Department of Biochemistry and Biophysics, UCSF MC 2240, Genentech Hall Room S416C, 600 16th Street, San Francisco, CA 94158-2517, USA.
J Mol Biol. 2011 Mar 18;407(1):79-91. doi: 10.1016/j.jmb.2010.11.053. Epub 2010 Dec 23.
We have used spin-labeled ADP to investigate the dynamics of the nucleotide-binding pocket in a series of myosins, which have a range of velocities. Electron paramagnetic resonance spectroscopy reveals that the pocket is in equilibrium between open and closed conformations. In the absence of actin, the closed conformation is favored. When myosin binds actin, the open conformation becomes more favored, facilitating nucleotide release. We found that faster myosins favor a more closed pocket in the actomyosin•ADP state, with smaller values of ΔH(0) and ΔS(0), even though these myosins release ADP at a faster rate. A model involving a partitioning of free energy between work-generating steps prior to rate-limiting ADP release explains both the unexpected correlation between velocity and opening of the pocket and the observation that fast myosins are less efficient than slow myosins.
我们使用自旋标记的 ADP 来研究一系列肌球蛋白中核苷酸结合口袋的动力学，这些肌球蛋白的速度范围不同。电子顺磁共振波谱显示口袋在开放和关闭构象之间处于平衡状态。在没有肌动蛋白的情况下，封闭构象占优势。当肌球蛋白结合肌动蛋白时，开放构象变得更加有利，促进核苷酸释放。我们发现，更快的肌球蛋白在肌球蛋白-ADP 状态下更倾向于一个更封闭的口袋，具有更小的 ΔH(0) 和 ΔS(0) 值，尽管这些肌球蛋白以更快的速度释放 ADP。一个涉及在限速 ADP 释放之前的产生工作步骤之间的自由能分配的模型，解释了速度和口袋打开之间的意外相关性，以及观察到快速肌球蛋白比慢速肌球蛋白效率更低的现象。
