Chaperonin-mediated protein folding

描述(由申请人提供)：这项提案寻求支持一个正在进行的长期项目，该项目涉及被称为伴侣蛋白的基本的百万吨大小的双环分子机器的作用机制，该分子机器在各种细胞间隔中介导ATP依赖的蛋白质折叠到自然状态。在这里，我们建议对细菌伴侣GroEL和它的协同伴侣GroES进行体内和体外的研究。体内的一项研究将旨在通过鉴定一种与GroEL ATP结合口袋结合并在体内立即产生功能丧失的化合物来解决GroEL的生理作用。由于缺乏紧密的条件突变体，GroEL的生理作用尚未被很好地了解。我们将检查蛋白质翻译机器是否因GroEL折叠机的停止而停止，并将检查新生多肽(如果产生)和先前存在的蛋白质的错误折叠/聚集。这将使我们能够观察伴侣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，将在我们早期对机器本身的研究基础上，在体外进行机制研究，指导对多肽底物的分析。我们将分析与GroEL结合的底物蛋白质的全球拓扑，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，解决该拓扑是唯一的和潜在的天然、随机还是有限的状态集合。我们还将讨论结合多肽是否占据了相对于GroEL的特征拓扑，其中特定部分结合在GroEL环的中心空腔内，而其余部分则定位在本体溶液中的外部，使用底物蛋白质暴露部分的外部标记和CryoEM的单粒子成像。我们还将讨论核苷酸/GROE结合在GroEL-底物二元复合体上的作用。一项研究将使用冷冻EM来检查通过将ADP/GROES添加到GroEL-罗丹尼斯形成的复合体，在该复合体中，顶端结构域的运动严重受阻，可能提供GroEL-GroES碰撞复合体的结构。在第二项研究中，将使用氚和氢/氚交换实验来研究ATP/GroES与GroEL底物复合体结合是否会在底物蛋白质释放到中央空腔之前产生活性的、强制的、展开的蛋白质的问题。

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.