The proposed project concerns the molecular mechanism of energy coupling by the Ca++ Mg++ ATPase of skeletal muscle sarcoplasmic reticulum (SR).
The aim i s to identify the functionally relevant regions of the polypeptide. The catalytic and the regulatory nucleotide sites will be labeled with photoaffinity nucleotide analogs and covalent site-directed probes. The regions of the polypeptide which contain the Ca++ sites will be identified by labeling with specific covalent reagents and Ca. Experiments will be done to determine the relation among the sites of phosphorylation and the catalytic and regulatory nucleotide sites will be determined by fluorescence energy transfer measurements. The functionally relevant polypeptide regions will be dissected by proteolytic enzymes and the resultant fragments separated by polyacrylamide slab gel electrophoresis. A two-dimensional peptide mapping technique will be implemented to allow the proteolytic fragments' alignment within the primary sequence of the ATPase polypeptide. The disposition of these specific regions relative to the internal and external aqueous compartments will be determined. The hydrophobic intramembranous segments of the polypeptide will be identified and placed within the primary sequence. Information concerning the pattern of protein folding within and through the bilayer will be deduced from the above results. Dynamic changes in the protein structure which occur concomitant with energy coupling will be specified by the simultaneous use of flash photoincorporation of probes and rapid mixing techniques. Changes in the structure of the functional sites will be determined at the critical reactions steps in which the enzyme interconverts between its two major conformational states. This will lead to new insights as to the relation between structure and function in the SR ATPase and the molecular basis of energy transduction. It is hoped that these results will enhance our understanding of the function of these systems in both normal and pathological conditions.
|Cao, C J; Lockwich, T; Scott, T L et al. (1991) Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase. Mol Cell Biochem 103:97-111|
|Scott, T L (1985) Distances between the functional sites of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum. J Biol Chem 260:14421-3|