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

有些蛋白质是通过自我剪接来加工的。内含子是一种反转的多肽序列，它便于自身从侧翼多肽(外显子)中切除，也便于外显子通过多肽键连接。该奖项支持的研究项目探索了关于提示(Hedgehog-INTein)域促进翻译后自动处理的机制的未回答的问题。这一有趣的生化催化案例将通过研究通过替代机制拼接的内含子来探索。例如，蛋白质剪接的第一步是酰胺到酯的重排，这个项目将通过深入的非规范内含子的动力学分析以及通过对刺猬蛋白质的核磁共振和定点突变研究来研究内含子如何促进这种热力学上的不利反应。蛋白质剪接的第三步，天冬酰胺环化与肽键切割，将通过表征一个可能通过谷氨酰胺环化进行剪接的内含子来进行研究。该研究计划的目标是详细了解提示结构域如何促进这两个不寻常的化学反应，以及它们如何协调这些单独的步骤，使剪接成为一个有效的过程，避免无效的副反应。这位获奖者还将对圣十字学院的生物化学专业和入门课程和高级课程做出重大的教育贡献。其中一个组成部分将是为发现化学课程设计和实施新的实验室，该课程通过实验室而不是仅仅通过教科书向学生介绍科学。高年级课程将通过整合初级文学并强调书面和口头报告来为学生做好终身学习的准备，而高年级实验室课程将把研究融入课堂。为了提高生物化学专业的生命力，将引入新的计划，以更好地为圣十字学生的研究生学习做准备，包括一个期刊俱乐部，一个新的研讨会系列，专业协会的成员资格，以及参加当地和全国的会议和研讨会。这个计划的更广泛的影响包括对研究和教育的贡献。这项研究计划将扩大对内毒素化学和一般生化催化的理解。这一知识可用于设计蛋白质工程的创新方案。这项计划将改善本科生，特别是生物化学专业的学生所接受的研究训练和教育准备。教育项目的目标是让这些学生更好地准备阅读文献，充分认识到他们研究经历的重要性和兴奋感，自信地口头和书面介绍他们的工作，并通过外联回馈科学界。这些项目将有助于确保圣十字学院强大的研究传统的持续活力，以及圣十字学生作为本科生所拥有的丰富经验将在他们作为研究生、科学家和教师的职业生涯中产生共鸣。

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.