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。我们将专注于两个合作寡聚酶，GroEL/ES和TRiC，这两个都是必不可少的各种新翻译的蛋白质和酶的细胞内折叠。通过比较批量测量与单分子的动态测序行为，我们将深入了解驱动这些酶的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，防止非途径反应，并以高度合作的ATP依赖性方式促进生产性蛋白质折叠至天然状态。同源寡聚E.大肠杆菌复合物GroEL与单环复合物GroES协同作用。与GroEL不同，真核细胞溶质伴侣蛋白（表示为CCT或TRiC）是异源寡聚的，并且在没有GroES样辅因子的情况下起作用。 所提出的工作的主要目标是探索这些酶的构象状态的动态测序的细节。这一目标将通过将研究人员的生物学和物理学专业知识应用于两个具体目标来实现，这两个目标侧重于了解伴侣蛋白作用的各个方面。通过选择伴侣蛋白复合物的单个拷贝，我们将消除处于不同构象状态的单个拷贝之间的异质性。通过这种方式，我们将能够感测和表征核苷酸循环期间发生的动态事件和局部环境变化的序列，因为分子伴侣有助于选定底物蛋白的折叠。目的1将调查有关的假设，在细菌GroEL/GroES伴侣蛋白系统中的辅助蛋白折叠的作用机制。目标2将解决类似的问题，研究较少，但同样重要的真核生物伴侣蛋白，三C。这种应用的探索性质产生于这样一个事实，即许多提出的测定法以前没有被用来探索伴侣蛋白机制。

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.