The general goal of the proposed research is to understand the role of molecular dynamics in the function of energy-transducing membrane-bound proteins. This effort involves two major components: (1) the development of spectroscopic methods for the measurement of protein and lipid motions and (2) the application of these methods to the study of specific membrane systems. (1) Electron paramagnetic resonance (EPR) techniques will be developed for the study of orientation and rotational dynamics of spin-labeled proteins and lipids, with particular emphasis on saturation transfer EPR, used for the study of microsecond motions of membrane proteins. A second major methodological area will be time-resolved optical spectroscopy, again focussing on methods for the microsecond time range, including transient phosphorescence (or absorption) anisotropy (for measuring rotational motions) and diffusion-enhanced energy transfer (for measuring translational motions and distances). The use of several complementary techniques on the same system is essential in minimizing the ambiguity of interpretation. (2) We will use these methods primarily to probe the relationship between molecular motion and energy-transduction in the Ca-ATPase of sarcoplasmic reticulum (SR), the active Ca pump that maintains the Ca gradient necessary for the function of skeletal muscle. We will perturb the system by varying conditions likely to affect molecular motions and/or function, e.g., lipid composition, temperature, anesthetics, and concentrations of substrates and other ligands. The spectroscopically detected effects on molecular motion and structure (of both lipid and protein components) will be compared with effects on defined kinetic steps in the ATPase and Ca transport reactions. Experiments will be designed to test specific models (proposed by us and others) for the mechanism of active Ca transport. We are particularly interested in the oligomeric state of the enzyme and in lipid dynamics, which may both play key roles in the mechanism. Analogous applications to other systems (cardiac SR, photoreceptor membranes, prothrombin, and cellular membranes) will be carried out through collaborations with other research groups. Besides providing essential information for the understanding of these important membrane systems, these studies, performed in the same laboratory where spectroscopic methods are being developed, should provide models for similar applications in a wide range of other systems.
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