Visualizing and Clarifying the Mechanical Properties of the Myosin V Brownian Search with Optical Tweezers
Visualizing and Clarifying the Mechanical Properties of the Myosin V Brownian Search with Optical Tweezers
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DOI：
10.1016/j.bpj.2010.12.848
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发表时间：
2011-02
影响因子：
3.4
通讯作者：
Keisuke Fujita;M. Iwaki;A. Iwane;T. Yanagida
中科院分区：
文献类型：
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作者：
Keisuke Fujita;M. Iwaki;A. Iwane;T. Yanagida
我们使用FRET研究了肌球蛋白V中与ADP释放相关的结构变化的动力学和热力学，这被认为是许多肌肉和非肌肉肌球蛋白中的应变敏感步骤。我们还使用了使用First/Froda的基本动力学方法，从三种不同的肌球蛋白V X射线晶体结构开始，来研究固有的柔韧性和相关的运动。我们的动力学和稳态FRET结果表明，在ADP释放之前，核苷酸结合口袋从闭合构象转变为开放构象，而肌动蛋白结合裂隙保持闭合。ADP与肌动球蛋白V结合的热力学分析表明，碰撞络合物的形成是由较大的热变和较小的熵变化驱动的。从开放式到封闭式肌球蛋白的过渡。ADP态伴随着一个较大的不利的熵降低，这表明闭袋构象比开袋构象更刚性。尽管目前还没有肌球蛋白V.ADP闭袋状态的晶体结构，但我们的FRET分析表明，这种构象可能类似于肌球蛋白V.ATP状态。First/Froda分析与这些结论一致，因为肌球蛋白V.ADP结构比载脂蛋白结构更灵活，而肌球蛋白V.ATP结构比肌球蛋白V.ADP更刚性。主成分分析表明，核苷酸结合口袋的打开和关闭与环1和转换区在所有三种晶体结构中的运动有关。有趣的是，我们发现肌动蛋白激活的最大肌球蛋白V ATPase速率的温度依赖性与口袋打开的步骤相关，这表明这是ATPase循环中的速率限制步骤。我们的结果为肌球蛋白中依赖应变的ADP释放的结构机制提供了洞察力。
We have used FRET to examine the kinetics and thermodynamics of the structural changes associated with ADP release in myosin V, which is thought to be a strain sensitive step in many muscle and non-muscle myosins. We also use essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine the intrinsic flexibility and correlated motions. Our kinetic and steady-state FRET results demonstrate that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP while the actin binding cleft remains closed. Thermodynamic analysis of ADP binding to actomyosin V suggests the collision complex formation is driven by a large enthalpy change and a small change in entropy. The transition from the open to closed pocket actomyosin. ADP state is associated with a large unfavorable decrease in entropy, which suggests the closed pocket conformation is more rigid than the open pocket conformation. Although no crystal structure is available of the closed pocket myosin V. ADP state, our FRET analysis reveals that this conformation may be similar to the myosin V. ATP state. FIRST/FRODA analysis is consistent with these conclusions as the myosin V. ADP structure is more flexible than the Apo structure, while the myosin V. ATP structure is more rigid than myosin V. ADP. Principal component analysis demonstrates that opening and closing of the nucleotide binding pocket correlates with the motions of loop 1 and the transducer region in all three crystal structures. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate correlates with the pocket opening step, suggesting this is the rate limiting step in the ATPase cycle. Our results provide insight into the structural mechanism of strain-dependent ADP release in myosins.