CAREER: Alternative Mechanisms of HINT Domain Autoprocessing: An Integrated Undergraduate Research and Education Program

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Some proteins are processed by self-splicing. Inteins are invervening polypeptide sequences that facilitate their own excision from flanking polypeptides (exteins) as well as the ligation of the exteins by a peptide bond. The research project supported by this award explores unanswered questions about the mechanism by which HINT (Hedgehog-INTein) domains facilitate post-translational autoprocessing. This interesting case of biochemical catalysis will be explored by the study of inteins that splice by alternate mechanisms. For instance, the first step of protein splicing is an amide to ester rearrangement, and this project will study how inteins promote this thermodynamically unfavorable reaction by in-depth kinetic analysis of a non-canonical intein and by NMR and site-directed mutagenesis studies of a hedgehog protein. The third step of protein splicing, asparagine cyclization coupled to peptide bond cleavage, will be studied by characterization of an intein that likely splices via cyclization of glutamine. The goal of the research program is to understand in detail the means by which HINT domains facilitate these two unusual chemical reactions, and how they coordinate these separate steps to make splicing an efficient process that avoids unproductive side-reactions. This awardee will also make significant educational contributions to the Biochemistry Concentration and both introductory and upper-level courses at the College of the Holy Cross. One component will be the design and implementation of new labs for the Discovery Chemistry curriculum, which introduces students to science through the lab rather than through textbooks alone. Upper division courses will prepare students to be life-long learners by integrating the primary literature and emphasizing written and oral presentations, and upper division lab courses will integrate research into the classroom. New programs will be introduced to improve the vitality of the Biochemistry Concentration to better prepare Holy Cross students for graduate studies, including a journal club, a new seminar series, membership in a professional society, and travel to local and national meetings and symposia.The broader impacts of this program include contributions to both research and education. The research program will broaden the understanding of intein chemistry in particular and biochemical catalysis in general. This knowledge can be applied in devising innovative schemes in protein engineering. This program will improve the research training and educational preparation received by the undergraduate student body, particularly students in the Biochemistry Concentration. The goal of the educational projects is to better prepare these students to read the literature, fully appreciate the importance and excitement of their research experiences, be confident with presenting their work orally and in writing, and give back to the scientific community through outreach. These projects will help to ensure the continued vitality of the strong tradition of research at Holy Cross, and the richness of the experience that Holy Cross students have as undergraduates will resonate in their careers as graduate students, scientists, and teachers.