GroEL: How Is Allostery Coupled to Assisted Folding?

GroEL：变构如何与辅助折叠结合？

• 批准号: 6892899
• 负责人: GEORGE H. LORIMER
• 金额: $ 30.07万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6892899
• 关键词: X ray crystallography; adenosine triphosphate; binding sites; calorimetry; computer simulation; fluorescence spectrometry; hydrolysis; molecular chaperones; molecular dynamics; protein folding; protein structure; site directed mutagenesis

DESCRIPTION (provided by applicant): The chaperonin system GroEL / GroES is the most intensely studied member of a growing family of cellular nano-machines. These ring shaped oligomers are functionally diverse but share an ability to disrupt molecular and super-molecular complexes, driven by cycles of ATP binding and hydrolysis. They appear to act as mechano-chemical transducers. A better understanding of the structure and mechanism of these nano-machines is imperative since a growing number of diseases have been attributed to their malfunction. The chaperonins, ubiquitous and indispensable proteins, play an important role in vivo, assisting their substrate proteins (SP) to achieve and to maintain their native states. E. coil GroEL, the archetypic chaperonin, comprises two heptameric rings, stacked back to back. Each sub-unit comprises three distinct domains; the equatorial domain (the site for ATP hydrolysis), an intermediate domain, and an apical domain (the site for SP binding). The catalytic cycle involves large, concerted domain movements in GroEL, triggered allosterically by the binding of key ligands (K+ ion and ATP to the equatorial domain, SP and GroES to the apical domain). Allosteric communication occurs between the domains, within and between the rings. But the coupling between these allosteric T state to R state transitions is poorly understood. In this proposal, experiments designed to determine the role of each of the allosteric effectors on the TT to TR to RR transitions are outlined. With this experimental data, a more robust, all-inclusive model for nested cooperativity will be developed. The location, stoichiometry and affinity constants for the binding of the allosterically and catalytically essential K+ ion will be determined. The identity of the base in the ATP binding site in both the T state and the R state will be clarified. The importance of connectivity between the peptide binding sites in stabilizing the T state will be explored with model peptides of defined length. Methods utilized in the proposed work include site-directed mutagenesis, inter-domain cross-linking, steadystate and stopped-flow kinetics, isothermal titration calorimetry, fluorescence spectroscopy, x-ray crystallography, peptide synthesis, kinetic and computational modeling. These combined studies will provide information of specific significance to the mechanism of the chaperonins, but also of general significance to the mechanism of other ring-shaped nano-machines.

描述（由申请人提供）：伴侣蛋白系统 GroEL / GroES 是不断发展的细胞纳米机器家族中研究最深入的成员。这些环状低聚物功能多样，但都具有破坏分子和超分子复合物的能力，由 ATP 结合和水解循环驱动。它们似乎充当机械化学传感器。由于越来越多的疾病归因于纳米机器的故障，因此必须更好地了解这些纳米机器的结构和机制。伴侣蛋白是普遍存在且不可或缺的蛋白质，在体内发挥着重要作用，协助其底物蛋白（SP）实现并维持其天然状态。 E.coil GroEL，原型伴侣蛋白，由两个背靠背堆叠的七聚环组成。每个子单元包含三个不同的域；赤道域（ATP 水解位点）、中间域和顶端域（SP 结合位点）。催化循环涉及 GroEL 中大的、一致的结构域运动，由关键配体（K+ 离子和 ATP 到赤道结构域，SP 和 GroES 到顶端结构域）的结合以变构方式触发。变构通讯发生在域之间、环内和环之间。但人们对这些变构 T 状态到 R 状态转变之间的耦合知之甚少。在该提案中，概述了旨在确定每个变构效应器在 TT 到 TR 到 RR 转变中的作用的实验。利用这些实验数据，将开发出更强大、更全面的嵌套协作模型。将确定变构和催化必需 K+ 离子结合的位置、化学计量和亲和常数。 T 态和 R 态下 ATP 结合位点的碱基身份将得到澄清。将通过确定长度的模型肽来探索肽结合位点之间的连接对于稳定 T 状态的重要性。拟议工作中使用的方法包括定点诱变、域间交联、稳态和停流动力学、等温滴定量热法、荧光光谱、X射线晶体学、肽合成、动力学和计算模型。这些综合研究将为伴侣蛋白的机制提供具有特定意义的信息，而且对其他环形纳米机器的机制也具有一般意义。