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

RUI：非经典内含肽蛋白质剪接步骤的催化和协调：综合研究和教育计划

• 批准号: 0950245
• 负责人: Kenneth Mills
• 金额: $ 46.5万
• 依托单位: College of the Holy Cross
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0950245&HistoricalAwards=false
• 关键词: RUI; Catalysis; Coordination; Steps; Protein

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.

这个综合研究和教育计划旨在让本科生参与假设驱动的研究项目。研究部分探讨了非规范内链对蛋白质剪接的催化作用。教育部分为圣十字学院的本科生提供了在课堂、实验室和科学会议上发展为年轻科学家学者的机会。智力优势：蛋白质剪接是自催化的，翻译后切除中间多肽（内链），同时连接两侧多肽（外链），以产生成熟的蛋白质。蛋白在没有辅助因子或辅助蛋白的情况下催化这一过程。虽然蛋白质剪接的化学机制已经确立，但是对于蛋白质如何促进这些步骤的理解还不太清楚。本项目重点研究了PolII和TerA两种非规范蛋白的催化机制，包括非规范蛋白如何促进蛋白质剪接的每一步，以及这些非规范蛋白如何在时间上协调每一步。深海焦球菌PolII蛋白的c端谷氨酰胺取代了高度保守的天冬酰胺，这直接参与了剪接的第三步。通过与伦斯勒理工学院的蛋白质核磁共振波谱学家王春宇合作，使用Mills团队开发的生化测定方法和核磁共振波谱法对模型肽进行研究，将解决PolII蛋白如何以及为什么改变剪接的第三步。特别感兴趣的问题包括保守残基如何催化剪接的每个步骤，以及内部如何在时间上协调剪接的步骤。热胞梭菌TerA蛋白的n端谷氨酰胺取代了保守的亲核丝氨酸或半胱氨酸，并且必须以某种方式绕过该蛋白剪接的第一步。Mills实验室将通过使用SDS-PAGE、Western blot和质谱技术比较野生型内联蛋白和定点突变体的剪接来解决这一问题，并启动结构测定，以确定保守的内联蛋白残基在没有n端亲核试剂的情况下促进剪接的作用。更广泛的影响：该项目将为本科生提供高质量、包容性的培训机会。米尔斯实验室的23名研究生中，有15名是女性。在实验室的校友中，有6名学生在医学院或拥有医学学位，2人正在申请医学院，1人是知识产权律师，5人在工业界。此外，该实验室的四位女校友正在攻读生物化学博士学位。该实验室将从222名一、二年级生物和化学专业的学生中选拔新学生，其中53%是女性。该实验室还将吸引来自美国国家科学基金会（nsf）资助的圣十字学院（Holy Cross）克拉维斯学者项目（Clavius Scholars program）的研究学生，该项目专注于指导来自代表性不足的少数民族或低收入家庭的理科生，尤其是来自伍斯特地区的学生。一旦进入实验室，每个学生将完成自己的论文项目，参加每周的小组会议和研讨会，出席国家ASBMB会议，参加圣十字？并希望以合著者的身份发表他们的研究成果。本项目将加强圣十字生物化学浓度的师徒活动。自1995年以来，81名学生参加了该项目，其中68%是女性。许多人进入了顶尖的医学和研究生课程，包括过去三个班的五名年轻女性博士课程。它还将涉及扩展项目，以提高伍斯特学童的科学素养和对科学的热情。此外，对非规范蛋白的研究将有助于理解酶在单个活性位点催化多步反应的策略。