CREST Sasaki Team, Japan Science and Technology Agency, The University of Tokyo, Kashiwa city, Chiba, Japan.
PLoS One. 2013 May 29;8(5):e64176. doi: 10.1371/journal.pone.0064176. Print 2013.
Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle. Recent structural studies suggest that the ring structure of the chaperonin twists to seal off the central cavity. In this study, we demonstrate ATP-dependent dynamics of a group II chaperonin at the single-molecule level with highly accurate rotational axes views by diffracted X-ray tracking (DXT). A UV light-triggered DXT study with caged-ATP and stopped-flow fluorometry revealed that the lid partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2-6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. Our analyses clearly demonstrate that the biphasic lid-closure process occurs with unsynchronized closure and a synchronized counterclockwise twisting motion.
II 类分子伴侣在真核细胞质和古菌中的蛋白质动态平衡中发挥重要作用。这些蛋白质协助新生多肽的折叠，并以 ATP 依赖性的方式重新折叠未折叠的蛋白质。伴侣介导的蛋白质折叠依赖于内置盖子的关闭和打开，这由 ATP 水解循环控制。最近的结构研究表明，伴侣蛋白的环结构扭曲以封闭中央腔。在这项研究中，我们通过衍射 X 射线跟踪（DXT）以高度精确的旋转轴视图在单分子水平上证明了 II 类分子伴侣的 ATP 依赖性动力学。用笼状-ATP 和停流荧光法进行的 UV 光触发 DXT 研究表明，在 ATP 结合后 1 秒内盖子部分关闭，从俯视到俯视，封闭的环随后逆时针扭曲 2-6 秒，扭曲的环以顺时针运动恢复到原始的开放状态。我们的分析清楚地表明，双相盖子关闭过程发生在盖子的非同步关闭和同步逆时针扭曲运动中。
