RUI: The Chemical Basis for Protein Self-assembly and Polymerization

本项目的目的是研究氨基酸谷氨酰胺在含有长谷氨酰胺重复序列的蛋白质的错误折叠和聚集过程中的分子和化学作用。 这种导致形成大纤维的异常过程是所有生物体共同的一般分子问题，并可导致代谢和结构缺陷。 最近，这种聚集的过程已经受到审查，因为在原纤维生长过程中形成的中间体本身可以在生物体中持续存在并干扰正常的细胞过程。 谷氨酰胺重复序列的延伸折叠成b-片层结构，然后通过进一步添加多肽链以形成长丝而作为聚集的模板。 单独的长丝可进一步缔合以形成复丝原纤。 谷氨酰胺被认为是通过氢键和疏水相互作用的机制来稳定这些结构，尽管这些相互作用的细节及其在自组装过程中各自的作用仍然存在争议。 PI开发了一种肽模型系统，可以形成一个原型的b-折叠，称为b-发夹，可以使用各种生物物理方法进行研究，以探测谷氨酰胺在促进b-折叠，细丝和原纤维形成中的作用。 三个具体目标是：（1）找到稳定原纤维组装途径中的中间体的条件，以便可以评估谷氨酰胺相互作用对生长和稳定性的作用;（2）测量氨基酸突变对这些过程的影响以探测化学机制;和（3）特定谷氨酰胺的同位素标记以直接探测各种中间体中的稳定氢键。 这些研究的结果将有助于理解蛋白质错误折叠的基本问题，并测试各种结构模型，已提出的蛋白质含有多聚谷氨酰胺repeats.This研究已开发为一套适合本科生的项目，因为主要的研究工作将由学生完成他们的生物学论文要求进行。 从事这项工作的学生将获得跨学科研究的培训。 这需要协作工作，学生将与化学系的同事和宾夕法尼亚大学的合作者密切合作。 在这个项目上的工作已经导致创建讲座蛋白质折叠和错误折叠的入门课程，更先进的讲座蛋白质结构和功能的高级课程，并开发了一个7周的初级文献为基础的蛋白质错误折叠和聚集的课程。 PI长期致力于改善代表性不足的群体获得研究的机会。 除了长期致力于为来自费城的K-12学生提供科学和写作的现有外展计划外，他还定期在夏季和学年期间在他的研究实验室中为至少一名学生预留空间。 更广泛地说，作为HHMI赞助的项目在哈弗福德学院的主任，PI负责授予学生跨学科研究奖学金，并为属于代表性不足的群体的学生提供内部和外部研究机会，管理几个外展计划，并支持教师发展研讨会和讲习班，以加强科学的整体研究计划。

The objective of this project is to study the molecular and chemical role of the amino acid, glutamine, in the process of misfolding and aggregation of proteins containing long glutamine repeats. This aberrant process, resulting in the formation of large fibrils, is a general molecular problem common to all organisms and can lead to metabolic and structural deficiencies. Recently, the process of this aggregation has come under scrutiny since the intermediates formed during fibril growth can themselves persist in the organism and interfere with normal cellular processes. Stretches of glutamine repeats fold into a b-sheet structure, which then acts as a template for aggregation by the further addition of polypeptide strands to form a long filament. Individual filaments can further associate to form multi-filament fibrils. The glutamines are thought to stabilize these structures through mechanisms involving hydrogen bonding and hydrophobic interactions, although the details of these interactions and their respective roles during the self-assembly process are still controversial. The PI has developed a peptide model system that can form a prototypical b-sheet, called a b-hairpin, that can be studied using a variety of biophysical methods to probe the role of the glutamines in fostering b-sheet, filament, and fibril formation. Three specific goals are: (1) finding conditions to stabilize intermediates in the fibril assembly pathway so that the role of glutamine interactions on growth and stability can be assessed; (2) measuring the effects of amino acid mutations on these processes to probe chemical mechanism; and (3) isotope-labeling of specific glutamines to probe directly for stable hydrogen bonding in the various intermediates. The outcome of these studies will help to understand a fundamental problem in protein misfolding and to test a variety of structural models that have been proposed for proteins containing poly-glutamine repeats.This research has been developed as a set of projects suitable for undergraduates since the primary research efforts will be undertaken by students working to complete their thesis requirements in biology. Students engaged in this work will gain training in interdisciplinary research. This necessitates collaborative work and students will be working closely with colleagues in the Chemistry Department and collaborators at the University of Pennsylvania. Work on this project has led to the creation of lectures in protein folding and misfolding for an introductory course, more advanced lectures for an upper-level course in protein structure and function, and the development of a 7-week primary-literature-based course on protein misfolding and aggregation. The PI has a long-standing commitment to improving access to research for under-represented groups. In addition to a long-term commitment to an existing outreach program for science and writing for K-12 students from Philadelphia, he has regularly reserved space in his research lab over the summer and during the academic year for at least one student self-identified as belonging to an under-represented group. More broadly, as the director of the HHMI-sponsored programs at Haverford College, the PI is responsible for awarding student interdisciplinary research fellowships and funding internal and external research opportunities for students belonging to under-represented groups, administering several outreach programs, and supporting faculty development seminars and workshops to enhance the overall research program in the sciences.