STRUCTURE/FUNCTION ANALYSIS OF MOLECULAR CHAPERONES

分子伴侣的结构/功能分析

• 批准号: 6384321
• 负责人: CELIA J HARRISON
• 金额: $ 25.84万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384321
• 关键词: X ray crystallography; adenosine diphosphate; adenosinetriphosphatase; allosteric site; calorimetry; conformation; guanine nucleotide exchange factors; molecular chaperones; protein folding; protein structure function; site directed mutagenesis; surface plasmon resonance; thermodynamics

DESCRIPTION: Molecular chaperones are proteins that interact with nascent proteins, denatured proteins or other macromolecular assemblies and help them achieve native tertiary or quaternary structure. The prokaryotic molecular chaperones DnaK, DnaJ and GrpE cooperate to sequester aggregation-sensitive nascent polypeptides and unfolded proteins in an ATP-dependent manner, effectively removing them from the crowded cellular environment. The recent X-ray crystal structure of the DnaK ATP-hydrolyzing (ATPase) domain complexed with the nucleotide exchange factor GrpE revealed that GrpE induces a nucleotide-complex destabilizing conformational change in the ADP-bound form o DnaK. GrpE is a very elongated, cruciform-shaped molecule that causes the deep nucleotide binding cleft of the DnaK ATPase domain (which resembles actin) to be opened up. In its role as a nucleotide exchange factor, the action of GrpE is related conceptually to that of other exchange factors that operate in a variety of cellular timing mechanisms. Some issues highlighted by the GrpE-DnaK-ATPase complex are fundamental questions that can be asked of all nucleotide exchange factors, such as how GrpE discriminates between the ADP-and the ATP-bound forms of DnaK, and how GrpE is displaced from DnaK by the binding of ATP. This proposal is concerned with structural and functional aspects of nucleotide exchange that govern the DnaK chaperone cycle. The specific aims of this proposal are to: 1) Determine how GrpE recognizes the ADP-state of DnaK, but binds and stabilizes the stereochemically non-equivalen nucleotide-free DnaK by alanine-scanning mutagenesis of the GrpE-DnaK interfac and X-ray crystallographic studies of nucleotide-bound DnaK. 2) Study the mechanism of displacement of GrpE from DnaK by exploiting the asymmetric natur of GrpE when bound to DnaK, and testing with an in vitro protein folding assay 3) Investigate the allosteric communication between DnaK ATPase domain and peptide binding domain via GrpE by X-ray crystallography and mutagenesis of th substrate-dissociating region of GrpE. The long-term goals of this proposal ar to understand the function of nucleotide exchange factors in a structural context, and to further structural knowledge of the molecular chaperones.

描述：分子伴侣是与新生的 蛋白质、变性蛋白质或其它大分子组装体， 它们获得天然的三级或四级结构。 原核 分子伴侣DnaK、DnaJ和GrpE协同螯合 聚集敏感的新生多肽和未折叠蛋白质， ATP依赖的方式，有效地将它们从拥挤的细胞中移除， 环境 DnaK ATP水解酶的最新X射线晶体结构 （ATP酶）结构域与核苷酸交换因子GrpE复合显示 GrpE诱导核苷酸复合物不稳定的构象变化 以ADP结合形式的DnaK。 GrpE是一个非常细长的十字形 引起DnaK ATP酶的深核苷酸结合裂缝的分子 （类似于actin的结构域）被打开。 作为一种核苷酸 交换因子，GrpE的作用在概念上与其他 在各种蜂窝定时机制中操作的交换因子。 GrpE-DnaK-ATP酶复合物突出的一些问题是根本性的 所有的核苷酸交换因子都可以被问到的问题，例如如何 GrpE区分ADP和ATP结合形式的DnaK，以及如何 GrpE通过ATP的结合而从DnaK置换。 这项建议是 涉及核苷酸交换的结构和功能方面， 控制着DnaK分子伴侣的循环 这项建议的具体目标是： 1)确定GrpE如何识别DnaK的ADP状态，但结合和 通过以下方式稳定立体化学上不等价的无核苷酸DnaK： GrpE-DnaK界面的丙氨酸扫描诱变和X射线 核苷酸结合的DnaK的晶体学研究。 2)机理研究 通过利用GrpE的不对称性质， 当与DnaK结合时，并用体外蛋白质折叠测定进行测试3） 研究DnaK ATP酶结构域与 肽结合域通过GrpE通过X射线晶体学和诱变 GrpE的底物解离区。 长期目标是 我们的建议是了解核苷酸交换因子在一个 结构背景，并进一步了解分子的结构 监护人