This project will study the chemical mechanism of protein splicing. Protein splicing is the means by which an intervening polypeptide, or intein, catalyzes its own removal from flanking polypeptides, or exteins. The intein also links the extein segments. The research project will investigate the following: 1. How the activity of an intein from an extreme thermophile is influenced by its unusual structural stability. The investigative approaches will examine how the intein balances the need to be stable at high temperatures with the need to be sufficiently flexible to be catalytically active; 2. The manner in which conserved residues in the intein catalyze each step of the reaction, providing insight into how enzymes catalyze and coordinate multi-step reactions at a single active site without cofactors; 3. The catalysis of cyclization of the amino acids asparagine or glutamine, coupled to peptide bond cleavage. This is an unusual reaction in protein chemistry which will be studied in the context of an intein as well as with a combination of model peptide experiments and computational modeling; 4. The regulation of intein activity, either by temperature or oxidation state. This process of regulation might suggest how inteins could control the activity of their flanking, interrupted proteins; 5. The autoprocessing of hedgehog-like domains, which resemble inteins in structure and sequence, including how conserved residues and the binding of cholesterol may influence the initial step of the reaction. These aspects of protein splicing will be investigated with the use of a combination of structural, biophysical, biochemical and computational techniques in projects suitable for undergraduates.
Broader Impact The research program will have a significant impact on the careers of undergraduate research students. The undergraduates will perform the experiments, present the results at national meetings and in peer-reviewed publications, and, through close mentoring, prepare to be future leaders in STEM. Students from the Biochemistry Concentration at the College of Holy Cross will be recruited to work on this project. In addition to research activities, the students will sponsor outreach in the community by hosting lectures aimed at broad audiences and by participating in the Science Ambassadors student outreach program. The project will broaden access to STEM by recruiting local students to the lab through the Worcester Pipeline which is a program that is conducted in conjunction with the University of Massachusetts Medical School
