The general goal of the proposed research is to understand the role of molecular dynamics in the function of membrane-bound enzymes. A large part of our effort is directed at the continuing development of instrumentation, experimental methodology, and theoretical analysis for the measurement of protein and lipid motion and spatial arrangements, by means of both magnetic resonance and optical spectroscopy. Electron paramagnetic resonance (EPR), both conventional and saturation transfer methods, will be used to study the orientation and rotational motion of spin-labeled proteins and lipids. Time-resolved phosphorescence will be used to study both rotational and translational diffusion, and intermolecular distances. We will use these methods primarily to probe the relationship between motion and function in the Ca-ATPase of sarcoplasmic reticulum (SR), the ATP-driven Ca++ pump that maintain's the Ca++ gradient necessary for muscle function. We will vary such parameters as lipid composition, lipid-to-protein ratio, temperature, and ion concentrations, correlating observed spectroscopic changes with changes in enzyme activity. In addition, the spectroscopic methods will be used to monitor molecular dynamics during enzyme action, to determine what motions may be coupled to function. We hope that these studies will help elucidate the molecular mechanism of action in this dynamic membrane system, and that the methods developed will stimulate similar progress in other systems.
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