Molecular Mechanism of Functional Expression of the Chaperonin

伴侣蛋白功能表达的分子机制

基本信息

批准号：
10480177
负责人：
KUWAJIMA Kunihiro
金额：
$ 8.38万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The objective of the present study is to elucidate the molecular mechanism of functional expression of the Escherichia coli chaperonin GroEL/ES. For this purpose, we investigated the interactions of the chaperonin with folding intermediates of three globular proteins, α-lactalbumin (αLA), staphylococcal nuclease (SNase) and green fluorescent protein (GFP), which have very different physical properties. We also investigated physicochemically the interactions of GroEL with the nucleotides (ATP and ADP) that are indispensable for the chaperonin function and the analogs of the nucleotides (ATPγS and AMP-PNP). The following results were obtained.(1) We used an SNase mutant, in which the refolding kinetics were significantly simplified, and apo-αLA that showed a simple single relaxation kinetics of refolding, as model target proteins of GroEL. We studied the effect of the chaperonin on the refolding kinetics of these target proteins by stopped-flow fluorescence spectroscopy. Especially for α … More LA, we succeeded the quantitative analysis of the reaction curves by computer simulations. When we added a nucleotide in the absence of GroES, only ATP was effective for reducing the affinity of GroEL for the target protein. However, when GroES was present, not only ATP but also the ATP analogs were found to effectively reduce the GroEL affinity for the target protein.(2) We constructed E. coli expression systems for GFP and its Cycle3 mutant and studied their in vitro refolding reactions by fluorescence spectroscopy. These proteins can also be used as model target proteins of the chaperonin.(3) We studied the interactions of GroEL with ADP and the ATP analog by titration calorimetry and fluorescence spectroscopy. In the latter method, we used the fluorescence intensity change of pyrenyl GroEL to monitor the interactions. We found that these nucleotides bound to GroEL in a non-cooperative manner and that there were two kinds of the binding sites with different affinities. Only ATP induced a cooperative fluorescence change of pyrenyl GroEL., suggesting that ATP hydrolysis was required for the cooperative change. From this results together with the above results in(1), the ATP hydrolysis and the resultant cooperative change are expected to by required for releasing the target protein form GroEL. Less

本研究的目的是阐明大肠杆菌伴侣蛋白 GroEL/ES 功能表达的分子机制为此，我们研究了伴侣蛋白与三种球状蛋白 α-乳白蛋白 (αLA)、葡萄球菌核酸酶 (SNase) 和绿色荧光蛋白 (GFP)，它们具有非常不同的物理特性，我们还研究了 GroEL 与绿色荧光蛋白 (GFP) 的相互作用。获得了以下结果。（1）我们使用了SNase突变体，其中重折叠动力学显着简化。，和apo-αLA，显示出简单的单重折叠动力学，作为GroEL的模型靶蛋白，我们研究了伴侣蛋白对重折叠的影响。通过停流荧光光谱法对这些目标蛋白进行重折叠动力学，特别是对于 α … More LA，我们通过计算机模拟成功地对反应曲线进行了定量分析。当我们在没有 GroES 的情况下添加核苷酸时，只有 ATP 能够有效还原。然而，当GroES存在时，不仅ATP而且ATP类似物都被发现能有效降低GroEL对靶蛋白的亲和力。(2)我们构建了GFP的大肠杆菌表达系统。及其Cycle3突变体，并通过荧光光谱研究了它们的体外重折叠反应，这些蛋白也可以用作伴侣蛋白的模型靶蛋白。(3)我们通过滴定量热法和荧光光谱研究了GroEL与ADP和ATP类似物的相互作用。在后一种方法中，我们利用芘基GroEL的荧光强度变化来监测相互作用，我们发现这些核苷酸以非合作方式与GroEL结合，并且有两种结合位点。只有 ATP 诱导芘基 GroEL 的协同荧光变化，表明 ATP 水解是协同变化所必需的，根据该结果以及上述 (1) 中的结果，预计 ATP 水解和由此产生的协同变化。从 GroEL 中释放靶蛋白所需的量较少。