Unfolded States and Folding Pathways by NMR

描述(申请人提供)：随着许多调节蛋白的功能展开形式的发现，未折叠蛋白质及其相互作用在细胞新陈代谢研究中越来越重要。许多疾病状态与未折叠和错误折叠的蛋白质有关。分子伴侣通常含有非结构或部分结构的结构域，其功能尚不清楚。这个项目的前三个具体目标是解决这些未折叠结构域在多肽和未折叠蛋白质结合中的假定功能。来自初步数据的一个新的观察是，在两个案例中，这些结构域似乎在溶液中形成了熔融的球状结构。第一个要研究的结构域(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，似乎形成了一个熔融的球状结构域。大肠杆菌伴侣触发因子(AIM 2)的C-末端结构域似乎也形成了熔化的球状结构域。在每一种情况下，都将通过核磁共振方法探索熔融球状状态的结构，并通过结合多肽和未折叠蛋白质来深入了解其功能。第三个结构域(Aim 3)是p23，它是一个人类Hsp90O辅助伴侣，其C末端的未折叠序列是伴侣功能所必需的。包含折叠的N-末端结构域和未折叠的C-末端序列的构建将通过核磁共振研究来确定Hsp9O和未折叠蛋白质的结合位点。第四个目的是关于群体感应因子SdiA的溶液结构研究，SdiA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，可以在细胞环境中折叠之前，已经发现了对小分子自动诱导剂的需求。将确定SdiA的结构和折叠行为，以发现这种行为在细菌群体感应中是否普遍存在。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成分的蛋白质系统中结构和功能的关系，以加深我们对折叠和去折叠反应在细胞代谢和疾病中的作用的理解。

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.