RUI: Catalysis and Coordination of the Steps of Protein Splicing by Non-canonical Inteins: An Integrated Research and Education Program

College Of The Holy Cross, Worcester MA

Investigators

Abstract

This integrated research and education program is designed to engage undergraduate students in hypothesis-driven research projects. The research component explores the catalysis of protein splicing by non-canonical inteins. The educational component provides undergraduates at Holy Cross the opportunity to develop as young scientist-scholars in the classroom, laboratory and as presenters at scientific meetings. Intellectual Merit: Protein splicing is the self-catalyzed, post-translational excision of an intervening polypeptide, the intein, concomitant with the ligation of the flanking polypeptides, the exteins, to produce the mature protein. The intein catalyzes this process without cofactors or auxiliary proteins. Although the chemical mechanism of protein splicing is established, the means by which the intein facilitates those steps is not well understood. The research program focuses on the catalytic mechanism of two non-canonical inteins, PolII and TerA, including how non-canonical inteins promote each step of protein splicing, and how these inteins temporally coordinate each step. The Pyrococcus abyssi PolII intein has a C-terminal glutamine in place of the highly conserved asparagine, which is directly involved in step three of splicing. How and why the PolII intein alters step three of splicing will be addressed by studies on model peptides using an established biochemical assay developed by the Mills group and by NMR spectroscopy, in collaboration with protein NMR spectroscopist Chunyu Wang of the Rensselaer Polytechnic Institute. Questions of particular interest include how conserved residues catalyze each step of splicing and how the intein temporally coordinates the steps of splicing. The Clostridium thermocellum TerA intein has an N-terminal glutamine in place of the conserved nucleophilic serine or cysteine, and must somehow bypass the first step of splicing in this intein. The Mills lab will address how this is done by comparing the splicing of wild type inteins and site-directed mutants using SDS-PAGE, Western blot and mass spectrometry, and initiate a structural determination to determine the role of conserved intein residues in promoting splicing without an N-terminal nucleophile. Broader Impacts: The project will provide high quality, inclusive training opportunities to undergraduate students. Of the 23 research students from the Mills lab, 15 were female. Of the lab alumni, six students are in medical school or have medical degrees, two are applying to medical schools, one is an intellectual property attorney, and five are in industry. In addition, four female alumni of the lab are in PhD programs in biochemistry. The lab will select its new students from the 222 first- and second-year biology and chemistry majors, 53% of whom are female. The lab also will draw research students from the NSF-funded Clavius Scholars program at Holy Cross, which focuses on mentoring science students who are members of underrepresented minorities or from lower income families, particularly from the Worcester area. Once in the lab, each student will complete their own thesis project, participate in weekly group meetings and seminars, present at the national ASBMB meeting, participate in Holy Cross?s summer research program, and hopefully publish their work as a co-author. The project will strengthen the mentoring activities of the Holy Cross biochemistry concentration. Since 1995, 81 students have participated in the program, 68% of them female. Many enter top medical and graduate programs, including five young women in PhD programs from the past three classes. It will also involve outreach programs to increase the scientific literacy and enthusiasm for science of Worcester schoolchildren. In addition, the study of non-canonical inteins will contribute to the understanding of strategies used by enzymes to catalyze multi-step reactions at single active sites.