Crossbridge Orientation and Dynamics in Muscle Contraction

这项研究的长期目标是确定肌肉收缩的分子机制。在这一提议中，我们希望为Huxley(1969)的“旋转肌球蛋白头部”假说提供直接证据，该假说是被广泛接受的收缩机制的工作模式。我们的方法是将肌球蛋白头部的结构变化(定向性和移动性)与力产生(机械循环)和ATP(生化循环)的利用联系起来。为了描述在收缩周期的不同步骤中发生的物理变化，稳态平衡将被机械和生物化学扰动，包括在某些步骤处“捕获”周期。在顺磁探针标记肌肉蛋白质后，将使用电子顺磁共振波谱(EPR)来测量这些蛋白质的取向和迁移率。EPR是唯一适合于这一目的的，因为它是位点特异性的，它对蛋白质的静态取向和旋转迁移率很敏感。我们刚刚开发了一些计算方法来从EPR谱中提取肌球蛋白头部取向(而不是自旋探针取向)。这项新技术将提供肌球蛋白头部在收缩周期中不同状态之间转换时的详细(3度分辨率)图像。我们将能够区分肌球蛋白头部的旋转和旋转运动。

The long term goal of this research is to determine the molecular mechanism of muscular contraction. In this proposal we hope to provide direct evidence for the "rotating myosin head" hypothesis of Huxley (1969), the widely accepted working model for the contractile mechanism. Our approach is to correlate the structural changes of the myosin head (orientation and mobility) with force production (mechanical cycle) and the utilization of ATP (biochemical cycle). In order to delineate physical changes taking place during different steps of the contractile cycle, steady- state equilibria will be perturbed mechanically and biochemically including "trapping" the cycle at certain steps. The orientation and mobility of muscle proteins will be measured using electron paramagnetic resonance spectroscopy (EPR) following the labeling of these proteins with paramagnetic probes. EPR is uniquely suited for this purpose because it is site-specific and it is sensitive to static orientation and rotational mobility of proteins. We have just developed a number of computational methods to extract the myosin head orientation (rather than the spin probe orientation) from EPR spectra. This new technology will give a detailed (3 degree resolution) picture of myosin head reorientation during transitions between the different states in the contractile cycle. We will be able to distinguish between pivoting and spinning motions of the myosin head.