MOLECULAR MECHANISM OF GROES/GROEL CHAPERONIN FUNCTION

从细菌到人类的所有有机体都会对热和其他压力作出反应 导致未折叠多肽的积累， 增加了少量高度保守的， 组成型表达的基因产物称为热休克或应激， 诱导蛋白质 越来越多的证据表明， 蛋白质通常参与多种必需的 生理过程，包括有效的细胞内蛋白 折页. GroES和GroEL伴侣蛋白是主要的热休克蛋白 从大肠杆菌中分离出来，通过调节 释放未折叠的多肽链， GroEL，在ATP水解偶联反应中，以最小化它们的 非生产性聚集。 尽管有大量的生化数据， 导致了描述性模型，很少有定量信息， 这些蛋白质是如何促进 重折叠 这项研究的目的是深入了解 GroES和GroEL组装和催化的分子机制 通过研究结构， 它们相互作用的动力学和热力学基础，以及 模型肽，以及这些过程如何与ATP结合， 水解 GroES与GroEL的相互作用将被表征 通过使用放射性标记的蛋白质的沉降平衡来测量它们的 强关联，并阐明它们相互作用的化学计量 在肽和核苷酸效应物的存在下。 的力 将通过滴定来描述与GroEL结合的稳定多肽 核糖核酸酶S肽与 监护人 将这些充满活力的驱动力与 结构和S肽与S蛋白的相互作用将产生 GroEL的多肽结合位点的热力学描述，和 可以解决多肽链结合中的明显协同效应， GroEL。 多肽荧光变化的快速动力学测量 结合和解离，再加上淬火流动实验，以测量 ATP结合、水解和产物释放的速率将阐明 ATP结合如何与GroEL释放的多肽链偶联。 这些实验将提供框架来测试一个简单的工作 通过GroES和肽调节GroEL ATP酶的假说 印刷受体. 通过以下方法测量的结构变化的相关性潜力 不同的沉降速度与功能扰动将是 利用工程化表达载体构建位点特异性 这些必需基因产物中的突变体，除了使它们的 从组成型水平纯化野生型GroES和GroEL， 一步。 将构建含半胱氨酸的突变体以制备 重原子衍生物，以帮助解决的结构， 从大的单晶体中分离出来的伴侣蛋白， 解决方案，使我们能够实现我们的长期目标， 伴侣蛋白与相关结构的分子相互作用 沿着它们的反应途径。

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.