University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Infrared Measurements of Protein Conformational Dynamics Robert Culik University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, Biophysics Commons, and the Physical Chemistry Commons Recommended Citation Culik, Robert, "Infrared Measurements of Protein Conformational Dynamics" (2014). Publicly Accessible Penn Dissertations. 1248. https://repository.upenn.edu/edissertations/1248 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1248 For more information, please contact [email protected]. Infrared Measurements of Protein Conformational Dynamics Abstract The topic of how a protein folds has been a major area of research for several decades; however, important details about this process are still undetermined. Experimental limitations in the study of protein folding are a result of no technique possessing both the necessary spatial and temporal resolution. This Thesis presents several studies conducted with the goal of expanding upon the experimentalist's toolbox, involving new methods of interrogating and/or perturbing protein systems of interest. The early chapters of this Thesis describe our efforts using established synthetic methods to extend the utility of infrared spectroscopy in the study of protein folding. Specifically, we show that, using the strategy of cysteine alkylation, we can incorporate novel vibrational probes into proteins in a site-specific manner. We also show that combined sidechain mutagenesis, probing at multiple frequencies, and isotopic labeling to obtain secondary structural resolution in infrared studies of protein folding, in the process uncovering details about the folding mechanism of the Trp-cage miniprotein. Similarly, we illustrated the use of thioamides as site-specific eporr ters of backbone-backbone hydrogen bonding, and applied this functionalization to the Trpzip2 beta-hairpin system to validate its proposed folding mechanism. Further work involved using D-amino acids to interrogate turn regions in proteins, specifically examining Trp-cage folding. The later chapters of this Thesis are focused on the effects of extrinsic molecules on the structural ordering of proteins. Taking advantage of the lack of tertiary structure of intrinsically disordered proteins, we examined the effect that trifluoroethanol has on protein folding, and found evidence that this cosolvent acts as a nano-crowder. We also introduced the idea of using phototriggers to modify the free energy landscape of folding for a given protein, and demonstrated that a peptide that typically folds in an activated (barrier-containing) manner can be made to fold in a downhill fashion upon irradiation. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Feng Gai Keywords Dynamics, Infrared, Protein Folding, Spectroscopy Subject Categories Biochemistry | Biophysics | Physical Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1248 INFRARED MEASUREMENTS OF PROTEIN CONFORMATIONAL DYNAMICS Robert M. Culik A DISSERTATION in Biochemistry and Molecular Biophysics 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 Graduate Group Chairperson __________________ __________________ Feng Gai Kathryn M. Ferguson Professor of Chemistry Associate Professor of Physiology Dissertation Committee S. Walter Englander (Chair) Jacob Gershon-Cohen Professor of Biochemistry, Biophysics, & Medical Science Jeffery G. Saven Associate Professor of Chemistry Sergei A. Vinogradov Associate Professor of Biochemistry and Biophysics Mark Goulian Edmund J. and Louise W. Kahn Professor of Biology, Physics & Astronomy Kim A. Sharp Associate Professor of Biochemistry and Biophysics Scott H. Brewer Associate Professor of Chemistry INFRARED MEASUREMENTS OF PROTEIN CONFORMATIONAL DYNAMICS COPYRIGHT 2014 Robert Mitchell Culik This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ To my grandfathers, Harold Edward Bellis and Rudolf Wenceslaus Čulík iii ACKNOWLEDGMENTS It is impossible to begin this Thesis without acknowledging the great swathe of people who have helped me get to this point. The most prominent influence in my scientific career thus far has been my thesis advisor, Dr. Feng Gai, whose passion, patience, and ceaseless scientific curiosity have been a great inspiration for me. I am incredibly thankful for his encouragement, his mentorship, and his ability to always believe in me. I also would be remiss not to thank all of the members of my thesis committee, Professors Walter Englander, Jeffery Saven, Sergei Vinogradov, and Mark Goulian, for their useful questions and comments at meetings, and for their efforts to make me realize my full potential. I owe an extra debt of appreciation to Drs. Englander and Saven for their efforts in helping me to receive external funding, and to Dr. Goulian for agreeing to be on my committee after a former committee member left the university. I would also like to thank Dr. Kim Sharp and Dr. Scott Brewer for agreeing to be on my final defense committee; I’d especially emphasize my gratitude to Dr. Brewer, who made the trip to Philadelphia for my defense. My attempts here at Penn would have been for naught if not for the helpful and supportive community that I have had the good fortune to land in. I am especially grateful to several people for helping to mold me into a better scientist and person. Dr. Arnaldo Serrano has been a mentor, peer, and friend who never flinched at giving me helpful advice or insight, even if the response was something I did not want to hear. Kyle Harpole has been a constant friend and fount of knowledge since the time we first interviewed at grad schools together, and I’m sure without him in my life I’d be a much iv blander person. I spent many a night chatting with Dr. Jacob Goldberg, whose unique perspective and strong work ethic left a deep impression on me. I also need to thank Dr. Bob Rarig, who I’ve shared countless experiences with, none of which haven’t contained a hearty chuckle or two. My labmates, both past and present, in the Gai group have been an incredible support net throughout the years, and the camaraderie we have had has been awesome. Thanks very much to Dr. Pramit Chowdhury, Dr. Smita Mukherjee, Dr. Julie Rodgers, Dr. Matthias Waegele, Dr. Lin Guo, Dr. Kate Smith-Dupont, Dr. Tom Troxler, Dr. Jian-Xin Chen, Dr. Lev Chuntonov, Dr. Ayanjeet Ghosh, Dr. Jianqiang Ma, Dr. Kwang-Im Oh, Lali Pazos, Chun-Wei Lin, Yi-Ju Chen, Beatrice Markiewicz, Paul Straus, Mary Rose Mintzer, Rachel Roesch, Jeff Rodgers, and Ismail Ahmed. I’d also like to thank the foosball crowd for the stress relief. In addition, I’ve had many collaborators over the years - I am grateful for their efforts and their desire to work with me. Thanks to Dr. Hyunil Jo for his hard work and his incredibly fast turnaround time and to his advisor, Dr. Bill DeGrado. I’d also like to thank Dr. Michelle Bunagan, Dr. Srinivas Annavarapu, and Dr. Vikas Nanda. I am also grateful to the staffs of the Chemistry and BMB Departments, and to the Chemistry Department as a whole for ‘adopting’ me and making me feel at home. Finally, I would like to thank my family for all of their love, support, and understanding over these years. My mom, dad, sister, extended family, and girlfriend Nicole have suffered a considerable amount of neglect over the years, and by their grace they still choose to associate with me. All of them have been bedrock for me, and helped me to bounce back time and again in my efforts presented here. v ABSTRACT INFRARED MEASUREMENTS OF PROTEIN CONFORMATIONAL DYNAMICS Robert M. Culik Feng Gai The topic of how a protein folds has been a major area of research for several decades; however, important details about this process are still undetermined. Experimental limitations in the study of protein folding are a result of no technique possessing both the necessary spatial and temporal resolution. This Thesis presents several studies conducted with the goal of expanding upon the experimentalist’s toolbox, involving new methods of interrogating and/or perturbing protein systems of interest. The early chapters of this Thesis describe our efforts using established synthetic methods to extend the utility of infrared spectroscopy in the study of protein folding. Specifically, we show that, using the strategy of cysteine alkylation, we can incorporate novel vibrational probes into proteins in a site-specific manner. We also show that combined sidechain mutagenesis, probing at multiple frequencies, and isotopic labeling to obtain secondary structural resolution in infrared studies of protein folding, in the process uncovering details about the folding mechanism of the Trp-cage miniprotein. Similarly, we illustrated the use of thioamides as site-specific reporters of backbone- backbone hydrogen bonding, and applied this functionalization to the Trpzip2 beta- hairpin system to validate its proposed folding mechanism. Further work involved using vi D-amino acids to interrogate turn regions in proteins, specifically examining