Analysis of Motor Protein Function

我将使用实验和理论相结合的方法来定义构象的变化， 肌球蛋白的核苷酸位点，与核苷酸和肌动蛋白的相互作用有关，沿着 这些构象变化与运动周期的关系。肌球蛋白的X射线结构在存在 肌动蛋白仍然是难以捉摸的，必须从无肌动蛋白的结构中推断出来。中的核心问题 要确定肌球蛋白的分子机制，还需要确定肌球蛋白的结构如何变化 当它与肌动蛋白弱或强结合时，以及在周期中是否出现其他重要结构 尚未解决的问题为了解决这个问题，我将使用顺磁性探针在核苷酸 监测肌球蛋白中核苷酸状态依赖性构象变化的位点，以及与肌球蛋白相关的位点。 弱结合和强结合肌动球蛋白状态。这些信息将与距离相结合 使用与蛋白质结合的光谱探针进行核苷酸位点的测量，沿着 肌动球蛋白功能的生化和机械观察，作为结构变化的进一步监测。 特别地，肌球蛋白II、肌球蛋白V和肌球蛋白VI已经以非常开放的结构结晶， 被广泛假设为代表肌动蛋白结合状态的肌球蛋白的核苷酸位点。这些肌球蛋白 将进行调查。自旋标记的核苷酸也将结合到晶体形式和性质 与上述体外观察相比，作为晶体结构是否，事实上， 代表肌动蛋白结合形式，以及它们是否发生在摩托车上。体外细胞培养的初步分析 光谱学和生物化学数据表明它们并不存在。驱动蛋白家族马达将被研究为 肌球蛋白的模型。一个主要的努力将致力于发展更多的定量解释的生产者责任报告 使用分子动力学建模的蛋白质结构的光谱。这些数据将导致一个 更详细的图片如何在核苷酸位点的构象变化参与力的产生。

I will use a combination of experimental and theoretical methods to define the conformational changes at the nucleotide site of myosin associated with the interactions with nucleotides and with actin, along with the relationship of these conformational changes to the motility cycle. Myosin x-ray structures in the presence of actin remain elusive, and must instead be extrapolated from actin-free structures. A central problem in defining the molecular mechanism of myosin remains to determine how the structure of myosin changes when it binds either weakly or strongly to actin, and whether additional important structures occur in the cycle that have not yet been resolved. To address this question, I will use paramagnetic probes at the nucleotide site to monitor nucleotide-state dependent conformational changes in myosin and those associated with the weakly and strongly bound actomyosin states. This information will be combined with distance measurements acrossthe nucleotide site using spectroscopic probes bound to the protein, along with biochemical and mechanical observations of actomyosin function, as a further monitor of structuralchanges. In particular, myosin II, myosin V, and myosin VI have been crystallized in structures with very open nucleotide sites that are widely hypothesized to represent the actin-bound states of myosin. These myosins will be investigated. Spin labeled nucleotides will also be bound to the crystal forms and the properties compared to in vitro observations, above, as an additional probe of whether the crystal structures, in fact, represent the actin bound forms, and whether they occur in the motor cycle. An initial analysis of in vitro spectroscopic and biochemical data suggests that they do not. Kinesin-family motors will be investigated as a model for myosin. A major effort will be devoted to developing more quantitative interpretations of EPR spectra in terms of protein structure using molecular dynamics modeling. Together these data will lead to a more detailed picture of how conformational changes at the nucleotide site are involved in force generation.