GroEL: How Is Allostery Coupled to Assisted Folding?

GroEL：变构如何与辅助折叠结合？

基本信息

批准号：
7239660
负责人：
GEORGE H. LORIMER
金额：
$ 28.51万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
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/7239660
关键词：
ATP Hydrolysis ATP phosphohydrolase Address Adopted Affinity Apical Back Binding Binding Sites Biological Process Calorimetry Catalysis Cereals Chemicals Collaborations Communication Complement Complex Computer Simulation Coupled Coupling Crystallography Data Data Set Disease Equilibrium Event Family Fluorescence Spectroscopy Goals GroEL Protein GroES Protein Hydrolysis Ions Kinetics Length Libraries Ligand Binding Ligands Link Location Measurement Methods Michigan Modeling Molecular Monovalent Cations Movement Nucleotides Numbers Peptide Synthesis Peptides Play Process Protein Binding Proteins Protocols documentation Rate Research Personnel Role Shapes Site Site-Directed Mutagenesis Structure Substrate Domain System Theoretical Studies Titrations Transducers Universities Work base chaperonin computer studies crosslink design in vivo insight member molecular dynamics mutant nanomachine polypeptide prevent protein folding research study response stoichiometry theories

项目摘要

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）：伴侣蛋白系统凹槽 /凹槽是不断增长的细胞纳米机器家族中最深入研究的成员。这些环形低聚物在功能上多样，但具有破坏分子和超分子复合物的能力，这是由ATP结合和水解循环驱动的。它们似乎充当机械化学传感器。必须更好地理解这些纳米机制的结构和机制，因为越来越多的疾病归因于它们的故障。无处不在和必不可少的蛋白质的伴侣蛋白在体内起着重要作用，帮助其底物蛋白（SP）实现并维持其本土状态。 E. COIL GROEL是原型伴侣蛋白，包括两个七聚环，背对背堆叠。每个子单元都包含三个不同的域。赤道结构域（ATP水解的位点），中间域和顶端结构域（SP结合的位点）。催化循环涉及凹槽中的大型，协同的域运动，通过关键配体的结合（K+离子和ATP与赤道域，SP和GROES与顶端域）的结合触发。变构通信发生在域之间，环之间和环之间。但是，这些变构状态与r状态过渡之间的耦合知之甚少。在此提案中，概述了旨在确定每个变构效应子在TT到RR转换的实验的实验。通过这些实验数据，将开发一个更健壮的全包合作模型。将确定变构和催化性必不可少的K+离子结合的位置，化学计量和亲和力常数。 t状态和R状态中ATP结合位点中基数的身份将被阐明。将使用定义长度的模型肽探索肽结合位点之间连通性在稳定t状态的重要性。拟议的工作中使用的方法包括位于定位的诱变，域间交联，稳态和停止流量动力学，等温滴定热量法，荧光光谱，X射线晶体学，肽合成，动力学和计算模型。这些合并的研究将为伴侣蛋白的机理提供特定意义的信息，但对其他环形纳米机制的机制具有一般意义。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Coupling between allosteric transitions in GroEL and assisted folding of a substrate protein.

GroEL 中的变构转变与底物蛋白的辅助折叠之间的耦合。

DOI：
10.1073/pnas.0700607104
发表时间：
2007
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Stan,George;Lorimer,GeorgeH;Thirumalai,D;Brooks,BernardR
通讯作者：
Brooks,BernardR

Allosteric transitions in the chaperonin GroEL are captured by a dominant normal mode that is most robust to sequence variations.

伴侣蛋白 GroEL 中的变构转变由对序列变化最稳健的主导正常模式捕获。