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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle
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Characterization of the pre-force-generation state in the actomyosin cross-bridge cycle

机译：肌动球蛋白跨桥循环中前生力状态的表征

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Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP·P_i) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer (FRET) to monitor conformational changes in the upper-50-kDa domain of the actin binding cleft in the weak and strong actin binding states. Steady-state and lifetime data monitoring the FRET signal suggest that the cleft is in a more open conformation in the weak actin binding states. Transient kinetic experiments suggest that a rapid conformational change occurs, which is consistent with cleft closure before actin-activated phosphate release. Our results have identified a pre-force-generation actomyosin ADP·P_i state, and suggest force generation may occur from a state not yet seen by crystallography in which the actin binding cleft and the nucleotide binding pocket are closed. Computational modeling uncovers dramatic changes in the rigidity of the upper-50-kDa domain in different nucleotide states, which suggests that the intrinsic flexibility of this domain allows myosin motors to accomplish simultaneous tight nucleotide binding (closed nucleotide binding pocket) and high-affinity actin binding (closed actin binding cleft).
机译：肌球蛋白是一种基于肌动蛋白的运动蛋白，在其ATPase循环过程中，通过在附着于肌动蛋白的状态（强结合：ADP或严格）和脱离于肌动蛋白的状态（弱结合：ATP或ADP·P_i）之间循环而产生力。然而，尚不清楚肌动蛋白结合位点在与肌动蛋白结合时发生了哪些特定的构象变化，以及这些结构变化如何导致产物释放以及产生力和运动。我们通过使用荧光共振能量转移（FRET）来监测肌动蛋白V的肌动蛋白结合区的动力学，以监测在弱和强肌动蛋白结合状态下肌动蛋白结合裂的上50 kDa域的构象变化。监测FRET信号的稳态和寿命数据表明，在弱肌动蛋白结合状态下，裂隙处于更开放的构型。瞬态动力学实验表明发生了快速构象变化，这与肌动蛋白激活的磷酸盐释放之前的裂隙闭合是一致的。我们的结果已经确定了力产生前的肌动球蛋白ADP·P_i状态，并暗示力的产生可能是由于尚未通过晶体学观察到的状态发生的，在该状态中肌动蛋白结合裂和核苷酸结合袋被封闭。计算模型揭示了在不同核苷酸状态下上50 kDa结构域的刚度发生了显着变化，这表明该结构域的固有柔韧性使肌球蛋白马达同时完成紧密的核苷酸结合（闭合的核苷酸结合口袋）和高亲和性肌动蛋白结合（封闭的肌动蛋白结合裂）。

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