ENERGY TRANSDUCTION BY SINGLE MYOSIN MOLECULES

单个肌球蛋白分子的能量转换

• 批准号: 6534474
• 负责人: HENRY SHUMAN
• 金额: $ 31.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-28 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534474
• 关键词: actinomycin; adenosine triphosphate; bioenergetics; cytoskeletal proteins; laboratory rabbit; microfilaments; muscle metabolism; myosins; nanotechnology; photolysis; protein protein interaction; striated muscles

The overall aims of this research are to understand the molecular mechanism by which muscle proteins convert chemical energy into mechanical work, and to obtain a precise correlation between the physiological and biochemical events of muscular contraction at the molecular level. Novel methods utilizing infrared optical traps (laser tweezers) will be applied capable of detecting mechanical events in individual myosin molecules and in individual interactions with filamentous actin. A new variant of the optical trap has been developed to effectively eliminate mechanical compliance in series with the measured contractile protein that normally obscures quantitative aspects of molecular events. Experiments are designed to probe the relations between biochemical reactions of the contractile proteins and the elementary mechanical steps of the cross-bridge cycle. Rapid changes in the chemical concentrations of pertinent biochemical species, such as ATP, will be made by laser pulse photolysis of photolabile "caged" precursors in the region of the interacting actomyosin. This method combined with the laser optical trap will resolve mechanical properties in various regions of the myosin molecule and the dependence of mechanical and biochemical reaction rates on mechanical strain imposed by sliding of the filament. The experiments will be carried out on single molecules of myosin from rabbit psoas muscle and on fragments of vertebrate myosin expressed in a heterologous expression system. Results from this project should significantly advance knowledge of the contractile process and thus bring a greater understanding of both normal and pathological states of striated muscle and other types of cell motility.

本研究的总体目的是了解肌肉蛋白将化学能转化为机械能的分子机制，并在分子水平上获得肌肉收缩的生理生化事件之间的精确关联。利用红外光学陷阱（激光镊子）的新方法将被应用于检测个体肌球蛋白分子中的机械事件以及个体与丝状肌动蛋白的相互作用。一种新的光学陷阱变体已经被开发出来，可以有效地消除与被测量的收缩蛋白串联的机械顺应性，这种机械顺应性通常会模糊分子事件的定量方面。实验旨在探讨收缩蛋白的生化反应与过桥循环的基本机械步骤之间的关系。通过激光脉冲光解作用于相互作用的肌动球蛋白区域的可光性“笼状”前体，可使相关生化物质（如ATP）的化学浓度发生快速变化。该方法与激光光阱相结合，将解析肌球蛋白分子各区域的力学特性，以及由丝的滑动施加的机械应变对机械和生化反应速率的依赖。实验将在兔腰肌肌球蛋白单分子和在异源表达系统中表达的脊椎动物肌球蛋白片段上进行。这个项目的结果将显著提高对收缩过程的认识，从而使我们对横纹肌的正常和病理状态以及其他类型的细胞运动有了更深入的了解。