Biophysical Aspects of Cotranslational Protein Folding

智力上的功绩。在细胞环境的背景下，蛋白质获得折叠结构的途径知之甚少。这个项目的目标是解决在生物相关的背景下管理蛋白质折叠的基本原则，因为新生的多肽在翻译过程中从核糖体隧道中出来。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远程折叠相互作用。因此，全长蛋白质的体外折叠路径可能不同于共翻译延长链的相应路径。利用在先前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长的函数的特定结构方面。该项目将专注于两种单域蛋白质(载脂蛋白和胸苷酸激酶)，它们主要是α-螺旋结构和大量的长距离天然接触。新生的多肽将通过氢/氢交换和飞行时间MALDI质谱仪进行分析。这种方法揭示了链紧凑度和主链氢键作为链延长的函数。在猝灭条件下，原位有限蛋白水解法可以获得高分辨率的信息。上述研究是在无细胞系统中进行的，无论是否含有核糖体相关的触发因子伴侣基因。Cavagnero小组坚定地致力于本科生参与研究，并积极参与学术界代表不足的群体。本科生对这项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。过去和现在的几名本科生都获得了享有盛誉的全校本科生研究奖。首席研究员与美国国家科学基金会赞助的威斯康星大学麦迪逊分校研究、教学和学习一体化中心(CIRTL)及其校园项目Delta合作，参与教育课程的开发。这包括一项在一般化学原理和日常生活科学应用之间建立联系的倡议。

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.