Analysis of Motor Protein Function

我将使用实验和理论相结合的方法来定义 肌球蛋白与核苷酸和肌动蛋白相互作用的核苷酸位置，以及 这些构象变化与运动周期的关系。肌球蛋白的X-射线结构 肌动蛋白仍然难以捉摸，必须从没有肌动蛋白的结构中推断出来。中国的一个中心问题 确定肌球蛋白的分子机制仍需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，我将在核苷酸上使用顺磁探针 监测肌球蛋白核苷酸状态依赖的构象变化以及与肌球蛋白相关的那些 肌动球蛋白弱结合态和强结合态。此信息将与距离相结合 使用与蛋白质结合的光谱探针测量核苷酸位置，以及 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。 特别是，肌球蛋白II、肌球蛋白V和肌球蛋白VI已经在非常开放的结构中结晶 被广泛假设为代表肌球蛋白肌动蛋白结合状态的核苷酸位点。这些肌球蛋白 将会被调查。自旋标记的核苷酸也将与晶体形式和性质结合在一起 与体外观察相比，作为额外的探针，上面的晶体结构，实际上， 表示肌动蛋白结合形式，以及它们是否出现在摩托车中。试管苗的初步分析 光谱和生物化学数据表明，它们并非如此。动蛋白家族马达将被研究为 肌球蛋白的模型。将致力于开发对EPR的更多量化解释 在蛋白质结构方面使用分子动力学建模的光谱。这些数据加在一起将导致 更详细地描绘了核苷酸位置的构象变化如何参与作用力的产生。

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.