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.
The Mills lab is interested in the chemical mechanism of protein splicing as the intellectual merit of the award. Protein splicing is catalyzed by an intein, which interrupts flanking polypeptides and promotes its own excision and the linking of the flanking proteins. We are also interested in how the structure and function of inteins from extreme thermophiles compares to that of inteins from organisms that grow under more typical conditions, and how the process of splicing is regulated. 1. In collaboration with Chunyu Wang and associates at Rensselaer Polytechnic Institute, we solved the structure by NMR spectroscopy of the PolII intein from Pyrococcus abyssi, an extreme thermophile. We learned that it has a folding pattern (a beta-hairpin) not present in the structures of mesophilic inteins, which we hypothesize might contribute to the enzyme’s stability at high temperatures. We also learned through a combination of biochemical assays and structural analysis which amino acid residues might control and catalyze the steps of splicing. 2. We learned that the P. abyssi PolII intein has a flexible loop that we suspect is not present in the closely related P. horikoshii intein. We have shown by biochemical and biophysical means that the P. horikoshii intein is more rigid, and have preliminary data to solve the structure by X-ray crystallography. 3. We learned that the activity of inteins from P. abyssi and Methanoculleus marisnigri are controlled by the oxidation state of a disulfide bond, which suggests a means by which intein activity can be triggered by different cellular conditions. Such a "trigger" for splicing is essential if inteins might play a role in regulation of protein function. 4. We showed that a Thermobifida fusca intein with protein sequence characteristics of inteins with unusual mechanisms follows the typical mechanism, a cautionary tale about over-interpreting bioinformatics data. We also showed that a Clostridium thermocellum intein does, in fact, follow an alternative mechanism of splicing. 5. We gathered insight into how inteins can promote multiple, different chemical steps at a single active site. For instance, the P. abyssi PolII intein appears to tightly regulate the relative rate of each step. On the other hand, the P. abyssi lon protease intein cannot promote downstream steps without the prior steps, suggesting mechanism-linked conformational changes order the steps of the reaction. 6. We have designed a model peptide system for the study of asparagine cyclization coupled to peptide bond cleavage. Because our peptide is small, our computational collaborators can model the entire peptide, and we are beginning to use our biochemical data to validate their QM/MM modeling of the transition state of the cyclization. 7. We have optimized the expression of hedgehog proteins in order to study the mechanism and structure of these intein-related enzymes. 7. We have continued to develop lab-based, guided inquiry laboratories for General Chemistry and have submitted one such exercise for publication. The Mills lab also has made the education of undergraduate students and outreach to the scientific community a priority in terms of broader impact. 1. Fourteen undergraduate research students worked in the Mills lab during the grant period. As a college that exclusively undergraduate, Holy Cross students have the opportunity to be in charge of their projects; they do the experiments, analyze the data and present the results. Each student in the lab has presented a poster at a national meeting of American Society for Biochemistry and Molecular Biology, and nine have co-authored peer-reviewed manuscripts during the grant period. Three students have started medical school, three have started PhD programs in biochemistry, and two have entered high school teaching to influence the next generation of STEM students. 2. The lab has supported the Science Ambassadors at Holy Cross, an outreach program by which Worcester elementary and middle school students participate in hands-on science demonstrations. We also started a twice-yearly educational program bringing biochemistry lessons to Tatnuck Elementary School in Worcester. 3. We have hosted two research students from the Worcester Pipeline Collaborative in the lab. The Collaborative mentors Worcester-area under-represented students with an interest in STEM and medicine. A third Pipeline student has started in the lab this year. 4. The lab hosted two biochemistry seminars a year in the department, and two science-in-the-liberal-arts talks, one by renowned painter Alexis Rockman and another by John Abramson on the role of scientific information in public policy.
