MECHANISMS OF CYTOSOLIC CHAPERONIN ACTION

描述：双环伴侣蛋白复合体是一种重要的神经递质 细胞蛋白质折叠。基于它们结合展开的能力 环腔内的多肽，它们防止了旁路反应 并促进生产性蛋白质折叠到自然状态 依赖于ATP的方式。部分折叠出现在伴侣素腔中 在底物蛋白释放到溶液中之前。功能界别 推导了细菌GroEL/Groes伴侣蛋白系统的原理 从体外重建研究来看，它的确切作用机制是 不管怎么说都不清楚。与同源低聚GroEL不同，最近唯一的 已发现的真核细胞胞浆伴侣蛋白trc是一种异源寡聚体 很复杂，而且似乎不依赖于Groes样的辅因子。鲜为人知 关于这种伴侣蛋白的功能和底物专一性。目标是 这一建议的关键是理解 伴侣素环复合体介导胞浆蛋白折叠。以下是 具体方面将涉及体外纯化的成分： 固定化伴侣蛋白络合物和诱捕伴侣蛋白的折叠分析 将用于实验以确定GroEL/GroES和 三位一体的行动。分析伴侣反应过程中ATP的水解性 周期与蛋白质的折叠和释放有关，修饰的伴侣蛋白 将对这一过程中存在的缺陷进行检查。这些研究将会完成 与原核生物和真核生物伴侣蛋白平行，了解是否 它们以类似的方式促进蛋白质折叠。 分子伴侣结合蛋白的构象将通过配基和 底物蛋白抑制物结合特性的监测 内源和外源底物荧光团的荧光变化。这个 TIC的底物光谱、底物折叠和释放动力学 将被分析，两个结构域的融合蛋白将作为模型系统 用于分析结构域折叠。 作为朝着重建细胞折叠的目标迈出的重要一步 在体外，建议的生化分析也将在 大分子挤压剂和浓缩细胞质的存在 紧密模拟细胞环境的蛋白质提取物，并影响 深刻地研究了伴侣系统的性质。 预计这些研究的结果将使人们深入了解 胞质蛋白质折叠的基本过程，可能对 更好地了解与疾病相关的异常蛋白质功能。

DESCRIPTION: Double-ring chaperonin complexes are essential mediators of cellular protein folding. Based on their ability to bind unfolded polypeptides within their ring cavities, they prevent off-pathway reactions and promote productive protein folding to the native state in an ATP-dependent manner. Partial folding appears in the chaperonin cavity before release of the substrate proteins into solution. The functional principles of the bacterial GroEL/GroES chaperonin system have been derived from in vitro reconstitution studies, the exact mechanism of its action is however unclear. Unlike the homo-oligomeric GroEL, the only recently discovered eukaryotic cytosolic chaperonin TRiC is a hetero-oligomeric complex and appears not to rely on a GroES-like cofactor. Little is known about the function and substrate specificity of this chaperonin. The goal of this proposal is to understand the molecular mechanism by which chaperonin ring complexes mediate cytosolic protein folding. The following specific aspects will be addressed with the purified components in vitro: Folding assays with immobilized chaperonin complexes and trap chaperonins will be used in experiments to determine the mechanism of GroEL/GroES and TRiC action. To analyze how ATP hydrolysis during the chaperonin reaction cycle is coupled to protein folding and release, the modified chaperonins with defects in this process will be examined. These studies will be done in parallel with the prokaryotic and eukaryotic chaperonins to learn whether they facilitate protein folding in a similar manner. The conformation of chaperonin-bound proteins will be assessed by ligand and inhibitor binding characteristics of substrate proteins by monitoring fluorescence changes of intrinsic and extrinsic substrate fluorophors. The substrate spectrum of TRiC, the kinetics of substrate folding and release will be analyzed, and two-domain fusion proteins will serve as model systems for the analysis of domain-wise folding. As a major step towards the goal of reconstituting cellular folding in vitro, the proposed biochemical assays will be performed also in the presence of macromolecular crowding agents and concentrated cytoplasmic protein extracts which closely mimic the cellular milieu and affect profoundly the properties of chaperonin systems. The results of these studies are expected to give insight into the fundamental process of cytosolic protein folding and may be useful for a better understanding of disease-related abnormal protein functions.