CAREER: Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids

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A major difficulty in experimental studies of protein folding is the inability to probe local structure and dynamics at the level of an individual residue in a non-perturbing fashion. Genetic incorporation of unnatural amino acids (UAAs) significantly enhances the ability to study local environments of large proteins, since defined spectroscopic labels can be incorporated site-specifically into proteins in vivo. The research goal of this proposal is the synthesis, characterization, and application of isotopically labeled UAAs, such as isotopic variants of 4-cyano-L-phenylalanine and 4-ethynyl-L-phenylalanine, to serve as sensitive spectroscopic probes of protein structure and dynamics with site-specificity in a relatively non-perturbing fashion. These UAAs will be paired with infrared (IR), nuclear magnetic resonance (NMR), and/or fluorescence spectroscopy to examine different local protein environments present in several protein systems including the dematin headpiece subdomain (DHP) and superfolder green fluorescent protein (sfGFP).The educational goal of this proposal is the integration of the proposed research into the undergraduate chemistry curriculum at Franklin & Marshall College and to mentor undergraduate research students investigating protein structure, thermodynamics, and kinetics. Aspects of the proposed research will be incorporated into both the general chemistry and physical chemistry courses. For instance, the ability to probe protein structure and unfolding with a variety of spectroscopic techniques will be explored in the Physical Chemistry 1 (Thermodynamics and Kinetics) course. At least ten undergraduate students will be involved in the research. Undergraduate research provides the opportunity for personalized teaching that greatly aids the intellectual development of the students. The training of undergraduate research students will position them for success in their future endeavors such as graduate school, medical school, or other career paths.