Single-Chain Insulin Analogs As Ultra-Stable Therapeutics and As Models of Protein (Mis)Folding: Stability, Structure, Dynamics, and Function of Novel Analogs
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SINGLE-CHAIN INSULIN ANALOGS AS ULTRA-STABLE THERAPEUTICS AND AS MODELS OF PROTEIN (MIS)FOLDING: STABILITY, STRUCTURE, DYNAMICS, AND FUNCTION OF NOVEL ANALOGS by MICHAEL D. GLIDDEN II Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Physiology and Biophysics Dissertation advisor: Dr. Michael A. Weiss CASE WESTERN RESERVE UNIVERSITY May 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Michael D. Glidden II candidate for the degree of Doctor of Philosophy*. Committee Chair Matthias Buck Committee Member Michael Weiss Committee Member Faramarz Ismail-Beigi Committee Member George Dubyak Committee Member Michael Zagorski Date of Defense December 8th, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication This Thesis is dedicated with honor and gratitude to the ONE THING and the mouse in my shirt pocket whose love, patience, and support helped me navigate the fog in calm and rough waters. i Table of Contents Dedication ..................................................................................................................................... i Table of Contents ......................................................................................................................... ii List of Tables .............................................................................................................................. iv List of Figures .............................................................................................................................. v Acknowledgements .................................................................................................................... vii List of Abbreviations .................................................................................................................. ix Abstract ........................................................................................................................................ x Chapter 1. Introduction 1.1. Historical overview and Thesis goals .............................................................................. 1 1.2. Insulin synthesis, structure, and function ......................................................................... 4 1.3. Clinical insulin analog pharmacology............................................................................ 11 1.3.1. Strategies for fast action ......................................................................................... 12 1.3.2. Strategies for intermediate to long (basal) action .................................................. 16 1.4. Mechanisms of insulin stabilization ............................................................................... 21 1.4.1. Helical stabilization ............................................................................................... 23 1.4.2. The reverse hydrophobic effect—untapped potential in insulin? .......................... 28 1.4.3. Stabilization by non-standard amino-acid substitution .......................................... 30 1.4.4. Biologically active, ultra-stable insulin analogs .................................................... 34 1.5. Conclusion ..................................................................................................................... 37 Chapter 2. Biophysical studies of a highly destabilized proinsulin mutant 2.1. Chapter summary ........................................................................................................... 39 2.2. Introduction .................................................................................................................... 40 2.3. Methods ......................................................................................................................... 44 2.4. Results ............................................................................................................................ 52 2.5. Discussion ...................................................................................................................... 75 2.6. Endnotes ......................................................................................................................... 82 Chapter 3. An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits native-like biological signaling duration 3.1. Chapter summary ........................................................................................................... 84 3.2. Introduction .................................................................................................................... 85 3.3. Methods ......................................................................................................................... 92 3.4. Results .......................................................................................................................... 114 3.5. Discussion .................................................................................................................... 140 3.6. Endnotes ....................................................................................................................... 164 ii Chapter 4. Solution structure of an ultra-stable insulin analog connects protein dynamics to a novel mechanism of receptor binding 4.1. Chapter summary ......................................................................................................... 166 4.2. Introduction .................................................................................................................. 167 4.3. Methods ....................................................................................................................... 173 4.4. Results .......................................................................................................................... 184 4.5. Discussion .................................................................................................................... 211 4.6. Endnotes ....................................................................................................................... 243 Chapter 5. Application of the reverse hydrophobic effect to therapeutic protein engineering 5.1. Chapter summary ......................................................................................................... 245 5.2. Introduction .................................................................................................................. 246 5.3. Methods ....................................................................................................................... 251 5.4. Results .......................................................................................................................... 256 5.5. Discussion .................................................................................................................... 278 Chapter 6. Fast-acting, ultra-stable single-chain insulin analogs obtained by standard amino acid substitutions 6.1. Chapter summary ......................................................................................................... 288 6.2. Introduction .................................................................................................................. 289 6.3. Methods ....................................................................................................................... 293 6.4. Results .......................................................................................................................... 295 6.5. Discussion .................................................................................................................... 304 Chapter 7. Summary and Discussion 7.1. Thesis summary ........................................................................................................... 308 7.2. Insulin folding, stability, and dynamics ....................................................................... 308 7.3. Potential therapeutic applications of biologically active SCIs ..................................... 318 7.4. Rational protein design and engineering challenges .................................................... 324 7.5. Final remarks ............................................................................................................... 328 Appendix A .............................................................................................................................. 330 Bibliography ............................................................................................................................ 345 iii List of Tables Table 1-1. Summary of clinical analog design elements and mechanisms of action ..................... 13 Table 2-1. Molecular properties of peptide models and clinical analogs ...................................... 54 Table 2-2. Estimated secondary structure contents obtained by CD deconvolution ...................... 60 Table 2-3. 1H chemical shifts for all isotopically labeled DesDi samples ..................................... 73 Table 2-4. 13C chemical shifts for all isotopically labeled DesDi samples .................................... 74 Table 3-1. SCI sequence design features and their associated rationale ........................................ 91 Table 3-2. Crystallographic data collection and refinement statistics for SCI-a ......................... 107 Table 3-3. Receptor-binding affinities ........................................................................................