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

职业：HINT 域自动处理的替代机制：综合本科研究和教育计划

• 批准号: 0447647
• 负责人: Kenneth Mills
• 金额: $ 79.45万
• 依托单位: College of the Holy Cross
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0447647&HistoricalAwards=false
• 关键词: CAREER; Alternative; Mechanisms; HINT; Domain

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.

有些蛋白质是通过自剪接加工的。内链是一种反转多肽序列，它有利于从侧翼多肽（外链）上切除自身，并通过肽键将外链连接起来。该奖项支持的研究项目探索了关于HINT （hedgehog - intin）结构域促进翻译后自动处理的机制的未解问题。这一有趣的生化催化案例将通过研究通过替代机制剪接的蛋白来探讨。例如，蛋白质剪接的第一步是酰胺到酯的重排，本项目将通过对非典型肠内蛋白的深入动力学分析以及对刺猬蛋白的核磁共振和定点诱变研究来研究肠内蛋白如何促进这种热力学上不利的反应。蛋白质剪接的第三步，天冬酰胺环化耦合肽键切割，将通过表征一个可能通过谷氨酰胺环化剪接的蛋白来研究。该研究计划的目标是详细了解HINT结构域促进这两种不同寻常的化学反应的方法，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，避免非生产的副反应。该获奖者还将对圣十字学院的生物化学专业以及入门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，通过实验室向学生介绍科学，而不仅仅是通过教科书。高年级的课程将整合主要文献，强调书面和口头报告，使学生成为终身学习者，高年级的实验课程将把研究融入课堂。将引入新的项目来提高生物化学专业的活力，使圣十字学院的学生更好地为研究生学习做准备，包括一个期刊俱乐部，一个新的系列研讨会，一个专业协会的会员资格，以及参加地方和国家会议和专题讨论会。该计划的广泛影响包括对研究和教育的贡献。该研究计划将扩大对蛋白质化学的理解，特别是对生物化学催化的理解。这些知识可以应用于设计蛋白质工程的创新方案。该项目将提高本科学生，特别是生物化学专业学生的研究训练和教育准备。这些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，对口头和书面展示他们的工作充满信心，并通过外展活动回馈科学界。这些项目将有助于确保圣十字大学强大的研究传统的持续活力，并且圣十字大学学生作为本科生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。