|Session Assignment: 402|
|POWER STROKE OF SKELETAL MUSCLE CROSS-BRIDGES CONTAINING PHOSPHORYLATED AND DE-PHOSPHORYLATED MYOSIN REGULATORY LIGHT CHAIN|
|Author: Krishna Midde||Presenter: Krishna Midde|
|Department: Molecular Biology and Immunology|
|Research Area: Cellular & Molecular Science|
|(1) Muscle Contraction, (2) Power Stroke, (3) Phosphorylation|
|K. Midde, D. Duggal, J. Nagwekar, R. Rich, I. Gryczynski and J. Borejdo Dept of Molecular Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107.|
|Short Description: The conventional measurements of orientation, such as fluorescence microscopy, Electron Paramagnetic Resonance (EPR) or X-ray diffraction or scattering, report only the average orientation of trillions of cross-bridges and provide no information about kinetics of an individual myosin cross-bridge. To avoid problems associated with averaging, it is necessary to decrease this enormous number to a small number where averaging of the signal is minimal and kinetics can be closely examined. Super-resolution microscopy can reduce this number to one, but each measurement may take minutes or hours depending on the strength of a signal. However, a power stroke is expected to occur in milliseconds. Here we describe a technique where the temporal resolution is enhanced while limiting the number of cross-bridges under observation to study the role of phosphorylation on power stroke.|
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.