High-throughput disulfide and FRET scanning to reveal protein conformational ensembles in vitro and in vivo.

PROJECT SUMMARY Research Strategy Two of the biggest challenges in molecular biomedical research are (1) the lack of technologies for detailed investigation of protein conformations in the cell or the native environment, and (2) a dearth of data on protein conformational dynamics for large proteins and across time scales. This proposal addresses both these grand

challenges head-on by developing two closely related technologies: high-throughput disulfide scanning, which will bridge the gap between in vitro and in vivo structural biology by revealing protein structures in both contexts, and single-molecule FRET scanning, which will reveal both average conformation and pairwise distance

fluctuations across thousands of residue pairs within a protein in a massively parallel format. I will not only develop these powerful techniques, but also apply them to key biomedical questions: the role of conformational plasticity in the function of chaperones; the role of conformational dynamics in antibiotic susceptibility of bacterial

transpeptidases; phenotypic effects of kinetically trapped and non-native protein conformations; and structural basis of genotype-phenotype relationships. Candidate and Environment I am a highly trained experimental protein biochemist and biophysicist with extensive experience investigating the roles of disulfide bonds and redox chemistry in protein structure, stability, misfolding, aggregation, and the

pathogenesis of disease. By carrying out the proposed research, I will acquire crucial technical expertise in emerging protein sequencing technologies and computational modeling and data analysis algorithms, as well as a deeper knowledge of statistical physics, microbial physiology, and experimental evolution. Harvard University

and the greater Boston-area academic community provides a stellar environment for academic professional development. A group of world-leading experts has been recruited as the Advisory Committee. The primary mentor has a proven track record of training future faculty members at top research-intensive institutions. I will

take full advantage of career-development courses, workshops on inclusive teaching and building communities of belonging, seminars on leading a research group, guidance and practice in mentorship, and a formal refresher on responsible conduct of research. I have maintained a high level of productivity throughout my research career

(14 publications in 8-years, starting as an undergraduate), and the work arising from this award will be widely disseminated and shared.