RUI: Protein Splicing and the Structure and Stability of Thermophilic Inteins

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The goal of the research program is to study the chemistry of proteins from thermophilic organisms. Thermophiles are microorganisms that live in places that are very hot and are often under very high pressure, such as deep-sea thermal vents. The program will investigate how these proteins can function at such extreme conditions, which could unfold or prevent the activity of proteins from organisms that grow under more hospitable conditions. This would allow insight into strategies that proteins use to stabilize their structure while remaining chemically active. In particular, the program will examine a class of enzymes (protein catalysts that facilitate chemical reactions) called inteins. Inteins interrupt other proteins, and must promote their own removal so that the interrupted proteins can function. Learning how this reaction is regulated may suggest roles for the splicing reaction and potentially new ways in which gene expression can be controlled. Inteins, and thermophilic enzymes more broadly, both have useful biotechnology applications, and a deeper understanding of structure and function will improve these tools. The broader impact of the program includes the development of the next generation of scientists and leaders in STEM, through direct training of undergraduate research students, through outreach to the youngest scientists in the Worcester community, and through increasing access to scientific research for under-represented groups both in the Worcester student community and with Holy Cross first year students.The research program seeks to understand the mechanism of protein splicing, a post-translational modification catalyzed by an intein. It will study unresolved questions about the catalytic mechanism at atomic-level resolution using both X-ray crystallography and NMR structure and dynamics experiments coupled with biochemical techniques. It will use a biophysical model peptide system, integrated with computational techniques, to investigate side chain cyclization coupled to peptide bond cleavage. It will examine how the structure of thermophilic inteins influences their activity and conformational flexibility, and investigate the physiological role of splicing. In addition, the program will have significant impact on our understanding of how enzymes catalyze multi-step reactions at a single active site and how thermophilic enzymes balance activity and stability through protein dynamics.