Single-Molecule Studies of Chaperonin Mechanism

描述（由申请人提供）：本探索性R21提案的目标是开发和使用单分子方法来了解伴侣蛋白功能的分子机制。我们将重点关注两个合作的低聚酶，GroEL/ES和TRiC，它们都是各种新翻译的蛋白质和酶的细胞内折叠所必需的。通过将整体测量结果与单分子的动态测序行为进行比较，我们将深入了解驱动这些酶功能的构象变化的时间和动力学。双环伴侣蛋白在其环腔内结合未折叠的多肽，防止通路外反应，并以高度合作，atp依赖的方式促进生产蛋白折叠到天然状态。同质低聚大肠杆菌复合体GroEL与单环复合体GroES协同作用。与groes不同的是，真核细胞质伴侣蛋白（CCT或TRiC）是异聚寡聚的，没有类似groes的辅助因子。

DESCRIPTION (provided by applicant): The goal of this exploratory R21 proposal is to develop and use single-molecule approaches to understand the molecular mechanisms of chaperonin function. We will focus on two cooperative oligomeric enzymes, GroEL/ES and TRiC, both of which are essential to the intracellular folding of a variety of newly-translated proteins and enzymes. By comparing bulk measurements with the dynamical sequencing behavior of single molecules, we will obtain insight into the timing and dynamics of the conformational changes that drive the function of these enzymes. Double-ring chaperonins bind unfolded polypeptides within their ring cavities, prevent off-pathway reactions, and promote productive protein folding to the native state in a highly cooperative, ATP-dependent manner. The homo-oligomeric E. coli complex GroEL acts in concert with the single-ring complex GroES. Unlike GroEL, the eukaryotic cytosolic chaperonin, denoted CCT or TRiC, is hetero-oligomeric and functions without a GroES-like cofactor. The primary objective of the proposed work is to explore the specifics of dynamical sequencing of the conformational states of these enzymes. This goal will be addressed by applying both biological and physical expertise of the researchers to two specific aims focused on understanding aspects of chaperonin action. By selecting individual copies of chaperonin complexes, we will remove heterogeneity between individual copies that are in different conformational states. In this way we will be able to sense and characterize the sequence of dynamical events and local environmental changes that occur during the nucleotide cycle, as chaperones assist in the folding of selected substrate proteins. Aim 1 will investigate hypotheses relating to the mechanism of action of assisted protein folding in the bacterial GroEL/GroES chaperonin system. Aim 2 will address similar questions for the less-studied but equally important eukaryotic chaperonin, TriC. The exploratory nature of this application arises from the fact that many of the assays proposed have not been previously utilized to explore chaperonin mechanisms.