Site Directed Spin Labeling to Probe Acto-myosin Binding

我研究的长期目标是了解肌球蛋白和调节的细丝(肌动蛋白-肌钙蛋白-原肌球蛋白)之间的动态相互作用，这是肌肉收缩的关键。目前的建议集中在肌动蛋白和肌球蛋白之间的相互作用。我将使用定点定向自旋标记来直接测试两个详细的结构模型，以了解肌动蛋白和肌球蛋白在肌肉收缩过程中相互作用的变化。该项目有三个目标：(1)将特定的肌动蛋白残基突变成半胱氨酸，并选择性地在这些残基上贴上自旋标记。(2)使用EPR检测这些位点与肌球蛋白之间的相互作用，并确定当核苷酸诱导肌动蛋白-肌球蛋白弱相互作用和强相互作用之间的转换时，这些相互作用如何变化。(3)突变肌球蛋白中选择的互补表面残基进行定点自旋标记，并使用EPR测试弱到强结构转变的模型。这项工作将为未来探索疾病的分子机制奠定基础，例如家族性肥厚型心肌病和扩张型心肌病，这些疾病被认为在某些情况下是由肌动蛋白-肌球蛋白界面突变引起的。

The long-term goal of my research is to understand the dynamic interactions between myosin and the regulated thin filament (actin- troponin-tropomyosin) that are critical for muscle contraction. The present proposal focuses on the interaction between actin and myosin. I will use site-directed spin labeling to test directly two detailed structural models for the changing interactions between actin and myosin during muscle contraction. This project has three aims: (1) Mutate specific actin residues, which are predicted to form the actin-myosin interface, into cysteine and attach spin labels selectively to these residues. (2) Use EPR to detect interactions between these sites and myosin, and determine how these interactions change when nucleotides induce the transition between weak and strong actin-myosin interactions. (3) Mutate selected complementary surface residues in myosin for site directed spin labeling, and use EPR to test models for the weak-to-strong structural transition. This work will set the stage for future work that probes the molecular mechanisms of diseases, such as familial hypertrophic cardiomyopathy and dilated cardiomyopathy, that are proposed to be caused, in some cases, by mutations in the actin-myosin interface.