Spin Labeling to Probe Actin/Myosin Binding

我研究的长期目标是了解肌凝蛋白和对肌肉收缩至关重要的受调节的细丝（肌动蛋白-肌钙蛋白-原肌凝蛋白）之间的动态相互作用。目前的建议侧重于肌动蛋白和肌凝蛋白之间的相互作用。我将使用定点自旋标记直接测试肌动蛋白和肌球蛋白在肌肉收缩过程中相互作用变化的两个详细结构模型。该项目有三个目标：(1)将预测形成肌动蛋白-肌球蛋白界面的特定肌动蛋白残基突变为半胱氨酸，并选择性地在这些残基上附加自旋标签。(2)利用EPR检测这些位点与肌凝蛋白之间的相互作用，并确定当核苷酸诱导肌动蛋白-肌凝蛋白相互作用在弱与强之间转变时，这些相互作用是如何变化的。(3)对肌球蛋白中选择的互补表面残基进行定向自旋标记，并使用EPR测试弱向强结构转变的模型。这项工作将为未来探索疾病的分子机制奠定基础，如家族性肥厚性心肌病和扩张性心肌病，这些疾病在某些情况下被认为是由肌动蛋白-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.