University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 The evelopmeD nt Of Unnatural Amino Acid- Based Probes And Methods For Biological Studies Ismail Abd Al Azim Ahmed University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biophysics Commons, and the Chemistry Commons Recommended Citation Ahmed, Ismail Abd Al Azim, "The eD velopment Of Unnatural Amino Acid-Based Probes And Methods For Biological Studies" (2019). Publicly Accessible Penn Dissertations. 3315. https://repository.upenn.edu/edissertations/3315 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3315 For more information, please contact [email protected]. The evelopmeD nt Of Unnatural Amino Acid-Based Probes And Methods For Biological Studies Abstract Proteins form a diverse ensemble of dynamic structures to carry out all life-sustaining functions. Therefore, many efforts have gone into studying the structure-dynamics-function relationship of proteins using a wide range of techniques, including fluorescence and infrared (IR) spectroscopies. While very useful, intrinsic fluorescence and IR signals arising from the natural amino acid side chains within the protein are often insufficient or unable to provide the information needed to understand the biological question of interest. To this end, various extrinsic spectroscopic probes, such as fluorescent dyes, have been used to increase the information content in specific measurements and applications. However, incorporation of a foreign moiety into any protein unavoidably affects its native structure and dynamics; hence effort must be made to reduce such perturbation. In this regard, the overarching aim of this thesis is to develop novel spectroscopic probes based on scaffolds of natural amino acids (NAAs). Because of their small size and similarity to NAAs, such unnatural amino acid-based (UAA-based) probes are expected to be minimally perturbing. Specifically, we show that (1) 4-cyanotryptophan (4CN-Trp) is a blue fluorescent amino acid useful for fluorescence microscopy applications; (2) 4CN-Trp and DiO (a common dye used to stain membranes) are a useful FRET pair to study peptide-membrane interactions; (3) 4CN-Trp, and tryptophan constitutes a dual FRET-PET pair which was used to study peptide end-to-end termini interactions and protein ligand-binding; and (4) the functional group of 4CN-Trp, 4-cyanoindole can be used in the form of a nucleoside as a dual fluorescence-IR reporter for DNA-protein studies. Furthermore, we extended applications of previously known UAAs and showed (5) p-cyanophenylalanine is useful as a fluorescence-based pH sensor which we used to determine peptide pKa’s and peptide membrane penetration kinetics and (6) we use a simple synthetic method for post- translationally installing an ester moiety on to proteins via cysteine alkylation as an UAA-based vibrational probe in proteins to study fibril formation and protein-ligand interactions Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Feng Gai Second Advisor Sergei Vinogradov Keywords fluorescence, GFP, nucleoside, probe, site-specific, unnatural amino acid This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3315 Subject Categories Biophysics | Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3315 THE DEVELOPMENT OF UNNATURAL AMINO ACID-BASED PROBES AND METHODS FOR BIOLOGICAL STUDIES Ismail A. Ahmed 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 2019 Supervisor of Dissertation _______________________ Feng Gai Ph.D. Edmund J. and Louise W. Kahn Endowed Term Professor of Chemistry Graduate Group Chairperson _______________________ Kim A. Sharp Ph.D. Associate Professor of Biochemistry and Biophysics Dissertation Committee Sergei Vinogradov Ph.D., Professor of Biochemistry and Biophysics P. Leslie Dutton Ph.D., Eldridge Reeves Johnson Professor of Biochemistry and Biophysics S. Walter Englander Ph.D., Professor of Biochemistry and Biophysics Lin Guo Ph.D., Assistant Professor of Biochemistry and Molecular Biology, Jefferson University Dedicated to My Beloved Family ii ACKNOWLEDGMENT Firstly, I would like to thank my Ph.D. advisor Dr. Feng Gai for the opportunity to join his lab. He welcomed me at a time when the doors of scientific research seemed to be closing in on me. He challenged me, he inspired me, and he supported me. He taught me to be rigorous, always ask questions, and to work hard¬–because like Kevin Durant always says, “hard work beats talent when talent fails to work hard.” I thank my thesis committee Dr. Sergei Vinogradov, Dr. P. Leslie Dutton, Dr. S. Walter Englander, and Dr. Lin Guo. It was an absolute pleasure having them serve on my committee. Our interactions were always pleasant, discussions always insightful, and they always supported me in any way I needed. Specifically, I wanted to recognize that Dr. Dutton has been a huge advocate for me since joining Penn and has helped me in more ways than I can enumerate. I am forever indebted. I thoroughly enjoyed my time in the Gai lab where I constantly shared science and laughter with my lab mates. I appreciated working with Mary Rose Hilaire on developing a blue fluorescent unnatural amino acid which served as the foundation for my Ph.D. thesis work. Our abilities perfectly complemented each other to bring something truly beautiful to life. In the last two years of my Ph.D., the lab significantly decreased in size, but that only brought us (Debopreeti Mukherjee and Arusha Acharyya) closer. We supported each other, and we were there for each other in good and bad times. It certainly has been a pleasure. One of the highlights of my graduate experience was serving as a mentor for Christina Eng, an undergraduate researcher in our lab. We shared our stories, laughs, and overall had a great time. Being a mentor taught me to be more responsible and to be selfless. I am so proud of her accomplishments, and I am looking forward to seeing what she does in the future. Dr. Thomas Troxler has served as a second mentor to me in the lab. He read all my grant proposals and gave me insightful feedback. He patiently fielded and answered all my questions in spectroscopy and physics. He helped me through various experiments and overall gave me valuable perception into the world of academia. iii Science is a collaborative effort where we share ideas, techniques, and expertise to the common goal of advancing knowledge. I have had the pleasure to work with outstanding collaborators such as Dr. William F. DeGrado and Dr. Hunyl Jo from UCSF. I have also worked extensively with Robert (Bobby) Micikas and Amos Smith III here at Penn. Bobby was not only a collaborator but a friend that will last a lifetime. I probably spent more time with Bobby than all other people at Penn combined. We constantly share our scientific ideas, ambitions, and life. To succeed as a graduate student, you need a strong support system. Former students and current professors Dr. Ishmail Abdus-Suboor and Dr. Jamaine Davis had a profound impact on me. They served as my day-to-day cheerleaders and made me believe in myself (and filter out the noise). They gave me the confidence to keep pushing forward and advised me how to navigate the field. Despite Dr. Abdus-Suboor having to run his lab, teach classes, give talks, take care of his kids, and more, he always has the time to go to lunch with me, pick up my phone calls, and answer my texts. I can’t even express my gratitude that would be worthy. Outside of the lab, I have been blessed with the best friends and family anyone can have. I am thankful for my childhood friends, college-mates, and the friends I made in my time in Philadelphia. We shared so many great times that helped me reset my mind so I could go back to lab afresh every single time. My family has been my sanctuary throughout life and graduate school. They have always believed in me and made my life easier. They make me feel proud that I am a scientist and always celebrate my contributions to science. From the bottom of my heart, I love you all! Lastly, I acknowledge the two women in my life that this Ph.D. would not have been possible without them. My mother, Angie has supported my scientific endeavors ever since I was a little kid. I remember that one time in second grade I was so enthusiastic about making my board for the science fair. It turned out hideously dilapidated. Mom, despite not knowing much science came to the rescue and helped me re-design an amazing board which got me 2nd place in the entire school. My mother is the most patient and resilient person I know; these iv characteristics helped me through graduate school. Momma, look at me now! It is all because of you. I cannot express enough the gratitude that I have for my dear wife, Jamila Hoque. She came into my life at a time where I had no idea if I could continue in science. She helped me stay the course and made me believe in myself through her unyielding belief in me and my dream. She grounded me; put up with the demanding hours I spent lab, loved and supported me unconditionally. We grew our young family together here at Penn, I will never forget this glorious chapter, and I am excited about our next chapters we will write together. v ABSTRACT THE DEVELOPMENT OF UNNATURAL AMINO ACID-BASED PROBES AND METHODS FOR BIOLOGICAL STUDIES Ismail A.