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Directed Evolution of Stabilized Peptides with Bacterial Display

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Directed Evolution of Stabilized Peptides with Bacterial Display Directed Evolution of Stabilized Peptides with Bacterial Display Tejas A. Navaratna A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Engineering) in the University of Michigan 2020 Doctoral Committee: Associate Professor Greg Thurber, Chair Professor James Bardwell Professor Erdogan Gulari Associate Professor Xiaoxia Nina Lin Associate Professor Fei Wen Tejas Navaratna [email protected] ORCID iD: 0000-0003-2976-0137 © Tejas Navaratna 2020 Acknowledgments I deeply thank Professor Greg Thurber for his guidance, trust, and support in me. I cannot count the number of times I came into his office unsure of future research paths and came away inspired and eager to probe new directions. He gave me the opportunity and freedom to explore my ideas, while also being an excellent mentor and advisor in this journey. I also thank my other committee members, Professors Jim Bardwell, Erdogan Gulari, Nina Lin, and Fei Wen for their support and productive discussions. I also would like to thank Professor Jim Van Deventer, Professor Cathy Fromen, Professor Kevin Solomon, and Dr. Marco Jost for helpful career advice. I would also like to acknowledge Andrew Min and Liz Goulston, two undergraduate students I have had the pleasure of mentoring. Working with them led me to realize my passion for mentorship. Other Thurber Lab members that I closely worked with are Lydia Atangcho, Liang Zhang, Marshall Case, and Mukesh Mahajan – I am deeply grateful for your advice and support. Finally, I would also like to thank Sumit Bhatnagar, Cornelius Cilliers, Bruna Menezes, Eshita Khera, Ian Nessler, Reggie Evans, Anna Kopp, Celia Dong, Haolong Huang, and Yinuo Chen for fostering a social, friendly, and wonderfully collaborative lab environment, even through the COVID-19 shutdown– I couldn’t have asked for better labmates. ii I am thankful for the generous support of the National Science Foundation and National Institutes of Health. I feel extremely lucky to have such great friends, both new connections made in Ann Arbor and old ones from Cambridge and San Jose. From rock climbing cliffs in Kentucky to scaling mountains in Washington, to virtual get togethers in these trying times, I will cherish these connections and memories for decades to come. You made me laugh when I needed it and I cannot thank you enough for your support. Finally, I am grateful to mom and dad and my sister for their support leading up to this point, and I could not have done it without you all. You encouraged me from childhood to pursue my curiosity – and this has led me to this point. iii Table of Contents Acknowledgments .......................................................................................................... ii List of Figures ............................................................................................................... vii List of Tables ................................................................................................................. xi List of Appendices ....................................................................................................... xii Abstract ........................................................................................................................ xiii Chapter I: Introduction and Background ..................................................................... 1 Abstract ......................................................................................................................... 1 Stapled Peptides ........................................................................................................... 2 Noncanonical amino acid incorporation ........................................................................ 9 Directed evolution and bacterial surface display ........................................................ 11 The MDM2-p53 interaction ......................................................................................... 16 Summary ..................................................................................................................... 18 References ................................................................................................................. 21 Chapter II: Directed Evolution Using Stabilized Bacterial Peptide Display ............ 30 iv Abstract ....................................................................................................................... 30 Introduction ................................................................................................................. 31 Results ........................................................................................................................ 35 Discussion ................................................................................................................... 46 Methods ...................................................................................................................... 54 Acknowledgements ..................................................................................................... 67 Supporting information ................................................................................................ 68 References ................................................................................................................. 82 Chapter III: Evolving MDM2-Binding Peptides with Staple Diversity ...................... 90 Abstract ....................................................................................................................... 90 Introduction ................................................................................................................. 91 Results ........................................................................................................................ 94 Discussion ................................................................................................................. 107 Methods .................................................................................................................... 110 Acknowledgements ................................................................................................... 115 Supporting information .............................................................................................. 115 References ............................................................................................................... 118 Chapter IV: A Tag-Free Two Color Approach to Sorting Displayed Libraries ...... 123 v Abstract ..................................................................................................................... 123 Introduction ............................................................................................................... 124 Results ...................................................................................................................... 127 Discussion ................................................................................................................. 135 Methods .................................................................................................................... 136 Acknowledgement .................................................................................................... 140 References ............................................................................................................... 140 Chapter V: Concluding Remarks and Future Directions ........................................ 142 Summary ................................................................................................................... 142 Future directions ....................................................................................................... 145 References ............................................................................................................... 150 Appendices ................................................................................................................. 151 References ............................................................................................................... 168 vi List of Figures Figure I - 1. Common chemistries used in peptide stapling approaches. ......................... 6 Figure I - 2. Structures of azidohomoalanine (AHA), and methionine (met), .................. 11 Figure I - 3. Beta barrel structure of eCPX (left) and histograms of expression. ............ 14 Figure I - 4. Partial map of signaling pathways involving the p53-MDM2 interaction. .... 17 Figure I - 5. Development of a p53 mimic for MDM2 inhibition ...................................... 19 Figure I - 6. Putting the pieces together ......................................................................... 20 Figure II - 1. Chemical Biology Enabled Stabilization. .................................................... 36 Figure II - 2. Stabilized Peptide Library Design and Selection. ...................................... 42 Figure II - 3. Chymotrypsin digest trajectories. ............................................................... 44 Figure II - 4. Structural characterization using solution NMR. ........................................ 46 Figure II - S1. Specific Surface Binding .......................................................................... 73 Figure II - S2. Logo plots for pooled sequences (A) and i, i+5 containing sequences (B). .......................................................................................................................................
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