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

这一综合研究和教育计划旨在让本科生参与到假设驱动的研究项目中。研究部分探索非规范内含子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作为演讲者发展为年轻科学家-学者的机会。智力上的优点：蛋白质剪接是一种自我催化的、翻译后切除的介入性多肽，内含子，伴随着侧翼多肽的连接，外显子，以产生成熟的蛋白质。内含素在没有辅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建立，但内含子促进这些步骤的方法还不是很清楚。该研究计划重点研究两个非正则内含子PolII和TERA的催化机制，包括非正则内含子如何促进蛋白质剪接的每一步，以及这些内含子如何在时间上协调每一步。深海热球菌PolII内含子有一个C-末端的谷氨酰胺，而不是高度保守的天冬酰胺，天冬酰胺直接参与了剪接的第三步。PolII内含子如何和为什么改变剪接的第三步将通过使用Mills小组开发的已建立的生化分析对模型肽进行研究，以及通过与伦斯勒理工学院的蛋白质核磁共振光谱学家王春雨合作进行的研究来解决。特别令人感兴趣的问题包括保守残基如何催化剪接的每一步，以及内含子如何在时间上协调剪接的步骤。温热梭状芽胞杆菌内含子有一个N-末端的谷氨酰胺来代替保守的亲核丝氨酸或半胱氨酸，并且必须设法绕过该内含子剪接的第一步。Mills的实验室将通过使用SDS-PAGE、Western印迹和质谱仪比较野生型内含子和定点突变体的剪接来解决这一问题，并启动结构测定，以确定保守的内含子残基在促进没有N-末端亲核试剂的剪接中的作用。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。在米尔斯实验室的23名研究生中，有15名是女性。在实验室的校友中，有六名学生在医学院或拥有医学学位，两名学生正在申请医学院，一名知识产权律师，五名工业界学生。此外，该实验室的四名女校友正在攻读生物化学博士学位。该实验室将从222名生物和化学专业一年级和二年级学生中挑选新生，其中53%是女性。该实验室还将从NSF资助的圣十字学院克劳维斯学者项目中吸引研究生，该项目的重点是指导那些来自少数族裔或低收入家庭的理科学生，特别是来自伍斯特地区的学生。进入实验室后，每个学生将完成自己的论文项目，参加每周一次的小组会议和研讨会，出席全国ASBMB会议，参加圣十字？S暑期研究项目，并有望以合著者的身份出版他们的作品。该项目将加强圣十字生物化学专业的辅导活动。自1995年以来，已有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，包括过去三个班级的五名年轻女性博士课程。它还将包括外展计划，以提高伍斯特学童的科学素养和对科学的热情。此外，对非正则内含子的研究将有助于理解酶在单一活性部位催化多步反应的策略。

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.