POWER STROKE OF SKELETAL MUSCLE CROSS-BRIDGES CONTAINING PHOSPHORYLATED AND DE-PHOSPHORYLATED MYOSIN REGULATORY LIGHT CHAIN

Purpose: Muscle contracts by cyclic, ATP-driven, reorientations of myosin cross-bridges while they are interacting with actin. It is widely recognized that during force generating step of this interaction, referred to as a power-stroke, myosin head forms a firm attachment to actin, while the torque is provided by the rotation of myosin neck (a cross-bridge consists of a head plus a neck). During contraction, the Regulatory Light Chain of myosin (RLC), which resides on the neck, gets phosphorylated. An important goal of muscle research is to elucidate the role of phosphorylation during active contraction. We set out to measure how the rate of power stroke is affected by phosphorylation in muscle ex vivo. Methods: The rate can only be established when small number of molecules is investigated. This is accomplished by Confocal Fluorescence Correlation Spectroscopy (CFCS). Results: Phosphorylation of RLC speeds up power stroke from ~0.64/s when RLC is de-phosphorylated to ~1.5/s when it is phosphorylated. Conclusions: Using this technique, we show that the state of phosphorylation of the Regulatory Light Chain (RLC) of myosin has a profound effect on kinetics of ex vivo rabbit psoas cross-bridges during contraction.