THE UN-DESIGN AND DESIGN OF INSULIN: STRUCTURAL EVOLUTION WITH APPLICATION TO THERAPEUTIC DESIGN By NISCHAY K. REGE Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biochemistry Dissertation Advisor: Dr. Michael A. Weiss CASE WESTERN RESERVE UNIVERSITY August, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Nischay K. Rege candidate for the degree of Doctor of Philosophy*. Committee Chair Paul Carey Committee Member Michael Weiss Committee Member Faramarz Ismail-Beigi Committee Member George Dubyak Date of Defense: June 26th, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication This thesis is dedicated to my mother, Dipti, whose constant love and faith have never failed, to my father, Kiran, who taught me of the virtue of curiosity, and to my wife, Shipra, whose kindness and companionship have given me enough strength for eight lifetimes. i Table of Contents Dedication ..................................................................................................................................... i Table of Contents ......................................................................................................................... ii List of Tables ............................................................................................................................... v List of Figures ............................................................................................................................ vii Acknowledgements ...................................................................................................................... x List of Abbreviations ................................................................................................................ xiv Abstract ..................................................................................................................................... xvi Chapter 1: Introduction ................................................................................................... 1 1.1 History of insulin research ..................................................................................................... 1 1.2 Purpose of Dissertation .......................................................................................................... 3 1.3 Insulin Structure ..................................................................................................................... 6 1.4 Insulin Oligomerization and Storage ..................................................................................... 8 1.5 Insulin Biosynthesis and Secretion: ..................................................................................... 15 1.6 Proinsulin Folding:............................................................................................................... 20 1.7 Insulin Fibrillation ............................................................................................................... 23 1.8 Insulin Physiology ............................................................................................................... 32 1.9 Insulin Receptor Structure ................................................................................................... 35 1.10 Insulin-IR Complexation ................................................................................................... 37 1.11 Diabetes Mellitus ............................................................................................................... 43 1.12 Development of Clinical Insulin Analogs: ......................................................................... 46 1.13 Conclusion ......................................................................................................................... 54 Chapter 2: Toxic Protein Misfolding Constrains the Evolution of Insulin Sequences ..................................................................................................................... 58 2.1 Chapter Summary ................................................................................................................ 58 2.2 Introduction .......................................................................................................................... 59 2.3 Results: ................................................................................................................................. 67 2.4 Discussion ............................................................................................................................ 93 2.5 Methods and materials ....................................................................................................... 105 Chapter 3: Identification of a Minimal Prodomain in Insulin: A Weak Hydrogen Bond is Critical for Proinsulin Folding, but Dispensible in the Mature Hormone ....................................................................................................... 111 3.1 Chapter Summary .............................................................................................................. 111 3.2 Introduction ........................................................................................................................ 112 Results ...................................................................................................................................... 117 ii 3.4 Discussion. ......................................................................................................................... 135 3.5 Methods. ............................................................................................................................ 142 Chapter 4: Protein Evolution at the Edge of Non-Foldability: Impaired Biosynthesis of an Insulin Variant with Native Structure and Function ............. 148 4.1 Chapter Summary .............................................................................................................. 148 4.2 Introduction ........................................................................................................................ 149 4.3 Results ................................................................................................................................ 160 4.4 Discussion .......................................................................................................................... 194 4.5 Methods ............................................................................................................................. 206 Chapter 5: Structure-Based Stabilization of Insulin as a Therapeutic Protein Assembly via Enhanced Aromatic-Aromatic Interactions .................................... 211 5.1 Chapter Summary .............................................................................................................. 211 5.2 Introduction ........................................................................................................................ 212 5.3 Results ................................................................................................................................ 217 5.4 Discussion .......................................................................................................................... 262 5.5 Methods ............................................................................................................................. 292 5.6 Endnotes ............................................................................................................................. 297 Chapter 6: 4SS-Insulin ................................................................................................. 299 6.1 Chapter Summary .............................................................................................................. 299 6.2 Introduction ........................................................................................................................ 300 6.3 Results ................................................................................................................................ 305 6.4 Discussion .......................................................................................................................... 327 6.5 Methods ............................................................................................................................. 333 6.6 Endnotes ............................................................................................................................. 336 Chapter 7: Towards a Glucose-Responsive Insulin Analog ...................................... 337 7.1 Chapter Summary .............................................................................................................. 337 7.2 Summary of previous GRI Research ................................................................................. 337 7.2 Design of Glucose-Responsive Insulin Analogs Containing Internal Glucose-Sensing Motifs ....................................................................................................................................... 365 7-3 Results ............................................................................................................................... 367 7-4 Discussion .......................................................................................................................... 394 7-5 Methods. ...........................................................................................................................