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.

伴侣蛋白凹槽在ATP驱动的gro groes关联分解过程的重复循环中介导底物蛋白折叠。我们已经成功地使用总内反射荧光显微镜（TIRFM）成功地检测了单个分子水平的过程。我们的结果表明，此过程由大约3秒和5秒的限制步骤组成。为了使这两个计时器过程低估，酸性化的GFP用作凹槽的底物蛋白，并观察到其折叠。这一观察结果的结果表明，底物折叠在前3秒时期被抑制，并揭示了一个顺式ADP*-Complex，这是一个新的复合体，在5秒的限制速率限制步骤中存在。furthermore，即所谓的足球形状的互构络合物的存在，由Groel双方与Gro by gros进行了调查，使用了一种新的方法，使用了新的方法。在这种成像中，将直径为100 nm的纳米孔在沉积在滑翔机上的金属膜中制造。这用于定位爆炸场，减少荧光背景，使我们能够在存在高浓度的荧光分子的情况下对单个荧光分子进行成像。使用这种方法，在标记为1μm的凹槽的荧光存在下，在单个分子的水平上揭示了GRO-GROES关联分解的过程。