Analysis of Motor Protein Function

描述（由申请人提供）：我将使用实验和理论相结合的方法来定义与核苷酸和肌动蛋白相互作用相关的肌球蛋白核苷酸位点的构象变化，以及这些构象变化与运动周期的关系。肌动蛋白存在时的肌球蛋白x线结构仍然难以捉摸，必须从无肌动蛋白的结构中推断出来。定义肌凝蛋白分子机制的一个核心问题仍然是确定当肌凝蛋白与肌动蛋白弱或强结合时，其结构是如何变化的，以及在这个循环中是否发生了其他尚未解决的重要结构。为了解决这个问题，我将在核苷酸位点使用顺磁探针来监测肌凝蛋白中核苷酸状态依赖的构象变化以及与弱和强结合的肌动球蛋白状态相关的构象变化。这些信息将与使用与蛋白质结合的光谱探针在核苷酸位点上的距离测量相结合，以及对肌动球蛋白功能的生化和机械观察，作为对结构变化的进一步监测。特别是，肌凝蛋白II、肌凝蛋白V和肌凝蛋白VI的结晶结构具有非常开放的核苷酸位点，被广泛假设为代表肌凝蛋白的肌动蛋白结合状态。这些肌凝蛋白将被研究。自旋标记的核苷酸也将结合到晶体形式和性质与体外观察相比较，如上所述，作为晶体结构是否实际上代表肌动蛋白结合形式的额外探针，以及它们是否发生在摩托车中。对体外光谱和生化数据的初步分析表明，它们不是。将研究肌球蛋白家族马达作为肌凝蛋白的模型。一个主要的努力将致力于开发更多的定量解释EPR光谱在蛋白质结构方面使用分子动力学建模。综合这些数据，我们将更详细地了解核苷酸位点的构象变化是如何参与力的产生的。

DESCRIPTION (provided by applicant): 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 across the nucleotide site using spectroscopic probes bound to the protein, along with biochemical and mechanical observations of actomyosin function, as a further monitor of structural changes. 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.