Chaperonin-mediated protein folding

伴侣蛋白介导的蛋白质折叠

• 批准号: 6815644
• 负责人: ARTHUR L HORWICH
• 金额: $ 29.74万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6815644
• 关键词: adenosine triphosphate; crosslink; cryoelectron microscopy; cysteine; high performance liquid chromatography; mass spectrometry; molecular chaperones; nucleotides; peptides; protein folding

DESCRIPTION (provided by applicant): This proposal seeks to support an ongoing Iongterm project concerning the mechanism of action of the essential megadalton-sized double ring molecular machines known as chaperonins, which mediate ATPdependent protein folding to the native state in a variety of cellular compartments. Here, we propose studies of the bacterial chaperonin, GroEL, and its cooperating co-chaperonin, GroES, both in vivo and in vitro. One study in vivo will aim to resolve the physiological action of GroEL, not well-understood due to lack of a tight conditional mutant, by identifying a chemical compound that binds to the GroEL ATP binding pocket and produces immediate-onset loss of function in vivo. We will examine whether the protein translation machinery is halted by such arrest of the GroEL folding machine, and will inspect for misfolding/aggregation both of nascent polypeptides, if they are produced, and of pre-existent proteins. This will allow us to observe the immediate physiologic consequences of disruption of the chaperonin system and should identify its authentic and essential substrates. In a second area, mechanistic studies will be carried out in vitro, following on our earlier studies of the machine itself, directing analysis to the polypeptide substrate. We will analyze the global topology of substrate proteins while bound to GroEL, using oxidative crosslinking between native and engineered cysteines in the substrates themselves, addressing whether the topology is unique and potentially native-like, random, or a limited ensemble of states. We will also address whether bound polypeptides occupy a characteristic topology relative to GroEL, with a particular portion bound inside the central cavity of a GroEL ring while the remainder is localized outside in the bulk solution, using external labeling of the exposed portion of substrate protein and single particle imaging with cryoEM. We will also address actions of nucleotide/GroES binding on GroEL-substrate binary complexes. One study will use cryoEM to examine complexes formed by addition of ADP/GroES to GroEL-rhodanese, in which apical domain movement is severely retarded, potentially providing a structure of a GroEL-GroES collision complex. In a second study, the question of whether ATP/GroES binding to GroEL-substrate complexes produces an active, forced, unfolding of substrate protein prior to its release into the central cavity will be studied using tritium and hydrogen/deuterium exchange experiments.

描述（由申请人提供）：本提案旨在支持一项正在进行的长期项目，该项目涉及被称为伴侣蛋白的基本巨噬子大小的双环分子机器的作用机制，伴侣蛋白在各种细胞区室中介导atp依赖性蛋白折叠到天然状态。在这里，我们建议在体内和体外对细菌伴侣蛋白GroEL及其协同伴侣蛋白GroES进行研究。一项体内研究将通过鉴定一种与GroEL ATP结合袋结合并在体内产生立即发作的功能丧失的化合物，旨在解决GroEL的生理作用，由于缺乏紧密条件突变体而不被很好地理解。我们将检查蛋白质翻译机制是否因GroEL折叠机器的停止而停止，并将检查新生多肽（如果产生的话）和预先存在的蛋白质的错误折叠/聚集。这将使我们能够观察到伴侣蛋白系统破坏的直接生理后果，并应该确定其真实和必要的底物。在第二个领域，机械研究将在体外进行，继我们早期对机器本身的研究之后，直接分析多肽底物。我们将分析底物蛋白与GroEL结合时的全局拓扑结构，使用底物本身中天然半胱氨酸和工程半胱氨酸之间的氧化交联，解决拓扑结构是否独特，是否可能与天然相似，随机或有限的状态集合。我们还将讨论结合的多肽是否占据相对于GroEL的特征拓扑结构，其中特定部分结合在GroEL环的中心腔内，而其余部分则定位在散装溶液的外部，使用底物蛋白暴露部分的外部标记和低温电子显微镜的单颗粒成像。我们还将讨论核苷酸/GroES结合在groel -底物二元配合物上的作用。一项研究将使用低温电子显微镜来检测添加ADP/GroES到GroEL-rhodanese中形成的配合物，其中顶端结构域的运动严重迟缓，可能提供GroEL-GroES碰撞配合物的结构。在第二项研究中，ATP/GroES与groel -底物复合物的结合是否会在底物蛋白释放到中心腔之前产生活性的、强制的、展开的问题将通过氚和氢/氘交换实验进行研究。