Biophysical Aspects of Cotranslational Protein Folding

智力优势。 蛋白质获得其折叠结构的途径在细胞环境中知之甚少。该项目的目标是解决在生物学相关背景下蛋白质折叠的基本原则，因为新生多肽在翻译过程中从核糖体隧道中退出。这些研究是基于一个工作假设，即在尚未合成的链部分之间不能建立长程折叠相互作用。因此，全长蛋白质的体外折叠途径很可能不同于共价延长链的相应途径。 利用先前NSF支持下开发的方法学进展，将阐明共翻译蛋白质折叠作为链延伸的函数的具体结构方面。 该项目将侧重于两个单域蛋白质（脱辅基肌红蛋白和胸苷酸激酶），主要具有α-螺旋结构和大量的长距离天然接触。 新生多肽将通过氢/氘交换，然后通过飞行时间MALDI质谱法进行分析。这种方法揭示了作为链伸长的函数的链压缩和主链氢键的程度。高分辨率的信息将获得原位有限的蛋白质水解条件下淬火。上述研究是在无细胞系统中进行的，该系统含有或缺乏核糖体相关触发因子伴侣的基因。Cavagnero集团坚定地致力于本科生参与研究，并积极参与学术界代表性不足的群体。本科生为研究做出了重大贡献，并经常作为研究出版物的合著者出现。几位过去和现在的本科生获得了著名的校园范围内的本科研究奖。首席研究员参与教育课程开发与NSF赞助的研究，教学和学习（CIRTL）在威斯康星大学麦迪逊分校及其校园计划，三角洲的整合中心合作。这包括一项旨在发展一般化学原理与日常生命科学相关应用之间联系的倡议。

Intellectual Merit. The pathways by which proteins attain their folded structure are poorly understood in the context of the cellular environment. The goal of this project is to address the fundamental principles governing protein folding in a biologically relevant context, as nascent polypeptides exit from the ribosomal tunnel during translation. The investigations are based on the working hypothesis that long-range folding interactions cannot be established among portions of the chain that have not been synthesized yet. Therefore, in vitro folding pathways of full length proteins are likely to be different from the corresponding pathways of cotranslationally elongating chains. Using methodological advances developed under prior NSF support, specific structural aspects of cotranslational protein folding as a function of chain elongation will be elucidated. The project will focus on two single-domain proteins (apo-myoglobin and thymidylate kinase) with predominantly alpha-helical structure and a large number of long-range native contacts. Nascent polypeptides will be analyzed by hydrogen/deuterium exchange followed by time-of-flight MALDI mass spectrometry. This approach unveils the degree of chain compaction and backbone hydrogen bonding as a function of chain elongation. High resolution information will be gained by in situ limited proteolysis under quenching conditions. The above studies are carried out in a cell-free system either containing or lacking the gene for the ribosome-associated Trigger Factor chaperone.Broader Impacts. The Cavagnero group is strongly committed to undergraduate participation to research and to the active involvement of underrepresented groups in academia. Undergraduate students contribute significantly to the research and regularly appear as coauthors of research publications. Several past and current undergraduate students have received prestigious campus-wide undergraduate research awards. The principal investigator is involved in educational curriculum development in collaboration with the NSF-sponsored Center for the Integration of Research, Teaching and Learning (CIRTL) at UW-Madison and its campus program, Delta. This includes an initiative to develop connections between general chemistry principles and everyday life-science-related applications.