GroEL: How Is Allostery Coupled to Assisted Folding?

描述（由申请人提供）：伴侣蛋白系统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。这些环状低聚物功能多样，但在ATP结合和水解循环的驱动下，具有破坏分子和超分子复合物的能力。它们似乎起着机械化学传感器的作用。由于越来越多的疾病被归因于纳米机器的故障，因此更好地了解这些纳米机器的结构和机制是必不可少的。伴侣蛋白是一种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）达到和维持其天然状态。E. coil GroEL是典型的伴侣蛋白，由两个七聚体环组成，背靠背堆叠。每个子单元包括三个不同的域；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中大的、协调的结构域运动，由关键配体（K+离子和ATP结合到赤道结构域，SP和GroES结合到顶端结构域）的变构性触发。变构通信发生在结构域之间、环内和环间。但是，这些变构T态到R态转变之间的耦合却知之甚少。在本提案中，实验旨在确定每个变构效应在TT到TR到RR转变中的作用。有了这些实验数据，我们将开发出一个更稳健、更全面的嵌套协作模型。将确定变构性和催化性必需K+离子结合的位置、化学计量学和亲和常数。在T态和R态下ATP结合位点上的碱基的身份将被澄清。肽结合位点之间的连通性在稳定T状态中的重要性将通过定义长度的模型肽来探索。所提出的工作中使用的方法包括定点诱变，区域间交联，稳态和停止流动动力学，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。这些联合研究将为伴侣蛋白的作用机制提供具有特定意义的信息，同时对其他环状纳米机器的作用机制也具有普遍意义。

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.