Unfolded States and Folding Pathways by NMR

通过 NMR 观察未折叠状态和折叠路径

• 批准号: 6546449
• 负责人: HELEN JANE DYSON
• 金额: $ 38.89万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6546449
• 关键词: DNA binding protein; Escherichia coli; bacterial proteins; circular dichroism; conformation; corticosteroid receptors; high performance liquid chromatography; molecular chaperones; myoglobin; nuclear magnetic resonance spectroscopy; plastocyanin; polymerase chain reaction; protein denaturation; protein folding; protein structure function; stop flow technique

DESCRIPTION (provided by applicant): Unfolded proteins and their interactions are assuming increasing importance in studies of cellular metabolism, as functional unfolded forms of many regulatory proteins are discovered. Many disease states are linked to unfolded and misfolded proteins. Molecular chaperones frequently contain unstructured or partly structured domains whose functions are poorly understood. The first three specific aims of this project address a postulated function of these unfolded domains in binding of peptides and unfolded proteins. A novel observation from preliminary data is that in two cases these domains appear to form molten globule-type structures in solution. The first domain to be studied (Aim 1) contains the cysteine-rich (CR) and C-terminal domains of Escherichia coli DnaJ, which appears to form a molten globule. A molten globule also appears to be formed by the C-terminal domain of the E. coli chaperone trigger factor (Aim 2). In each of these cases, the structure of the molten globule state will be probed by NMR methods, and insights into the function will be provided by binding of peptides and unfolded proteins. The third domain (Aim 3) is p23, a human Hsp9O co-chaperone, whose unfolded C-terminal sequence is required for chaperone function. Constructs containing the folded N-terminal domain and the unfolded C-terminal sequence will be studied by NMR to determine the sites of binding of Hsp9O and unfolded proteins. The fourth Aim is concerned with a solution structural study of a quorum-sensing factor, SdiA, which has been implicated in virulence of pathogenic E. coil strains. A requirement for a small molecule autoinducer has been found before another quorum-sensing protein, TraR from Agrobacterium tumefaciens, can fold in the cellular environment. The structure and folding behavior of SdiA will be determined, in order to discover whether this behavior is general in bacterial quorum-sensing. These four specific aims are directed towards the overall goal of this research project, to elucidate the relationship of structure and function in protein systems with one or more unfolded components, in order to further our understanding of the role of folding and unfolding reactions in cellular metabolism and disease.

描述（由申请人提供）：随着许多调节蛋白的功能性未折叠形式的发现，未折叠蛋白及其相互作用在细胞代谢研究中变得越来越重要。许多疾病状态与未折叠和错误折叠的蛋白质有关。分子伴侣经常包含非结构化或部分结构化的结构域，其功能人们知之甚少。该项目的前三个具体目标解决了这些未折叠结构域在肽和未折叠蛋白质结合中的假定功能。初步数据的一个新观察结果是，在两种情况下，这些域似乎在溶液中形成熔球型结构。要研究的第一个结构域（目标 1）包含大肠杆菌 DnaJ 的富含半胱氨酸 (CR) 和 C 末端结构域，它似乎形成熔球。熔球似乎也是由大肠杆菌伴侣触发因子的 C 末端结构域形成的（目标 2）。在每种情况下，都将通过核磁共振方法探测熔球状态的结构，并通过肽和未折叠蛋白质的结合来深入了解其功能。第三个结构域 (Aim 3) 是 p23，一种人 Hsp9O 辅助伴侣，其未折叠的 C 端序列是伴侣功能所必需的。将通过 NMR 研究包含折叠的 N 端结构域和未折叠的 C 端序列的构建体，以确定 Hsp9O 和未折叠蛋白的结合位点。第四个目标涉及群体感应因子 SdiA 的溶液结构研究，该因子与致病性大肠杆菌菌株的毒力有关。在另一种群体感应蛋白（来自根癌农杆菌的 TraR）在细胞环境中折叠之前，我们发现需要小分子自诱导剂。我们将确定SdiA的结构和折叠行为，以发现这种行为是否在细菌群体感应中具有普遍性。这四个具体目标是针对本研究项目的总体目标，阐明蛋白质系统中的结构和功能与一种或多种未折叠成分的关系，以进一步了解折叠和未折叠反应在细胞代谢中的作用。和疾病。