GroEL: How Is Allostery Coupled to Assisted Folding?

GroEL：变构如何与辅助折叠结合？

• 批准号: 6752462
• 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/6752462
• 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是细胞纳米机器不断增长的家族中研究最深入的成员。这些环状低聚物在功能上是多样的，但都具有破坏分子和超分子复合物的能力，这是由ATP结合和水解的循环驱动的。它们似乎充当机械化学转换器。更好地了解这些纳米机器的结构和机制是必要的，因为越来越多的疾病已归因于它们的故障。伴侣蛋白是生物体内普遍存在的、不可缺少的蛋白质，在生物体内起着重要的作用，帮助其底物蛋白（SP）达到并维持其天然状态。E.卷曲GroEL，原型伴侣蛋白，包含两个背对背堆叠的七聚体环。每个亚单位包含三个不同的结构域：赤道结构域（ATP水解位点）、中间结构域和顶端结构域（SP结合位点）。催化循环涉及GroEL中的大的协同结构域运动，通过关键配体的结合（K+离子和ATP到赤道结构域，SP和GroES到顶端结构域）变构触发。变构通讯发生在结构域之间、环内和环之间。但是这些变构T状态到R状态转变之间的耦合知之甚少。在这个建议中，实验设计，以确定TT到TR到RR过渡的每个变构效应的作用进行了概述。有了这些实验数据，将开发一个更强大的，包罗万象的嵌套协同模型。的位置，化学计量和亲和常数的结合的变构和催化必需的K+离子将被确定。将阐明T状态和R状态下ATP结合位点中碱基的身份。肽结合位点之间的连接在稳定T状态的重要性将探讨与模型肽的定义长度。在拟议的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，动力学和计算建模。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，但对其他环形纳米机器的机制也具有普遍意义。