Geeves M A, Conibear P B
Max Planck Institute for Molecular Physiology, Dortmund, Germany.
Biophys J. 1995 Apr;68(4 Suppl):194S-199S; discussion 199S-201S.
It has been shown that in solution myosin subfragment 1 binds to actin in three principal steps: [formula: see text] The nucleotide bound to myosin has a major influence on the equilibrium constant of the third of these steps but little effect on the other two. The third step is thought to be coupled to the force-generating event. Three-step binding and structure: The formation of the collision complex is strongly ionic strength dependent but independent of temperature. The isomerization to the A state is not strongly dependent on ionic strength but is affected by organic solvent and temperature. In contrast the isomerization to the R state-is affected by both ionic strength and organic solvent but little affected by temperature. The recent docking of the three-dimensional structures of actin and S1 suggest possible structural correlates of these events. These studies lead to predictions for the docking process, which may be tested using site-directed mutagenesis or peptide inhibitors. Three-step binding and head-head interactions: Studies of HMM binding to actin compared with S1 binding show that binding of two heads in the A state are unlikely presumably because of strain effects. However, binding of two heads as one A and one R state shows little evidence of strain while the isomerization of the second head to give two R states is fivefold weaker than for an isolated S1 head. These results suggest that in a rapidly shortening muscle only one head is likely to be attached at a time. Under isometric conditions, although it is possible for both heads to bind to adjacent actins, it is unlikely that both will be in the force holding R state simultaneously. Three-step binding and regulation by tropomyosin-troponin:Our recent solution studies have established that the thin filament can exist in three calcium-dependent states which we termed blocked, closed and open. A blocked state cannot form the A state with S1 and a closed state cannot form the force holding R state nor accelerate product release from S1. Thus control operates at two distinct points in the docking process. The docking process itself is coupled to hydrolysis of ATP (the A-to-R isomerization is inhibited by the presence of the gamma Pi on ATP), and therefore all of these events are interrelated.The coming together of these different strands provides a biochemical framework that should allow the dynamic properties of the crossbridge in muscle to be understood.
研究表明，在溶液中肌球蛋白亚片段1与肌动蛋白的结合分三个主要步骤：[公式：见正文]与肌球蛋白结合的核苷酸对这些步骤中第三步的平衡常数有重大影响，但对其他两步影响很小。第三步被认为与力产生事件相关联。三步结合与结构：碰撞复合物的形成强烈依赖离子强度，但与温度无关。向A状态的异构化对离子强度的依赖性不强，但受有机溶剂和温度影响。相比之下，向R状态的异构化既受离子强度影响，也受有机溶剂影响，但受温度影响较小。最近肌动蛋白和S1三维结构的对接表明了这些事件可能的结构关联。这些研究对对接过程进行了预测，可通过定点诱变或肽抑制剂进行验证。三步结合与头对头相互作用：与S1结合相比，对HMM与肌动蛋白结合的研究表明，两个处于A状态的头不太可能结合，这可能是由于应变效应。然而，一个处于A状态和一个处于R状态的两个头的结合几乎没有应变迹象，而第二个头异构化为两个R状态的能力比单个S1头弱五倍。这些结果表明，在快速缩短的肌肉中，一次可能只有一个头附着。在等长条件下，虽然两个头有可能与相邻的肌动蛋白结合，但它们不太可能同时处于产生力的R状态。三步结合与原肌球蛋白 - 肌钙蛋白调节：我们最近的溶液研究表明，细肌丝可以存在三种依赖钙的状态，我们称之为封闭、关闭和开放状态。封闭状态不能与S1形成A状态，关闭状态不能形成产生力的R状态，也不能加速S1释放产物。因此，控制在对接过程的两个不同点起作用。对接过程本身与ATP水解相关联（ATP上γ磷酸根的存在会抑制A到R的异构化），因此所有这些事件都是相互关联的。这些不同线索的汇聚提供了一个生化框架，应该能够让我们理解肌肉中横桥的动态特性。
