Chaperonin-mediated protein folding

伴侣蛋白介导的蛋白质折叠

• 批准号: 7108010
• 负责人: ARTHUR L HORWICH
• 金额: $ 27.49万
• 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/7108010
• 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）：该提案旨在支持一项正在进行的长期项目，该项目涉及称为伴侣蛋白的基本兆道尔顿大小的双环分子机器的作用机制，该分子机器在各种细胞隔室中介导ATP依赖性蛋白质折叠至天然状态。在这里，我们提出了研究的细菌伴侣蛋白，GroEL，和它的合作伙伴伴侣蛋白，GroES，在体内和体外。一项体内研究旨在通过鉴定与GroEL ATP结合口袋结合并在体内产生立即发作的功能丧失的化合物来解决GroEL的生理作用，由于缺乏紧密条件突变体而未被充分理解。我们将检查蛋白质翻译机器是否被GroEL折叠机的这种逮捕所停止，并将检查新生多肽（如果它们产生的话）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中断的直接生理后果，并应确定其真实和必要的底物。在第二个领域，将在体外进行机械研究，在我们早期对机器本身的研究之后，将分析导向多肽底物。我们将分析全球拓扑结构的底物蛋白质，而绑定到GroEL，使用天然和工程半胱氨酸之间的氧化交联在基板本身，解决拓扑结构是否是独特的，潜在的天然样，随机的，或有限的合奏状态。我们还将解决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环的中心空腔内，而其余部分位于本体溶液的外部，使用底物蛋白的暴露部分的外部标记和cryoEM的单颗粒成像。我们还将讨论GroEL-底物二元复合物上的核苷酸/GroES结合的作用。一项研究将使用cryoEM来检查通过向GroEL-罗丹酸盐添加ADP/GroES形成的复合物，其中顶端结构域运动严重受阻，可能提供GroEL-GroES碰撞复合物的结构。在第二项研究中，ATP/GroES结合GroEL-底物复合物是否会在底物蛋白释放到中心腔之前产生活性的、强制的、底物蛋白的解折叠的问题将使用氚和氢/氘交换实验进行研究。