ENERGY TRANSDUCTION BY SINGLE MYOSIN MOLECULES

本研究的总体目标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相关性。 利用红外光学陷阱（激光镊子）的新方法将被应用能够检测单个肌球蛋白分子中的机械事件，并在个别与丝状肌动蛋白的相互作用。 已经开发了一种新的光阱变体，以有效地消除与测量的收缩蛋白质串联的机械顺应性，所述收缩蛋白质通常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和跨桥循环的基本力学步骤之间的关系。 快速变化的化学浓度的相关的生化物种，如ATP，将由激光脉冲光解的光不稳定的“笼”前体的相互作用肌动球蛋白的区域。 这种方法结合激光光阱将解决在肌球蛋白分子的各个区域的机械性能和机械和生物化学反应速率对由细丝滑动施加的机械应变的依赖性。 实验将在来自兔腰肌的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的片段上进行。从这个项目的结果应显着推进收缩过程的知识，从而带来了更好的理解横纹肌和其他类型的细胞运动的正常和病理状态。

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.