Analysis of Motor Protein Function

运动蛋白功能分析

• 批准号: 7176086
• 负责人: EDWARD F PATE
• 金额: $ 23.95万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-16 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7176086
• 关键词: Actins; Actomyosin; Address; Binding; Biochemical; Data; Family; Generations; In Vitro; Kinesin; Lead; Measurement; Mechanics; Methods; Modeling; Molecular; Monitor; Motor; Myosin ATPase; Myosin Type II; Myosin Type V; Nucleotides; Property; Proteins; Site; Spin Labels; Structure; cell motility; molecular dynamics; myosin VI; protein function; protein structure

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.

描述（由申请人提供）：我将使用实验和理论方法的组合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，沿着这些构象变化与运动周期的关系。肌动蛋白存在下的肌球蛋白的X射线结构仍然难以捉摸，而必须从肌动蛋白的结构外推。定义肌球蛋白分子机制的一个中心问题仍然是确定当肌球蛋白与肌动蛋白弱结合或强结合时，其结构如何变化，以及在该周期中是否出现其他尚未解决的重要结构。为了解决这个问题，我将使用顺磁探针在核苷酸的网站，以监测核苷酸状态依赖肌球蛋白的构象变化和那些与弱，强结合肌动球蛋白状态。该信息将与使用结合到蛋白质的光谱探针的核苷酸位点的距离测量结合，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。特别是，肌球蛋白II，肌球蛋白V和肌球蛋白VI已结晶的结构非常开放的核苷酸位点，被广泛假设为代表肌动蛋白结合状态的肌球蛋白。将对这些肌球蛋白进行研究。自旋标记的核苷酸也将与晶体形式结合，并且与上述体外观察相比，其性质作为晶体结构是否实际上代表肌动蛋白结合形式以及它们是否出现在摩托车中的额外探针。对体外光谱和生化数据的初步分析表明，它们并不存在。驱动蛋白家族马达将作为肌球蛋白的模型进行研究。一个主要的努力将致力于开发更多的定量解释EPR光谱的蛋白质结构，使用分子动力学建模。总之，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。