Molecular Dynamics of Muscle Contraction

描述（由申请人提供）：该项目的目的是确定肌动球蛋白结构动力学在肌肉收缩机制中的作用。该项目之前的工作已经证明了EPR和光谱学在检测肌球蛋白和肌动蛋白在溶液和皮肤肌纤维中的运动方面的力量和互补性质。这项工作将继续进行，并增加重点在(1)使用肌动蛋白和肌球蛋白突变体获得新的位点定向光谱探针附着位点和功能和结构扰动，(2)检测瞬态生化条件下的结构动力学，以及(3)我们的光谱测量与x射线晶体学和电子显微镜的结构分析的直接相关性。总体方法是检测肌凝蛋白和肌动蛋白在相互作用和与ATP的生理相互作用过程中的结构动力学，设计实验来测试和完善特定的分子模型来产生力。目的1着重于发展改进的光谱方法来研究肌球蛋白和肌动蛋白在溶液和肌肉纤维中的结构动力学。在剩下的目标中，这些方法将用于测试关于结构动力学在肌动蛋白结合从弱到强的力产生转变中的功能作用的特定假设：在目标2中，探针附着在溶液中的肌动蛋白上，以测试其与肌动蛋白和核苷酸相互作用时的结构动力学模型。在Aim 3中，探针附着在溶液中的肌动蛋白上，以测试其与肌球蛋白-核苷酸复合物相互作用时的结构动力学模型。在Aim 4中，探针在溶液中连接到肌动蛋白和肌凝蛋白上，直接探测肌动蛋白-肌凝蛋白界面的结构动力学。在Aim中，5探针附着在皮肤肌纤维中的肌球蛋白上，直接探测结构动力学与力产生的耦合。这些光谱研究将与x射线晶体学和低温电镜的结构分析相协调，以提供3D结构与生理功能分子动力学之间的联系。

DESCRIPTION (provided by applicant): The goal of this project is to determine the role of actomyosin structural dynamics in the mechanism of muscle contraction. Previous work on this project has demonstrated the power and complementary nature of EPR and optical spectroscopy in detecting the motions of myosin and actin in solution and in skinned muscle fibers. This work will continue, with increased emphasis on (1) the use of actin and myosin mutants to obtain new site-directed spectroscopic probe attachment sites and functional and structural perturbations, (2) the detection of structural dynamics under transient biochemical conditions, and (3) direct correlation of our spectroscopic measurements with structural analysis by x-ray crystallography and electron microscopy. The overall approach is to detect the structural dynamics of myosin and actin during their physiological interactions with each other and with ATP, designing experiments to test and refine specific molecular models for force generation. Aim 1 focuses on the development of improved spectroscopic methods for studying structural dynamics of myosin and actin in solution and in muscle fibers. In the remaining aims, these methods will be used to test specific hypotheses about the functional role of structural dynamics in the force-generating transition from weak to strong actomyosin binding: In Aim 2, probes are attached to myosin in solution, to test models for its structural dynamics during interaction with actin and nucleotides. In Aim 3, probes are attached to actin in solution, to test models for its structural dynamics during interaction with myosin-nucleotide complexes. In Aim 4, probes are attached to both actin and myosin in solution, to probe directly the structural dynamics of the actin-myosin interface. In Aim 5 probes are attached to myosin in skinned muscle fibers, to probe directly the coupling of structural dynamics to force generation. These spectroscopic studies will be coordinated with structural analysis by x-ray crystallography and cryo-EM, in order to provide a connection between 3D structure and the molecular dynamics of physiological function. The proposed research brings together a powerful combination of techniques, from molecular genetics to biochemistry to biophysics, to solve the molecular mechanism of muscle contraction. In particular, this work is of fundamental importance for understanding muscle function, and the technology generated by this work is already being used to provide molecular insight into muscle malfunction. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in motor proteins, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field.