INTERACTION OF MYOSIN WITH ACTIN

之间形成的交叉桥的循环缔合和解离 肌球蛋白分子在粗纤维中，肌动蛋白分子在细纤维中 细丝与三磷酸腺苷的水解反应相结合。耦合的循环可能会导致 细丝的滑动从而产生力和 作品的生产。对化学物质作用机理的理解 三磷酸腺苷水解释放的自由能转化为机械功 (收缩)需要详细的分子相互作用知识 都参与了滑动运动。有证据表明 亚片段L(S1)区域内的一个能量转导“环” 肌球蛋白。生物相关站点之间的站点间通信 (肌动蛋白结合，ATP)在这个环内是能量的一个重要特征 肌肉中的转导。 拟议工作的第一部分涉及协会和 肌动蛋白与荧光标记Sl的解离动力学我们会 检查这些反应的动力学与未调节的和 利用停流和跳温调节肌动蛋白细丝 松弛荧光法。第二个项目旨在了解 站点间通信与动态特性的关系 Sl的重链。我们将通过以下方式确定分子距离 荧光共振能量转移(FRET)，并使用这些距离 阐明S1中发生的结构变化和形成的络合物 肌动蛋白和S1之间的关系，由肌肉激活引起。分子内 交联的S1和分子间交联的蛋白质将用于 这些研究。生物相关性引起的FRET的变化 微扰将在快速动力学实验中得到动力学解决。 动力学和光谱信息都将被合并到 收缩模型。合成的多肽将被用作研究的模型 一小段S1重链的柔韧性。他们将会是 通过核磁共振、荧光和计算机模拟等手段进行了研究。最后，我们会 获得具有特定序列的协同突变的NSL蛋白 结构/功能研究的变更。

Cyclic association and dissociation of cross-bridges formed between myosin molecules in the thick filament and actin molecules in the thin filament are coupled to ATP hydrolysis. The coupled cycle can lead to sliding of the filaments thus resulting in generation of force and production of work. An understanding of the mechanism by which chemical free energy released from ATP hydrolysis is converted to mechanical work (contraction) requires detailed knowledge of molecular interactions that are involved in the sliding motions. Evidence points to the presence of an energy transduction "loop" within the subfragment l(Sl) region of myosin. Intersite communication between biologically relevant sites (actin-binding, ATP) within this loop is an important feature of energy transduction in muscle. The first part of the proposed work addresses the association and dissociation kinetics of actin with fluorescently-labeled Sl. We will examine the kinetics of these reactions with both unregulated and regulated actin filaments by using stopped-flow and temperature-jump relaxation fluorometry. The second project is designed to understand the relationship between intersite communication and the dynamic properties of the heavy chain of Sl. We will determine molecular distances by fluorescence resonance energy transfer (FRET) and use these distances to elucidate structural changes that occur in Sl and the complex formed between actin and Sl resulting from muscle activation.. Intramolecularly crosslinked Sl and intermolecularly crosslinked proteins will be used for these studies. The changes in FRET induced by biologically relevant perturbations will be kinetically resolved in rapid kinetic experiments. Both kinetic and spectroscopic information will be incorporated into contractile models. Synthetic peptides will be used as models to study the flexibility of a short segment of Sl heavy chain. They will be studied by NMR, fluorescence and computer simulation. Finally, we will obtain collaboratively mutant nSl proteins that have specific sequence alteration for structure/function studies.