The long-term goal of this research is to understand the role of protein rotational motions in the generation of force during muscle contraction. Site-selective molecular probes (spin labels or luminescent dyes) will be attached to myosin or actin, and spectroscopy experiments (electron paramagnetic resonance (EPR) and time-resolved optical anisotropy) will provide direct information about the orientation and rotational motions of he labeled proteins. These experiments will be performed on purified proteins, in which biochemical conditions can be precisely controlled and monitored, and also on skinned muscle fibers, in which mechanical performance can be controlled and monitored. Experimental conditions will be chosen to provide direct tests for the proposed motions of myosin heads (cross-bridges) and actin subunits during the generation of force. The high orientational resolution of EPR and time resolution of optical anisotropy will be used to resolve the multiple conformations present under complex physiological conditions. Although the primary emphasis will be on these applications, an integral apart of this project is the development of the spectroscopic methods. The five closely related principal aims of the project are (1) development of EPR techniques (both conventional and saturation-transfer) (2) application of these EPR techniques to detect the orientations and rotations of myosin and actin, (3) development of time-resolved optical anisotropy techniques (transient absorption, phosphorescence, and fluorescence), (4) application of these optical techniques to resolve the rotational motions of myosin and actin, and (5) correlation of the molecular dynamics with structural, mechanical, and biochemical measurements on the same preparations. These studies should provide direct insight into the role of molecular dynamics in this fundamental physiological process, and the technology we are developing should be applicable to a wide range of other biophysical system in which conformational dynamics are coupled to enzyme action.
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