MOLECULAR MECHANISM OF GROES/GROEL CHAPERONIN FUNCTION

从细菌到人类的所有有机体都会对高温和其他压力做出反应 这导致未折叠的多肽通过快速积累 增加少量高度保守的合成， 结构性表达的基因产物称为热休克或应激- 诱导蛋白。迅速积累的证据表明，压力引起的 蛋白质通常参与一系列不同的必需 生理过程，包括有效的细胞内蛋白质 折叠。Groes和GroEL伴侣蛋白是主要的热休克蛋白 来自大肠杆菌，通过调节细胞内的蛋白质折叠来控制蛋白质折叠 释放与其强烈结合的未折叠多肽链 GroEL，在三磷酸腺苷水解偶联反应中，使其最小化 非生产性聚合。尽管有丰富的生化数据 导致了描述性模型，几乎没有定量信息 可用于为这些蛋白质如何促进 重新折叠。这项研究的目标是深入了解 GroES和GroEL组装催化的分子机制 有效的细胞蛋白质折叠通过研究结构， 它们相互作用的动力学和热力学基础，以及 模型多肽，以及这些过程如何与ATP结合和 水解液。将表征GroES与GroEL的相互作用 通过使用放射性标记蛋白质的沉降平衡来测量它们的 并阐明它们相互作用的化学计量学 在多肽和核苷酸效应物存在的情况下。这些力量 与GroEL结合的稳定多肽将通过滴定来描述 量热法测定核糖核酸酶S多肽与核糖核酸酶的结合 守护神。将这些充满活力的驱动力与 S多肽的结构及其与S蛋白的相互作用 GroEL多肽结合部位的热力学描述，以及 可通过以下方式解决多肽链结合中的明显协同效应 格罗尔。多肽荧光变化的快速动力学测量 结合和解离，结合急冷流实验测量 ATP结合、水解和产物释放的速率将被阐明 三磷酸腺苷结合如何与GroEL释放的多肽链偶联。 这些实验将为测试一个简单的工作提供框架 GroES和多肽对GroEL-ATPase的调控假说 底物。关联结构变化的可能性由以下指标衡量 具有泛函摄动的沉积速度差 利用工程表达载体构建特定部位 这些重要基因产物中的突变体，除了使他们的 从组织水平纯化野生型GroE和GroEL 只需一步。含有半胱氨酸的突变体将被构建以准备 重原子导数，以帮助解决结构 将X射线衍射到原子的大单晶中的伴侣蛋白 分辨率，使我们能够实现我们的长期目标，将 具有相关结构的伴侣蛋白的分子相互作用 沿着它们的反应路径。

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.