Dr. Gabrielle Valles recently received her Ph.D. from the University of Connecticut, where she used NMR spectroscopy and X-ray crystallography to study the structure and function of a eukaryotic deubiquitinase. We are requesting funding for a Research Supplement to Promote Diversity in Health-Related Research because Dr. Valles is an African-American woman and US citizen and thus underrepresented in the biomedical sciences. Dr. Valles will join Dr. Aimee Shen’s laboratory to work both on the parent grant (R01 GM140361-02) and an extension of this grant based on recent unpublished work. The central focus of the parent grant is to delineate the mechanisms by which spore-forming bacteria control the activity of a highly conserved, spore-specific amidase, CwlD. Our lab previously showed that CwlD activity in the important nosocomial pathogen, Clostridioides difficile, depends on a binding partner conserved exclusively in the Peptostreptococcaceae family, the GerS lipoprotein. Our recent crystal structure of the CwlD:GerS complex (solved in collaboration with Co- Investigator Dr. Sylvie Doublié) reveals that GerS binds at a site distal from CwlD’s active site in this structure (Aim 1a). Accordingly, the revised objective of the parent grant is to determine how GerS binding at an allosteric site is communicated to CwlD’s active site. To accomplish this goal, Dr. Valles will conduct structure-function analyses of CwlD, GerS, and the CwlD:GerS complex using NMR spectroscopy, X-ray crystallography, mutagenesis analyses in C. difficile, B. subtilis, and E. coli, and co-immunoprecipitation and fluorescence microscopy analyses. Most of these techniques were proposed in the parent grant, but the NMR analyses will leverage Dr. Valles’ training during her graduate work. Through this work, Dr. Valles will learn bacterial genetic, cell biology, and peptidoglycan analysis techniques for which she has no prior experience. She will take two immersive, hands-on short courses to supplement this training on bacterial genetics and microscopy. The proposed research training and career development plan will allow Dr. Valles to establish a productive research program for her future independent academic position. By the end of her research training, Dr. Valles will be uniquely positioned to conduct bona fide structure-function analyses in bacteria because few scientists have the combined training needed to solve structures and conduct structure-guided mutational analyses in the organism of interest. This interdisciplinary training will enhance Dr. Valles’ competitiveness for faculty jobs because most structure-function studies involve a collaboration between a biologist and a structural biologist. To prepare for this future career, Dr. Shen will help Dr. Valles further develop the non-research skills that will allow Dr. Valles to thrive in her future academic research position, e.g. manuscript preparation, oral presentation, grant writing, mentoring, teaching, networking ...