ENERGY TRANSDUCTION BY SINGLE MYOSIN MOLECULES

本研究的总体目的是了解肌肉蛋白质将化学能转化为机械功的分子机制，并在分子水平上获得肌肉收缩的生理和生化事件之间的精确关联。利用红外光学陷阱(激光镊子)的新方法将能够检测单个肌球蛋白分子中的机械事件，以及在单个肌球蛋白与丝状肌动蛋白的相互作用中。光学陷阱的一种新变种已经被开发出来，以有效地消除与所测量的收缩蛋白串联的机械顺应性，这些机械顺应性通常掩盖了分子事件的定量方面。实验旨在探索收缩蛋白的生化反应与跨桥循环的基本机械步骤之间的关系。在相互作用的肌球蛋白区域，通过激光脉冲光解不耐光的“笼子”前体，将迅速改变相关生化物种的化学浓度，如ATP。这种方法与激光光学陷阱相结合，将解决肌球蛋白分子不同区域的机械性质，以及机械和生化反应速度对细丝滑动造成的机械应变的依赖。这些实验将在兔腰肌肌球蛋白的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。该项目的结果将大大提高对收缩过程的了解，从而更好地了解横纹肌的正常和病理状态以及其他类型的细胞运动。

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.