STRUCTURE/FUNCTION ANALYSIS OF MOLECULAR CHAPERONES

分子伴侣的结构/功能分析

• 批准号: 6019511
• 负责人: CELIA J HARRISON
• 金额: $ 23.27万
• 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/6019511
• 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：分子伴侣是一种与新生细胞相互作用的蛋白质。 蛋白质、变性蛋白质或其他大分子组装和帮助 它们实现了天然的三级或四级结构。原核生物 分子伴侣DNAK、DNAJ和GRPE协同隔离 聚集性敏感的新生多肽和未折叠蛋白 以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水解物的最新X射线晶体结构 与核苷酸交换因子GRPE复合的(ATPase)结构域被揭示 GRPE诱导核苷酸复合体的不稳定构象变化 在与ADP结合的形式中。GRPE是一种非常细长的十字形 导致DNAK-ATPase深核苷酸结合裂解的分子 要打开的域(类似肌动蛋白)。在其作为核苷酸的作用中 交换因素，GRPE的作用在概念上与其他 在各种细胞计时机制中运行的交换因子。 GRPE-DNAK-ATPase复合体强调的一些问题是根本的 可以对所有核苷酸交换因子提出的问题，例如如何 GRPE区分DNAK的ADP结合形式和ATP结合形式，以及如何 GRPE被三磷酸腺苷结合取代了DNAK。这项建议是 与核苷酸交换的结构和功能方面有关的 管理DNAK陪护循环。这项建议的具体目的是： 1)确定GRPE如何识别DNAK的ADP状态，但绑定和 通过以下方式稳定立体化学上不等价的无核苷酸DNAK GRPE-DNAK界面和X射线的丙氨酸扫描突变 核苷酸结合的DNAK的结晶学研究。2)机制研究 利用GRPE的非对称性将GRPE从DNaK置换出来 当与DNAK结合时，并用体外蛋白质折叠试验进行测试3) 研究DNAK-ATPase结构域和变构通讯 X射线结晶学研究GRPE的多肽结合域及其诱变作用 GRPE的TH底物解离区。这样做的长期目标是 建议了解核苷酸交换因子在生物多样性中的作用 结构背景，并进一步了解分子的结构知识 监护人。