The long-term goal of the proposed research is to understand the role of molecular dynamics in the function of membrane proteins. This effort involves the development of complementary spectroscopic methods for the measurement of protein and lipid motions, and the application of these methods to muscle membrane systems, with emphasis on the Ca-ATPpase of sarcoplasmic reticulum (SR). Experiments are planned to obtain biophysical information about specific molecular mechanisms, and to help define the general principles that govern membrane molecular dynamics. The following aims will be pursued: (1) Improved spectroscopic methods will be developed for studying membrane molecular dynamics, and the Ca-ATPase will be used as a model system to demonstrate their use, obtaining a more quantitative and detailed description of molecular motions and interactions of this integral membrane protein and its associated phospholipids in the unperturbed """"""""resting state"""""""" in SR. (1a) Electron paramagnetic resonance (EPR) measurements will be made on the orientations and rotational motions of nitroxide spin labels bound to the CA-ATPase or lipids in SR. Saturation transfer EPR will be used to study microsecond motions of membrane proteins or protein- restricted lipid chains. Conventional EPR will be used to measure orientation and nsec motions. (1b) Time-resolved optical spectroscopy, using pulsed lasers, will received increased emphasis. Phosphorescence anisotropy will be used to measure microsecond rotations, while fluorescence will be used to measure distances and nsec motions. The use of several complementary techniques on the same system is essential for minimizing the ambiguity of interpretation. (2) To determine what changes in molecular motions and interactions are coupled to the Ca-ATPase reaction cycle, spectroscopic measurements will be performed in the presence of substrates and ligands that play key roles in the mechanism (e.g., Ca++ and ATP), including measurements during active Ca++ transport. The results will be correlated with quantitative transient biochemical kinetic measurements. (3) To determine which molecular motions are important in the Ca-ATPase mechanism, molecular dynamics and Ca-ATPase activity will be measured as a function of perturbations designed to to affect these properties (e.g., temperature, anesthetics, lipid composition, cross-linking, and ionic conditions). (4) The effects of peripheral membrane proteins and peptides on the molecular dynamics and function of SR will be studied, with initial studies focussed on melittin. this is an example of aim (3). One of the goals is to developed SR as a model system for the regulation of integral membrane proteins by peptides. (5) The studies will be extended to cardiac SR. This connects with aim (4), since the cardiac Ca-ATPase is regulated by phospholamban, which shares some of melittin's physical and functional properties. (6) the calcium-release channel (ryanodine receptor) of SR will be studied, including experiments on a mutant that is the basis for malignant hyperthermia. This system offers an ideal opportunity to extend the biophysical studies of membrane molecular dynamics into the areas of molecular genetics and pathobiology.
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