INTERACTION OF MYOSIN WITH ACTIN

肌球蛋白与肌动蛋白的相互作用

• 批准号: 3156006
• 负责人: HERBERT C CHEUNG
• 金额: $ 17.93万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-07 至 1995-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3156006
• 关键词: actins; adenosine diphosphate; adenosine triphosphate; bioenergetics; chemical association; chemical kinetics; circular dichroism; conformation; crosslink; fluorescence polarization; fluorescent dye /probe; fluorimetry; laboratory rabbit; light scattering; molecular site; muscle contraction; myosins; nuclear magnetic resonance spectroscopy; nucleotide analog; oligopeptides; protein structure function; site directed mutagenesis; stop flow technique; synthetic peptide; temperature jump

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.

形成的交叉桥的循环缔合和解离 粗肌丝中的肌球蛋白分子和细丝中的肌动蛋白分子 丝与ATP水解偶联。 耦合循环可导致 细丝的滑动从而导致力的产生， 工作的生产。 了解化学物质 ATP水解释放的自由能转化为机械功 （收缩）需要分子相互作用的详细知识， 参与了滑动运动。 证据表明 在亚片段I（S1）区域内的能量转导“环”， 肌球蛋白。 生物学相关研究中心之间的研究中心间通信 （肌动蛋白结合，ATP）是能量的重要特征 肌肉中的转导。 建议工作的第一部分涉及协会， 肌动蛋白与荧光标记的S1的解离动力学。 我们将 研究这些反应的动力学， 利用停流和温度跃变调节肌动蛋白丝 弛豫荧光法 第二个项目旨在了解 站点间通信与动态特性之间的关系 Sl的重链的一部分。 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复合物 肌肉激活引起的肌动蛋白和Sl之间的关系。 分子内 交联的S1和分子间交联的蛋白质将用于 这些研究。 生物学相关蛋白诱导的FRET变化 扰动将在快速动力学实验中动力学地解决。 动力学和光谱信息都将被纳入 收缩模型 合成肽将被用作研究的模型 S1重链的短片段的柔性。 他们将 通过核磁共振、荧光和计算机模拟进行研究。 最后我们将 获得具有特定序列的协同突变nS 1蛋白， 改变结构/功能研究。