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: 6604262
• 负责人: HELEN JANE DYSON
• 金额: $ 37.04万
• 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/6604262
• 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）：随着发现许多调节蛋白的功能展开形式，展开的蛋白质及其相互作用在细胞代谢的研究中的重要性越来越重要。许多疾病状态与展开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域，其功能知之甚少。该项目的前三个特定目的涉及这些展开的域在肽和展开蛋白质结合中的假设功能。来自初步数据的新观察结果是，在两种情况下，这些结构域似乎在溶液中形成了熔融球体型结构。要研究的第一个结构域（AIM 1）包含大肠杆菌DNAJ的富含半胱氨酸的（CR）和C末端结构域，似乎形成了熔融球。熔融球似乎也由大肠杆菌伴侣触发因子的C末端结构域形成（AIM 2）。在每种情况下，将通过NMR方法探测熔融球状状态的结构，并且将通过肽和展开的蛋白质结合提供对该功能的见解。第三个结构域（AIM 3）是P23，一种人类HSP9O副酮，其展开的C末端序列是伴侣功能所必需的。 NMR将研究包含折叠N末端结构域和展开的C末端序列的构建体，以确定Hsp9O和展开的蛋白质的结合位点。第四个目标与对群体感应因子SDIA的溶液结构研究有关，该研究与致病性E.卷积菌株的毒力有关。在另一种法定感应蛋白（来自农杆菌Tumefaciens的Trar）可以在细胞环境中折叠之前，已经发现了小分子自动诱导剂的需求。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中是一般的。这四个具体目标是针对该研究项目的整体目标，旨在阐明蛋白质系统中具有一个或多个展开的成分的结构和功能的关系，以进一步了解我们对折叠和不展开反应在细胞代谢和疾病中的作用的理解。