and Discussion………………………………………………...……….219 6.4 Conclusions……………………………………………………………………..226 vii VII. SUMMARY…………………………………………………………...…………..227 REFERENCES………………………………………………………………………....230 viii LIST OF TABLES 1.1: Biologically Relevant NMR Active Nuclei….…………………………..…………22 1.2: Resonance frequencies and chemical sifts for a set of bromomethanes…………….24 1.3: Spin systems and their peak ratio intensities………………………………………..27 3.1: Dissociation constants and ligand efficiency for best hits…………………………150 6.1: Crystallographic data collection and structure parameters for PDB entry 6CIW….226 ix LIST OF FIGURES 1.1: Structure and nomenclature for the 20 common biological amino acids………...….7 1.2: Summary of the first three levels of protein structure………………………………11 1.3: Diagram of the effect of magnetic field on the transition energy between spin states……………………………………………………………………………………...22 1.4: Chemical shifts for ethyl acetate…………………...………………………………..27 1.5: The NMR coordinate frames…………………………………...…………………...29 1.6: The inversion recovery pulse sequence………………………………...…………...31 1.7: The Carr-Purchell-Meiboom-Gill pulse sequence………………………..………...33 1.8: 15N HSQC spectrum for the glutaredoxin from Pseudomonas aeruginosa………....36 1.9: Diagram of chemical exchange as it would appear in a 1D NMR spectrum…….….39 1.10: 15N-HSQC of the glutaredoxin from Pseudomonas aeruginosa with progressively higher concentration titrations of a polymer ligand……………………………..………40 1.11: Diagram of the STD effect and calculation of a difference spectrum………….….45 1.12: Correlations between backbone resonance assignments among the six standard backbone triple resonance experiments…………………………..……………………..54 2.1: STD spectra of proteins without fragments (upfield region)………………..………73 2.2: Solvent mapping of BrmGRX via FTMap…………………………..……………...77 2.3: 1H-NMR spectrum for compound 7 or RK464 in deuterated methanol………..…...80 x 2.4: HRMS spectrum for compound 7 or RK464 in methanol………………..…………81 2.5: IR for methyl 4-(1H-imidazol-1-yl) benzoate or compound 7 or RK464…………..81 2.6: X-ray crystallography structure for compound 7 or RK464 (CCDC code: CCDC 1471207), with a R value of 2.49..……………………………………………………….82 2.7: 1H-NMR for compound 8 or