GroEL: How Is Allostery Coupled to Assisted Folding?

描述(由申请人提供)：伴侣系统GroEL/GroES是不断增长的细胞纳米机器家族中研究最深入的成员。这些环状低聚物在功能上是多样化的，但在ATP结合和水解的循环驱动下，它们都具有破坏分子和超分子复合体的能力。它们似乎扮演着机械力化学传感器的角色。由于越来越多的疾病被归因于它们的故障，更好地了解这些纳米机器的结构和机制是当务之急。伴侣蛋白是一种普遍存在的、不可或缺的蛋白质，在体内发挥着重要的作用，帮助其底物蛋白(SP)实现和维持其天然状态。E.coil GroEL是典型的伴侣蛋白，由背靠背堆叠的两个七聚体环组成。每个亚单位包括三个不同的结构域：赤道结构域(ATP水解区)、中间域和顶端结构域(SP结合位点)。催化循环涉及GroEL中大的、协调的结构域移动，由关键配体(K+离子和ATP与赤道域，SP和GroES与顶区)的结合变构触发。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态到R态的跃迁之间的耦合还不是很清楚。在这项提案中，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。有了这些实验数据，将开发出一个更稳健、更全面的嵌套协作性模型。将确定变构和催化必需的K+离子结合的位置、化学计量比和亲和常数。T状态和R状态下的ATP结合位点中的碱基的同一性将得到澄清。将用确定长度的模型多肽来探索多肽结合位点之间的连通性在稳定T状态中的重要性。所采用的方法包括定点突变、结构域间交联、稳态和停流动力学、等温滴定量热法、荧光光谱、X射线结晶学、多肽合成、动力学和计算模拟。这些结合的研究将为伴侣蛋白的机制提供具有特定意义的信息，也将对其他环状纳米机器的机制提供普遍意义。

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.