Unfolded States and Folding Pathways by NMR

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的重要性日益增加。许多疾病状态与未折叠和错误折叠的蛋白质有关。分子伴侣通常包含非结构化或部分结构化的结构域，其功能知之甚少。该项目的前三个具体目标是解决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。从初步数据的一个新的观察是，在两种情况下，这些域似乎形成熔融球型结构的解决方案。待研究的第一个结构域（目标1）含有大肠杆菌DnaJ的富含半胱氨酸（CR）和C-末端结构域，其似乎形成熔融球。一个熔化的小球似乎也是由E.大肠杆菌伴侣触发因子（Aim 2）。在每一种情况下，熔融球状态的结构将通过NMR方法进行探测，并通过肽和未折叠蛋白质的结合来了解功能。第三个结构域（Aim 3）是p23，一种人Hsp 9 O共伴侣蛋白，其未折叠的C-末端序列是伴侣蛋白功能所需的。将通过NMR研究含有折叠的N-末端结构域和未折叠的C-末端序列的构建体，以确定Hsp 9 O和未折叠蛋白质的结合位点。第四个目的是关于一个群体感应因子SdiA的溶液结构研究，它与致病性大肠杆菌的毒性有关。线圈应变。在另一种群体感应蛋白（来自根癌农杆菌的TraR）可以在细胞环境中折叠之前，已经发现需要小分子自诱导物。SdiA的结构和折叠行为将被确定，以发现这种行为是否在细菌群体感应中普遍存在。这四个具体目标是针对本研究项目的总体目标，阐明具有一个或多个未折叠组分的蛋白质系统中结构和功能的关系，以进一步了解折叠和未折叠反应在细胞代谢和疾病中的作用。

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.