MOLECULAR MECHANISM OF GROES/GROEL CHAPERONIN FUNCTION

GROES/GROEL 伴侣蛋白功能的分子机制

• 批准号: 2186897
• 负责人: Edward Eisenstein
• 金额: $ 18.97万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186897
• 关键词: adenosine diphosphate; adenosine triphosphate; adenosinetriphosphatase; bioenergetics; biophysics; calorimetry; chemical association; chemical binding; chemical kinetics; conformation; molecular chaperones; molecular site; mutant; pancreatic ribonuclease; peptides; protein folding; protein structure function; sedimentation equilibrium; sedimentation velocity; site directed mutagenesis; stoichiometry; stop flow technique; stress proteins; thermodynamics; ultracentrifugation

All organisms from bacteria to man respond to heat and other stresses that lead to an accumulation of unfolded polypeptides by rapidly increasing the synthesis of a small number of highly conserved, constitutively expressed gene products called heat-shock or stress- induced proteins. Evidence is rapidly accumulating that stress-induced proteins are normally involved in a diverse set of essential physiological processes, including efficient intracellular protein folding. The GroES and GroEL chaperonins are major heat-shock proteins from Escherichia coli that control protein folding in cells by regulating the release of unfolded polypeptide chains that are strongly bound to GroEL, in an ATP hydrolysis coupled reaction, to minimize their nonproductive aggregation. Despite the wealth of biochemical data that has led to descriptive models, little quantitative information is available to base a molecular mechanism for how these proteins facilitate refolding. The goal of this research is to gain insight into the molecular mechanism whereby GroES and GroEL assemble and catalyze efficient cellular protein folding by investigating the structural, kinetic and thermodynamic basis of their interaction with each other, and model peptides, and how these processes are coupled to ATP binding and hydrolysis. The interaction of GroES with GroEL will be characterized by sedimentation equilibrium using radiolabeled proteins to measure their strong association and to clarify the stoichiometry of their interaction in the presence of peptide and nucleotide effectors. The forces stabilizing polypeptide binding to GroEL will be described by titration calorimetry measurements of the association of ribonuclease S peptide to the chaperonin. A comparison of these energetic driving forces to the structure and interactions of the S peptide with the S protein will yield a thermodynamic description of the polypeptide binding site of GroEL, and may resolve apparent cooperative effects in polypeptide chain binding by GroEL. Rapid kinetic measurements of fluorescence changes upon peptide binding and dissociation, coupled with quench flow experiments to measure the rates of ATP binding, hydrolysis and product release, will elucidate how ATP binding is coupled to polypeptide chain release from GroEL. These experiments will provide the framework to test a simple working hypothesis for the regulation of the GroEL ATPase by GroES and peptide substrates. The potential to correlate structural changes measured by difference sedimentation velocity with functional perturbations will be exploited with engineered expression vectors to construct site-specific mutants in these essential gene products, in addition to enabling their purification from constitutive levels wild-type GroES and GroEL in a single step. Cysteine containing mutants will be constructed to prepare heavy atom derivatives to aid in solving the structures of the chaperonins from large, single crystals that diffract x rays to atomic resolution, enabling us to achieve our long range goal of correlating the molecular interactions of chaperone proteins with relevant structures along their reaction pathway.

从细菌到人的所有生物都会应对热量和其他压力 这导致通过迅速的 增加少数高度保守的合成， 组成型表达的基因产品，称为热震或应力 - 诱导蛋白质。 证据迅速积累了压力引起的 蛋白质通常参与一系列重要的一组 生理过程，包括有效的细胞内蛋白 折叠式的。 凹槽和凹槽伴侣蛋白是主要的热冲蛋白 来自通过调节细胞中蛋白质折叠的大肠杆菌 释放展开的多肽链，与 在ATP水解偶联反应中，凹槽以最小化其 非生产性聚集。 尽管有大量的生化数据 导致了描述性模型，几乎没有定量信息是 可用于以分子机制为基础这些蛋白质如何促进 重新折叠。 这项研究的目的是深入了解 分子机制，凹槽和凹槽组装和催化 通过研究结构，有效的细胞蛋白折叠 它们相互作用的动力学和热力学基础， 模型肽，以及如何将这些过程与ATP结合和 水解。 凹槽与凹槽的相互作用将被表征 通过使用放射性标记的蛋白质进行沉降平衡来测量其 牢固的关联并阐明其相互作用的化学计量学 在存在肽和核苷酸效应子的情况下。 部队 稳定多肽与凹槽的结合将通过滴定描述 核糖核酸酶S肽与 伴侣蛋白。 这些能量驱动力与 S肽与S蛋白的结构和相互作用将产生 凹槽多肽结合位点的热力学描述， 可以通过通过 凹槽。 肽上荧光变化的快速动力学测量值 结合和解离，与淬灭流实验结合以测量 ATP结合，水解和产品释放的速率将阐明 如何将ATP结合与从凹槽释放多肽链释放。 这些实验将为测试简单工作的框架提供 假设通过GRO和肽调节凹槽ATPase 基材。 相关的结构变化的潜力 功能扰动的差异沉积速度将是 用工程表达向量利用以构建特定地点 这些基本基因产物中的突变体，除了能够 构成水平的野生型凹槽和凹槽的纯化 单步。 将构建含有半胱氨酸的突变体的准备 沉重的原子衍生物有助于解决 来自衍射X射线到原子的大型单晶的伴侣蛋白 解决方案，使我们能够实现将关联的远程目标 伴侣蛋白与相关结构的分子相互作用 沿着他们的反应途径。