Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin
Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin
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DOI：
10.1529/biophysj.106.097618
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发表时间：
2007-03-01
影响因子：
3.4
通讯作者：
Warshaw, David M.
中科院分区：
文献类型：
--
作者：
Kad, Neil M.;Patlak, Joseph B.;Warshaw, David M.
肌肉的ATP水解率和缩短速度具有负荷依赖性。在分子水平上，肌球蛋白通过ATP水解耦合杠杆臂旋转产生力和运动。当使用激光陷阱对表达的平滑肌肌球蛋白（S1）的单头施加负载时，AIDP释放动力学在辅助负载下加速，在阻力负载下减慢;然而，ATP结合基本不受影响。为了研究负载如何在马达内传递，将位于杠杆臂的假定支点处的甘氨酸突变为缬氨酸（G709V）。在没有负载的情况下，停流和激光陷阱的研究表明，突变显着减缓ADP释放和ATP结合的速率，占肌动蛋白滑动速度下降了270倍。突变体的AIDP释放速率的负荷依赖性与野生型S1（WT）的相同，尽管速率较慢。相比之下，无论加载方向如何，加载均加速ATIP结合20倍。通过克服升高的活化能屏障，向突变马达传递机械能部分逆转了减慢的ATP结合。这些结果表明，保守的G709附近的构象变化是至关重要的肌球蛋白的活性位点和杠杆臂之间的机械化学信息的传输。
The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the AIDP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the similar to 270-fold decrease in actin sliding velocity. The load dependence of the mutant's AIDP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATIP binding by similar to 20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.