University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Chemical Modification Methods for Protein Misfolding Studies Yanxin Wang University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Chemistry Commons Recommended Citation Wang, Yanxin, "Chemical Modification Methods for Protein Misfolding Studies" (2015). Publicly Accessible Penn Dissertations. 2082. https://repository.upenn.edu/edissertations/2082 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2082 For more information, please contact [email protected]. Chemical Modification Methods for Protein Misfolding Studies Abstract Protein misfolding is the basis of various human diseases, including Parkinson’s disease, Alzheimer’s disease and Type 2 diabetes. When a protein misfolds, it adopts the wrong three dimensional structures that are dysfunctional and sometime pathological. Little structural details are known about this misfolding phenomenon due to the lack of characterization tools. Our group previously demonstrated that a thioamide, a single atom substitution of the peptide bond, could serve as a minimalist fluorescence quencher. In the current study, we showed the development of protein semi-synthesis strategies for the incorporation of thioamides into full-length proteins for misfolding studies. We adopted the native chemical ligation (NCL) method between a C-terminal thioester fragment and an N-terminal Cys fragment. We first devised strategies for the synthesis of thioamide-containing peptide thioesters as NCL substrates, and demonstrated their applications in generating a thioamide/Trp-dually labeled α-synuclein (αS), which was subsequently used in a proof-of-concept misfolding study. To remove the constraint of a Cys at the ligation site, we explored traceless ligation methods that desulfurized Cys into Ala, or β- and γ- thiol analogs into native amino acids after ligation in the presence of thioamides. We further demonstrated that selective deselenization could be achieved in the presence of both Cys residues and thioamides, expanding the scope of thioamide incorporation through traceless ligation to proteins with native Cys. Finally, we showed that hemiselenide protected selenocysteines (Sec) can be incorporated onto the protein N-terminus through chemoenzymatic modification by aminoacyl transferase (AaT) as ligation handles. Further developments are underway in our laboratory to expand the AaT substrate scope for β- and γ- thiol amino acid analogs. In summary, we developed a set of methods that allowed the incorporation of thioamide probes into full-length protein, which enabled the application of this minimalist probe in protein misfolding studies. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor E. James Petersson Keywords Chemical biology, Parkinson's disease, Protein engineering, Protein semi-synthesis, Synuclein, Thioamide Subject Categories Biochemistry | Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2082 CHEMICAL MODIFICATION METHODS FOR PROTEIN MISFOLDING STUDIES Yanxin Wang 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 2015 Supervisor of Dissertation ________________________ E. James Petersson Associate Professor of Chemistry Graduate Group Chairperson ________________________ Gary A. Molander, Hirschmann-Makineni Professor of Chemistry Dissertation Committee Tobias Baumgart, Associate Professor of Chemistry Donna Huryn, Adjunct Professor of Chemistry Jeffery G. Saven, Professor of Chemistry CHEMICAL MODIFICATION METHODS FOR PROTEIN MISFOLDING STUDIES COPYRIGHT 2015 Yanxin Wang For my parents and mentors iii ACKNOWLEDGMENT First and foremost, I would like to thank Professor E. James Petersson for allowing me to join his laboratory and for his generous guidance throughout my graduate career. He has been a very knowledgeable and supportive mentor, who introduced me to the field of chemical biology and helped me develop my professional skills as a scientist. He gave me much freedom in intellectual exploration, while always making himself available for feedback and discussions. I would not have been where I am without his support. I would also like to thank my dissertation committee members − Professors Tobias Baumgart, Donna Huryn and Jeffery G. Saven – for consistently providing useful suggestions and fresh perspectives that helped me improve my research. I am fortunate to have worked with the many members and alumni of the Petersson group, who have been great colleagues, collaborators, and friends: Dr. Tomohiro Tanaka, Dr. Yun Huang, Dr. Conor Haney, Dr. Jacob Goldberg, Dr. Anne Wagner, Dr. John Warner, Dr. Lee Speight, Dr. Solongo Batjargal, Dr. Rebecca Wissner, Mark Fegley, Christopher Walters, X. Stella Chen, John J. Ferrie, D. Miklos Szantai-Kis, Itthipol Sung- weinwong, Christina Cleveland, J. M. Vicky Jun, Eileen Moison, Colin Fadzen, Anand Muthusamy, E. Keith Keenan, Eileen Hoang, and Jimin Yoon. We worked together to keep the instruments functional, the supplies well-stocked, and the everyday life exciting. I am particularly appreciative of Dr. Jacob Goldberg, Dr. Anne Wagner and Dr. Solongo Batjargal for their invaluable inputs on my research, and for getting me started in the lab. iv I owe my gratitude for the many people in and outside the department for helping me obtain good quality data and for maintaining a care-free research environment: Dr. Rakesh Kohli for mass spectrometry, Dr. George Furst and Dr. Jun Gu for NMR spectroscopy, Dr. Patrick Carroll for X-ray crystallography, Dr. Christopher Lanci for biological chemistry instrumentation, Dr. Dewight Williams for electron microscopy, Dr. Dustin Covell for cell culture, Judith N. Currano for the numerous times that she helped me find references and information, Mandy Swope and Kristen Muscat for watching my back on administrative business, and all other staff of the chemistry department. Finally, I would like to express my sincere appreciation for my family and friends. With me being the only child, my parents made tremendous sacrifices by allowing me to study here half way across the globe for five years. I owe my achievements and growth to their continuous inspiration, cultivation and unconditional support. I am also thankful for having a group of caring and supportive friends; we had much fun together and helped each other maintain our sanity in graduate school. v ABSTRACT CHEMICAL MODIFICATION METHODS FOR PROTEIN MISFOLDING STUDIES Yanxin Wang E. James Petersson Protein misfolding is the basis of various human diseases, including Parkinson’s disease, Alzheimer’s disease and Type 2 diabetes. When a protein misfolds, it adopts the wrong three dimensional structures that are dysfunctional and sometime pathological. Little structural details are known about this misfolding phenomenon due to the lack of characterization tools. Our group previously demonstrated that a thioamide, a single atom substitution of the peptide bond, could serve as a minimalist fluorescence quencher. In the current study, we showed the development of protein semi-synthesis strategies for the incorporation of thioamides into full-length proteins for misfolding studies. We adopted the native chemical ligation (NCL) method between a C-terminal thioester fragment and an N-terminal Cys fragment. We first devised strategies for the synthesis of thioamide-containing peptide thioesters as NCL substrates, and demonstrated their applications in generating a thioamide/Trp-dually labeled α-synuclein (αS), which was subsequently used in a proof-of-concept misfolding study. To remove the constraint of a Cys at the ligation site, we explored traceless ligation methods that desulfurized Cys into Ala, or β- and γ- thiol analogs into native amino acids after ligation in the presence of thioamides. We further demonstrated that selective deselenization could be achieved in vi the presence of both Cys residues and thioamides, expanding the scope of thioamide incorporation through traceless ligation to proteins with native Cys. Finally, we showed that hemiselenide protected selenocysteines (Sec) can be incorporated onto the protein N- terminus through chemoenzymatic modification by aminoacyl transferase (AaT) as ligation handles. Further developments are underway in our laboratory to expand the AaT substrate scope for β- and γ- thiol amino acid analogs. In summary, we developed a set of methods that allowed the incorporation of thioamide probes into full-length protein, which enabled the application of this minimalist probe in protein misfolding studies. vii TABLE OF CONTENTS ACKNOWLEDGMENT ................................................................................................. iv ABSTRACT ...................................................................................................................... vi LIST OF TABLES ............................................................................................................ x LIST OF ILLUSTRATIONS .......................................................................................... xi Chapter 1 . Introduction ................................................................................................. 1 1.1 Protein Misfolding