University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Multifunctionalization of Proteins: Strategies for Combining Semi- Synthetic and Bioorthogonal Techniques John Blaine Warner University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Chemistry Commons Recommended Citation Warner, John Blaine, "Multifunctionalization of Proteins: Strategies for Combining Semi-Synthetic and Bioorthogonal Techniques" (2014). Publicly Accessible Penn Dissertations. 1494. https://repository.upenn.edu/edissertations/1494 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1494 For more information, please contact [email protected]. Multifunctionalization of Proteins: Strategies for Combining Semi-Synthetic and Bioorthogonal Techniques Abstract Proteins play important roles in biological processes including muscle movement, initiating immune responses, and memory formation. Full understanding of protein function requires determining its structure, assignment of function, and elucidation of interactions with other proteins or metabolites. Protein modification is useful for installing chemical entities that allow esearr chers to specifically probe function, attach fluorophores for localization, covalently trap protein interactions, or perform affinity enrichment from a complex milieu. Installation of functional groups otherwise not accessible from canonical protein expression requires a toolbox of protein modification strategies. We have developed strategies for using a combination of techniques to functionalize proteins, including E. coli aminoacyl transferase (AaT) mediated N-terminal protein modification, Amber codon suppression for incorporation of unnatural amino acids (Uaas), auxotrophic strain incorporation of Uaas, and click chemistry reactions. A chemoenzymatic approach is used with AaT mediated N-terminal transfer of an azide functionality and subsequent click- chemistry to fluorescently label protein N-termini. We also use AaT to install a unique N- terminal protein semi-synthesis ligation site. Additionally, Amber codon suppression is used to site- specifically incorporate Uaa side chains with click-chemistry handles, intrinsic fluorophores, and photocrosslinkers into proteins. Click-chemistry is used with Amber codon suppression to control protein function in a highly selective manner. We also use auxotrophic strain incorporation to globally replace a natural amino acid with an isostere Uaa to introduce azide and alkyne functional groups. Protein multifunctionalization is achieved by combining these modification methods in a single expressed protein. Effectively achieving this combination has required careful consideration of both molecular biology and chemical reactivity. We demonstrate the combination of these techniques to label alpha- synuclein, a protein which is involved in neurodegenerative diseases. Moreover, the protocols for multiple labeling that we have developed should be generally practicable for a variety of proteins. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor E. James Petersson Keywords alpha synuclein, amber codon suppression, aminoacyl transferase, click chemistry, multifunctionalization, unnatural amino acids Subject Categories Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1494 MULTIFUNCTIONALIZATION OF PROTEINS: STRATEGIES FOR COMBINING SEMI-SYNTHETIC AND BIOORTHOGONAL TECHNIQUES John B. Warner A DISSERTATION in Chemistry Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2014 Supervisor of Dissertation _____________________ E. James Petersson Assistant Professor of Chemistry & Assistant Professor of Biochemistry and Molecular Biophysics, Perelman School of Medicine Graduate Group Chairperson ______________________ Gary A. Molander Hirschmann-Makineni Professor of Chemistry Dissertation Committee: Christopher B. Murray, Richard Perry University Professor Ivan J. Dmochowski, Associate Professor of Chemistry Tobias Baumgart, Associate Professor in Physical & Biological Chemistry MULTIFUNCTIONALIZATION OF PROTEINS: STRATEGIES FOR COMBINING SEMI-SYNTHETIC AND BIOORTHOGONAL TECHNIQUES COPYRIGHT 2014 John B. Warner DEDICATION To Jiná, for being there for me. iii ACKNOWLEDGEMENTS Firstly, it goes without saying that I would not have come this far if it had not been for the guidance and mentorship of my advisor Professor E. James Petersson. I had the fortune to work on many projects in the lab and acquire a plethora of skills I didn’t think I ever would. Though we are no longer ‘essential personnel’, I would still trek in through a blizzard to give group meeting. My undergraduate advisor, Professor Silvana Andreescu, was also very influential to shaping my views and practices as a young scientist and without that training I certainly would not be where I am today. I would also like to acknowledge the support and guidance of my committee members: Professor Christopher Murray, Professor Ivan Dmochowski, and Professor Tobias Baumgart. Throughout my years in the Petersson Lab I have had the privilege to work with many talented undergraduate, graduate, and post-doctoral students: Nick Marotta, Anne Wagner, Eileen Moison, Mark Fegley, Lee Vandivier, Chrissie Grindley, Alyssa Klein, Rebecca Wissner, Lee Speight, Solongo Batjargal Ziraldo, Jerri Wang, Colin Fadzen, Anand Muthusamy, Dr. Tomohiro Tanaka, Haviva Garrett, Dr. Moumita Samanta, Keith Keenan, Stella Chen, Chris Walters, Benson Chen, and Dr. Yun Huang. As well as new members joining the lab and extending our legacy: Jack Ferrie and Itthipol Sungwienwong. My lab mates have made coming to work every morning enjoyable and exciting. I have also had the fortune to collaborate with an amazing group of scientists at the Perelman School of Medicine at the University of Pennsylvanian’s Center for Neurodegenerative Disease Research. Professor Virginia M.Y. Lee, Dr. Kelvin Luk, and iv Dr. Dustin Covell have been invaluable in my research endeavors. UPenn Chemistry is fortunate to have an amazing support staff that keeps the building running and the research going. My sincerest gratitude goes out to: Judith Currano, George Furst, Jun Gu, Rakesh Kohli, Chris Lanci, Tom Troxler, Mandy Swope, Kristen Muscat, Candice Adams, Yvonne Kline, Leslie Shinn, Dan Burke, Bruce Dolliver, Rico Vargas, Josh Henry, Andrei Korchynsky, Cuong Nguyen, Claire Din, and Tony Baruffi. I came to UPenn with a great cohort of fellow graduae students, many of which have since moved into new phases of their careers. The Chemistry program is a close and tight knit community that freely doles advice and reagents (often when desperately needed). Matthew ‘Will’ Eibling, Brittany Riggle, Julie Griepenburg, Julia Richards, Garry Seward, Chris Butts, Joe Swift, Ian Farrell, Chih-Jung Hsu, Ben Capraro, Danielle Reifsnyder Hickey, Rob Hickey, Julia Lehman, and many others who helped make UPenn so great. Finally, I would like to thank my families for their love and support through the long hours, missed holidays, and fancy jargon. v ABSTRACT MULTIFUNCTIONALIZATION OF PROTEINS: STRATEGIES FOR COMBINING SEMI-SYNTHETIC AND BIOORTHOGONAL TECHNIQUES . John B. Warner E. James Petersson Proteins play important roles in biological processes including muscle movement, initiating immune responses, and memory formation. Full understanding of protein function requires determining its structure, assignment of function, and elucidation of interactions with other proteins or metabolites. Protein modification is useful for installing chemical entities that allow researchers to specifically probe function, attach fluorophores for localization, covalently trap protein interactions, or perform affinity enrichment from a complex milieu. Installation of functional groups otherwise not accessible from canonical protein expression requires a toolbox of protein modification strategies. We have developed strategies for using a combination of techniques to functionalize proteins, including E. coli aminoacyl transferase (AaT) mediated N- terminal protein modification, Amber codon suppression for incorporation of unnatural amino acids (Uaas), auxotrophic strain incorporation of Uaas, and click chemistry reactions. A chemoenzymatic approach is used with AaT mediated N-terminal transfer of an azide functionality and subsequent click- chemistry to fluorescently label protein N- termini. We also use AaT to install a unique N-terminal protein semi-synthesis ligation vi site. Additionally, Amber codon suppression is used to site-specifically incorporate Uaa side chains with click-chemistry handles, intrinsic fluorophores, and photocrosslinkers into proteins. Click-chemistry is used with Amber codon suppression to control protein function in a highly selective manner. We also use auxotrophic strain incorporation to globally replace a natural amino acid with an isostere Uaa to introduce azide and alkyne functional groups. Protein multifunctionalization is achieved by combining these modification methods in a single expressed protein. Effectively achieving this combination has required careful consideration of both molecular biology and chemical reactivity. We demonstrate the combination of these techniques to label α-synuclein, a protein which is involved in neurodegenerative diseases. Moreover,