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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在atp驱动的GroEL- groes结合-解离过程的重复循环中介导底物蛋白折叠。我们已经成功地利用全内反射荧光显微镜（TIRFM）检测了单分子水平的过程。我们的结果表明，该过程由两个定时器组成，分别为3秒和5秒的速率限制步骤。为了更好地理解这两个时间过程，酸变性GFP被用作GroEL的底物蛋白，并观察其折叠。结果表明，底物折叠在前3秒被抑制，并在5秒限速步骤中发现一个顺式ADP*复合物，即一个新的复合物。此外，用一种新的单分子荧光成像方法检测了所谓的足球状中间复合物的存在，该复合物由两侧与GroES结合的GroEL组成。在这张图像中，直径为100纳米的纳米孔被制作在沉积在玻片上的金属薄膜中。这被用来定位消失场，减少荧光背景，使我们能够在高浓度荧光分子存在的情况下成像单个荧光分子。利用该方法，在荧光标记的1 μM的GroES存在下，在单分子水平上揭示了Gro-GroES缔合-解离过程。