Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Biol Chem. 2012 Nov 30;287(49):41118-25. doi: 10.1074/jbc.M112.398628. Epub 2012 Oct 9.
The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding.
伴侣蛋白 GroEL 是一种重要的分子伴侣，与它的辅助因子 GroES 一起在大肠杆菌中介导蛋白质折叠。人们普遍认为，在反应循环中，GroEL 的两个环会交替与 GroES 结合处于折叠活跃状态。换句话说，在整个循环中形成不对称的 GroEL-GroES 复合物（子弹形复合物），而不会形成对称的 GroEL-(GroES)(2)复合物（橄榄球形复合物）。我们最近表明，在反应循环中，橄榄球形复合物与子弹形复合物共存。然而，蛋白质在橄榄球形复合物中的折叠过程仍知之甚少。在这里，我们使用 GFP 作为底物，通过单分子荧光技术可视化橄榄球形复合物中的蛋白质折叠。我们直接表明 GFP 折叠发生在橄榄球形复合物的两个环中。值得注意的是，折叠是一个顺序的两步反应，动力学与子弹形复合物非常吻合。这些结果表明，相同的反应在橄榄球形复合物的两个环中独立发生，以促进蛋白质折叠。
