The long-term goal of this research is to understand the coupling of molecular dynamics to ATPase enzyme action, focussing on the two principal ATP-driven energy transduction systems in vertebrate skeletal muscle: the myosin-actin system (contraction, force-generation), and the Ca-ATPase of sarcoplasmic reticulum (relaxation, active Ca transport). Because these processes are essentially dynamic, we focus on direct detection of protein motions, using site-specific spectroscopic probes: electron paramagnetic resonance (EPR) with nitroxide spin labels, and optical anisotropy with fluorescent and phosphorescent dyes. These methods are chosen because they have sufficient sensitivity and selectivity to detect specific molecular motions in a complex system under physiological conditions. The present project focuses on spectroscopic measurements during the transient phase of the ATPase reaction cycle. This is accomplished by initiating spectroscopic data acquisition, then using laser flash photolysis of """"""""caged"""""""" compounds (e.g., caged ATP, caged Ca), then observing the millisecond-time-resolved spectroscopic response to the resulting step change in ligand (e.g., ATP, Ca) concentration. The following specific aims will be pursued: (1) Develop improved methods for obtaining and analyzing transient spectroscopic (EPR and optical) signals from muscle fibers and membranes, using flash photolysis of caged compounds. (2) Use this technology to probe molecular dynamics and interactions in purified myosin, actin, and their complexes. (3) Probe molecular orientation, motion, and interaction in skinned muscle fibers, during the transient phase of contraction. (4) Detect transient rotational dynamics of the Ca-ATPase during calcium transport in SR membranes. Collaborative studies will be carried out with experts in the transient biochemical and mechanical kinetics of both systems, in an effort to obtain direct correlations between molecular dynamics and physiological transitions. We will use several different caged compounds, to probe different parts of the ATPase cycle, and we will perform spectroscopic experiments under conditions known to affect the physiological transients. While our central goal is to provide new and essential information about the molecular events in muscle contraction and relaxation, we also hope that the technology we are developing will prove effective in a wide range of biophysical energy transduction problems.
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