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Using Molecular Constraints and Unnatural Amino Acids to Manipulate and Interrogate Protein Structure, Dynamics, and Self- Assembly University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2016 Using Molecular Constraints and Unnatural Amino Acids to Manipulate and Interrogate Protein Structure, Dynamics, and Self- Assembly Beatrice Markiewicz University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biophysics Commons, and the Physical Chemistry Commons Recommended Citation Markiewicz, Beatrice, "Using Molecular Constraints and Unnatural Amino Acids to Manipulate and Interrogate Protein Structure, Dynamics, and Self-Assembly" (2016). Publicly Accessible Penn Dissertations. 1879. https://repository.upenn.edu/edissertations/1879 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1879 For more information, please contact [email protected]. Using Molecular Constraints and Unnatural Amino Acids to Manipulate and Interrogate Protein Structure, Dynamics, and Self-Assembly Abstract Protein molecules can undergo a wide variety of conformational transitions occurring over a series of time and distance scales, ranging from large-scale structural reorganizations required for folding to more localized and subtle motions required for function. Furthermore, the dynamics and mechanisms of such motions and transitions delicately depend on many factors and, as a result, it is not always easy, or even possible, to use existing experimental techniques to arrive at a molecular level understanding of the conformational event of interest. Therefore, this thesis aims to develop and utilize non-natural chemical modification strategies, namely molecular cross-linkers and unnatural amino acids as site-specific spectroscopic probes, in combination with various spectroscopic methods to examine, in great detail, certain aspects of protein folding and functional dynamics, and to manipulate protein self-assemblies. Specifically, we first demonstrate how strategically placed molecular constraints can be used to manipulate features of the protein folding free energy landscape, thus, allowing direct measurement of key components via temperature-jump kinetic studies, such as folding from a transition-state structure or the effect of internal friction on the folding mechanism. Secondly, we utilize a photolabile non-natural amino acid, Lys(nvoc), to probe the mechanism of protein misfolding in a β-hairpin model and identify an aggregation gatekeeper that tunes the aggregation propensity. We further develop a method where the induced-charge produced by photocleavage of Lys(nvoc) can be used to target and destabilize hydrophobic regions of amyloid fibril assemblies, esultingr in complete disassembly, Finally, we highlight new useful properties of a site-specific spectroscopic probe, 5-cyanotryptophan (TrpCN), by demonstrating (1) how the frequency and linewidth of the infrared nitrile stretching vibration is sensitive to multiple hydrogen bonding interactions and solvent polarity, (2) that the fluorescence emission, quantum yield, and lifetime is extremely sensitive to hydration, and serves as a convenient fluorescence probe of protein solvation status, and (3) that the unique characteristics of TrpCN can be used to target the structure, local environment, and mechanism of the tryptophan gate in the M2 membrane proton channel of the influenza A virus. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor Feng Gai Subject Categories Biophysics | Chemistry | Physical Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1879 USING MOLECULAR CONTSTRAINTS AND UNNATURAL AMINO ACIDS TO MANIPULATE AND INTERROGATE PROTEIN STRUCTURE, DYNAMICS, AND SELF-ASSEMBLY Beatrice N. Markiewicz 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 2016 Supervisor of Dissertation ____________________ Feng Gai Edmund J. and Louise W. Kahn Endowed Term Professor of Chemistry Graduate Group Chairperson _____________________ Gary A. Molander Hirschmann-Makineni Professor of Chemistry Dissertation Committee Marsha Lester (Chair), Edmund J. Kahn Distinguished Professor Ivan Dmochowski, Professor of Chemistry Sergei Vinogradov, Associate Professor of Biochemistry and Biophysics USING MOLECULAR CONTSTRAINTS AND UNNATURAL AMINO ACIDS TO MANIPULATE AND INTERROGATE PROTEIN STRUCTURE, DYNAMICS, AND SELF-ASSEMBLY COPYRIGHT 2016 Beatrice N. Markiewicz This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/ DEDICATION To my family iii ACKNOWLEDGEMENTS Looking back on the last several years, my time at Penn has been enriching, rewarding, and of course challenging. I consider myself to be extremely fortunate to have had such a strong support system, and there many people to thank. The person most instrumental in developing and shaping my scientific career is my thesis advisor, Professor Feng Gai. His constant dedication to not only me, but all of his students is really admirable. One of the most memorable lessons he has taught me, which has been motivating me throughout my graduate career, is that success in science may involve a little bit of luck, but it mainly comes from your own perseverance and hard work. His mentorship, enthusiasm toward science, willingness to teach and discuss how to approach research, and words of encouragement, has created an environment where I was able to mature both intellectually and individually. Professor Feng Gai truly devoted an immense amount of time, passion, and support to help me grow as a scientist and as a person, and for that I am extremely grateful and thankful. I also want to thank my committee members, Professors Marsha Lester, Ivan Dmochowski, and Sergei Vinogradov, for their guidance and encouragement over the years. Their valuable input has allowed me to improve my abilities as a scientist. Also, I greatly appreciate the efforts of Dr. Lester, Dr. Dmochowski, and Dr. Tobias Baumgart in helping me receive external funding. I thank my collaborators Professor Bill DeGrado, Dr. Hyunil Jo, Dr. Thomas Lemmin, Dr. Giacomo Fiorin, Dr. Steven Brown, and Professor Amos B. Smith for their help with protein samples, molecular dynamics simulations, synthesis of compounds, and scientific inquiry. iv Also, I sincerely thank the former and current Gai lab members and the UOPL Staff Members: Dr. Robert Culik, Dr. Ileana Pazos, Dr. Wenkai Zhang, Dr. Thomas Measey, Dr. Bei Ding, Dr. Thomas Troxler, Dr. Jian-Xin Chen, Dr. Kwang Im Oh, Dr. Natalia Rubtsova, Rachel Abaskharon, Mary Rose Hilaire, Chun-Wei Lin, Ismail Ahmed, Debopreeti Mukherjee, and Jeffrey Rodgers. None of the work here would have been possible without the support of the group and I thank everyone for contributing to such a collaborative environment. I consider myself so lucky to have worked with such a brilliant group of people. I especially want to thank Robert Culik who has been an exceptional mentor and friend, and who played a major role in my training when I first started. Also, I want to thank Lali, whose vibrant personality made each day in the lab an enjoyable one. I would also like to thank my collaborator Wenkai, who has an amazing excitement toward science, a natural ability to mentor, and imparts humor and wisdom simultaneously, makings for very delightful conversations. I want to specially thank Tom Troxler as well for his collaboration and mentorship. I would also like to take the time to thank all the friends I have made in the department. Most of all, I need to thank my family: my parents, Krystyna Markiewicz, Jack Wieckowski, and my grandma, Maria Ryczaj, for all their love, understanding, support, and all that they have done to help me succeed in life. To my boyfriend Walter Dorfner, we entered this journey together, experienced the ups and downs of graduate school together, and now can celebrate our achievements together. I thank him for the unwavering love and support. v ABSTRACT USING MOLECULAR CONTSTRAINTS AND UNNATURAL AMINO ACIDS TO MANIPULATE AND INTERROGATE PROTEIN STRUCTURE, DYNAMICS, AND SELF-ASSEMBLY Beatrice N. Markiewicz Feng Gai Protein molecules can undergo a wide variety of conformational transitions occurring over a series of time and distance scales, ranging from large-scale structural reorganizations required for folding to more localized and subtle motions required for function. Furthermore, the dynamics and mechanisms of such motions and transitions delicately depend on many factors and, as a result, it is not always easy, or even possible, to use existing experimental techniques to arrive at a molecular level understanding of the conformational event of interest. Therefore, this thesis aims to develop and utilize non- natural chemical modification strategies, namely molecular cross-linkers and unnatural amino acids as site-specific spectroscopic probes, in combination with various spectroscopic methods to examine, in great detail, certain aspects of protein folding and functional dynamics, and to manipulate protein self-assemblies. Specifically, we first demonstrate how strategically placed molecular constraints can be used to manipulate features of the protein folding free energy landscape, thus, allowing direct measurement of key components via temperature-jump kinetic studies, such as folding
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