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

该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错误折叠和聚集过程中的分子和化学作用。这种异常过程导致大原纤维的形成，是所有生物体共同的一般分子问题，可导致代谢和结构缺陷。最近，由于在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，这种聚集过程受到了密切关注。谷氨酰胺重复序列的延伸折叠成b-sheet结构，然后通过进一步添加多肽链形成长丝，作为聚集的模板。单个细丝可以进一步结合形成多细丝原纤维。谷氨酰胺被认为通过涉及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的各自作用仍然存在争议。PI已经开发了一种肽模型系统，可以形成一个原型b-sheet，称为b-hairpin，可以使用各种生物物理方法来研究，以探索谷氨酰胺在促进b-sheet，细丝和原纤维形成中的作用。三个具体目标是：(1)寻找稳定原纤维组装途径中中间体的条件，以便评估谷氨酰胺相互作用对生长和稳定性的作用；(2)测定氨基酸突变对这些过程的影响，探讨其化学机制；(3)对特定谷氨酰胺进行同位素标记，直接探测各种中间体中稳定的氢键。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，并测试含有多谷氨酰胺重复序列的蛋白质的各种结构模型。本研究已发展为一套适合本科生的项目，因为主要的研究工作将由努力完成生物学论文要求的学生承担。从事这项工作的学生将获得跨学科研究方面的训练。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。在这个项目中，我们开设了入门课程的蛋白质折叠和错误折叠课程，为蛋白质结构和功能的高级课程开设了更高级的课程，并开设了为期7周的基于初级文献的蛋白质错误折叠和聚集课程。PI长期致力于改善代表性不足的群体获得研究的机会。除了长期致力于现有的针对费城K-12学生的科学和写作拓展项目外，他还在夏季和学年期间定期为至少一名自认为属于弱势群体的学生保留研究实验室的空间。更广泛地说，作为哈弗福德学院hhmi赞助项目的负责人，PI负责授予学生跨学科研究奖学金，为属于代表性不足群体的学生提供内部和外部研究机会，管理几个外展项目，并支持教师发展研讨会和讲习班，以加强科学领域的整体研究项目。

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.