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Molecular mechanism of chaperonin-assisted protein folding studied by single molecule imaging.

单分子成像研究伴侣蛋白辅助蛋白质折叠的分子机制。

基本信息

批准号：
14380320
负责人：
FUNATSU Takashi
金额：
$ 10.82万
依托单位：
The University of Tokyo (2004)Waseda University (2002-2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Chaperonin GroEL mediates substrate protein folding in a repeated cycle of ATP-driven GroEL-GroES association-dissociation processes. We have succeeded in detecting the processes at the level of a single molecule using total internal reflection fluorescence microscopy (TIRFM). Our results indicated that this process consists of two timers approximately 3 sec and 5 sec of the rate-limiting steps. In order to gain understating of this two timer process, acid-denatured GFP was used as a substrate protein for GroEL and its folding was observed. The result from this observation demonstrated that the substrate folding was inhibited in the first 3 sec period, and revealed a cis ADP*-complex, that is a new complex existed in the 5 sec rate-limiting step.Furthermore, the existence of a so called football-shaped intermediate complex, consisting of a GroEL bound both sides with GroES, was examined using a new single molecule fluorescence imaging method. In this imaging, nano-holes with 100 nm diameter were fabricated in a metal film deposited on a slide-glass. This was used to localize evanescent field, reducing the fluorescence background, enabling us to image a single fluorescent molecule in the presence of a high concentration of fluorescence molecules. Using this method, processes of Gro-GroES association-dissociation was revealed at the level of a single molecule in the presence of fluorescently labeled 1 μM of GroES.

伴侣蛋白GroEL介导底物蛋白质折叠在ATP驱动的GroEL-GroES缔合-解离过程的重复循环中。我们已经成功地检测到在一个单一的分子水平上使用全内反射荧光显微镜（TIRFM）的过程。我们的研究结果表明，这个过程包括两个定时器约3秒和5秒的限速步骤。为了了解这两个计时器过程，使用酸变性的GFP作为GroEL的底物蛋白并观察其折叠。结果表明，底物折叠在前3秒内受到抑制，在5秒限速步骤中发现了一种新的复合物--cis ADP*-复合物。此外，通过新的单分子荧光成像方法，还发现了一种由GroEL和GroES结合而成的所谓足球状中间复合物的存在。在该成像中，在沉积在载玻片上的金属膜中制造直径为100 nm的纳米孔。这被用来定位倏逝场，减少荧光背景，使我们能够在高浓度荧光分子存在下对单个荧光分子进行成像。使用该方法，在荧光标记的1 μM GroES存在下，在单分子水平上揭示了Gro-GroES缔合-解离过程。