USE OF PARAMAGNETIC PROBES TO STUDY MUSCLE CROSS BRIDGES

三维结构最近被确定为 肌动蛋白和肌球蛋白，导致了一个原子级的力模型 制作。在此模型中，顺序打开和关闭两个 肌球蛋白结构中的深裂缝被认为是驱动 被称为颈部的长杠杆臂的旋转。一条裂隙 形成核苷酸结合位点(称为口袋)，另一个 横跨肌动蛋白结合部位。虽然这种模式看似合理，但直接 这三个元素中任何一个元素运动的证据是 缺乏。我们建议同时使用荧光和顺磁探针 监测这两个裂隙的打开和关闭情况。我们还将 将顺磁性探针连接到肌球蛋白轻链上，并使用它们来 测量肌球蛋白头颈部的方位 肌肉纤维，以确定该区域在用力时是否旋转 一代。这些实验将在肌肉纤维中进行， 而在经过基因改造的孤立肌球蛋白头部中， 在肌动蛋白循环的不同时间点将其捕获。在一个 单独但相关的一组实验，我们将扩展这些 微管型电机的研究方法。数据将是 根据肌动蛋白、肌球蛋白S1和肌球蛋白的分子模型进行分析 他们的建筑群的模型。将类似物对接到ATP结合中 肌球蛋白的位置将有助于设计和解释实验。

Three dimensional structures have recently been determined for both actin and myosin, leading to an atomic level model for force production. In this model, the sequential opening and closing of two deep clefts in the myosin structure has been proposed to drive the rotation of a long lever arm known as the neck region. One cleft forms the nucleotide binding site (known as the pocket), the other spans the actin binding site. While this model is plausible, direct evidence for the motion of any one of the three of these elements is lacking. We propose to use both fluorescent and paramagnetic probes to monitor the opening and closing of the two clefts. We will also attach paramagnetic probes to myosin light chains and use them to measure the orientation of the neck region of the myosin head in muscle fibers to determine if this region rotates during force generation. These experiments will be carried out in muscle fibers, and in isolated myosin heads that have been genetically altered, to arrest them at different points in the acto-myosin cycle. In a separate but related set of experiments, we will extend these approaches to a study of microtubular based motors. The data will be analyzed in terms of molecular models of actin, myosin S1 and of models of their complexes. Docking of analogs into the ATP binding site of myosin will aid in designing and interpreting experiments.