STRUCTURE/FUNCTION ANALYSIS OF MOLECULAR CHAPERONES

分子伴侣的结构/功能分析

• 批准号: 6519922
• 负责人: CELIA J HARRISON
• 金额: $ 29.74万
• 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/6519922
• 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体并帮助 它们获得天然的三级或四级结构。 原核生物 分子伴侣 DnaK、DnaJ 和 GrpE 合作隔离 聚集敏感的新生多肽和未折叠蛋白 ATP依赖方式，有效地将它们从拥挤的细胞中清除 环境。 DnaK ATP 水解的最新 X 射线晶体结构 (ATPase) 结构域与核苷酸交换因子 GrpE 复合 GrpE 诱导核苷酸复合物不稳定的构象变化 以 ADP 结合形式 o DnaK。 GrpE 是一种非常细长的十字形 导致 DnaK ATP 酶深核苷酸结合裂隙的分子 要打开的域（类似于肌动蛋白）。 作为核苷酸的作用 交换因子，GrpE 的作用在概念上与其他的作用相关 在多种细胞计时机制中运作的交换因子。 GrpE-DnaK-ATPase 复合物强调的一些问题是根本性的 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，以及如何区分 通过 ATP 的结合，GrpE 从 DnaK 中被取代。 这个提议是 涉及核苷酸交换的结构和功能方面 控制 DnaK 伴侣循环。 该提案的具体目标是： 1) 确定 GrpE 如何识别 DnaK 的 ADP 状态，但结合并 通过以下方式稳定立体化学非等价的无核苷酸 DnaK GrpE-DnaK 界面和 X 射线的丙氨酸扫描诱变 核苷酸结合 DnaK 的晶体学研究。 2）研究机理 利用 GrpE 的不对称性质从 DnaK 中置换 GrpE 当与 DnaK 结合时，并使用体外蛋白质折叠测定进行测试 3) 研究 DnaK ATPase 结构域和 通过 X 射线晶体学和诱变通过 GrpE 肽结合域 GrpE 的第 3 个底物解离区。 本次活动的长期目标 建议 ar 来了解核苷酸交换因子在 a 中的功能 结构背景，并进一步了解分子的结构知识 伴侣。

项目成果

Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor.

• DOI: 10.1021/bi034416b
• 发表时间: 2003-08
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: A. Gelinas;J. Toth;Kelley A. Bethoney;K. Langsetmo;W. Stafford;C. Harrison

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation.

GrpE.DnaK 结合界面的能量突变分析：通过沉降速度分析超速离心的平衡关联常数。

• DOI: 10.1016/j.jmb.2004.03.074
• 发表时间: 2004
• 期刊: Journal of molecular biology.
• 作者: Gelinas,AmyD;Toth,Joseph;Bethoney,KelleyA;Stafford,WalterF;Harrison,CeliaJ
• 通讯作者: Harrison,CeliaJ