Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto 603-8555, Japan.
J Mol Biol. 2012 Sep 14;422(2):291-9. doi: 10.1016/j.jmb.2012.05.026. Epub 2012 May 25.
Chaperonin GroEL and its partner GroES assist the folding of nascent and stress-damaged proteins in an ATP-dependent manner. Free GroES has a flexible "mobile loop" and binds to GroEL through the residues at the tip of the loop, capping the central cavity of GroEL to provide the substrate polypeptide a cage for secure in-cage folding. Here, we show that restriction of the flexibility of the loop by a disulfide cross-linking between cysteines within the loop results in the inefficient formation of a stable GroEL-polypeptide-GroES ternary complex and inefficient folding. Then, we generated substrate proteins with enhanced binding affinity to GroEL by fusion of one or two SBP (strongly binding peptide for GroEL) sequences and examined the effect of disulfide cross-linking on the assisted folding. The results indicate that the higher the binding affinity of the substrate polypeptide to GroEL, the greater the contribution of the mobile loop flexibility to efficient in-cage folding. It is likely that the flexibility helps GroES capture GroEL's binding sites that are already occupied by the substrate polypeptide with various binding modes.
伴侣蛋白 GroEL 及其搭档 GroES 以依赖于 ATP 的方式协助新生和应激损伤蛋白的折叠。游离的 GroES 具有灵活的“移动环”，通过环尖端的残基与 GroEL 结合，覆盖 GroEL 的中心腔，为底物多肽提供一个安全的笼状折叠环境。在这里，我们表明，通过环内半胱氨酸之间的二硫键交联限制环的灵活性会导致不稳定的 GroEL-多肽-GroES 三元复合物的形成效率降低和折叠效率降低。然后，我们通过融合一个或两个 SBP（与 GroEL 强结合的肽）序列生成与 GroEL 结合亲和力增强的底物蛋白，并研究二硫键交联对辅助折叠的影响。结果表明，底物多肽与 GroEL 的结合亲和力越高，移动环的灵活性对有效笼内折叠的贡献就越大。这种灵活性可能有助于 GroES 以各种结合模式捕获已经被底物多肽占据的 GroEL 的结合位点。
