The goal of this project is to determine the actual mechanism of active (energy dependent) ion transport across biological membranes to at least the submolecular level. The sarcoplasmic reticulum Ca+2ATPase is employed as the prototypical ion pump. Recent time-resolved synchrotron x-ray diffraction studies determined the cylindrically-averaged profile structure for the calcium pump to moderate resolution (ca. 15 percent) within fully-functional isolated sarcoplasmic reticulum membranes for three important, transiently-trapped enzyme intermediates occurring within the cyclic series of partial reactions responsible for active calcium transport, the so-called E1, (Ca+2)xE1 and (Ca+2)xE1-P intermediates. These studies were extended to determine the locations of lanthanide ions (La+3, Tb+3), replacing calcium ions on the enzyme high-affinity metal binding sites, within the profile structure of the calcium pump AND the effect of enzyme phosphorylation on the positions and metal ion occupancies of these sites. Collectively, these studies suggest a novel mechanism for active ion transport. As a result of this work, together with the critical developments of high count-rate, rapid time-framing x-ray detector and vectorially-oriented single monolayers of the detergent-solubilized Ca+2ATPase, Dr. Blasie is now poised to investigate, not only the profile structures of all enzyme intermediates within the dynamic active calcium transport process (as opposed to only a few selected transiently trapped intermediates), but also the positions and metal ion occupancies of the calcium binding sites themselves within the profile structures of each of these enzyme intermediates. This key time-resolved structural information, combined with the emerging high-resolution 3-dimensional structure for the enzyme, will undoubtedly provide deep insight into the mechanism of active calcium transport by the Ca+2ATPase, most likely high relevant to the transport mechanism for other members of the P-type family of ion pumps as well, including the plasma membrane Na+1/K+1ATPases and Ca+2ATPases. Such knowledge concerning the actual mechanism for the formation of transmembrane ionic gradients is an important step toward understanding their regulation as central to cardiac bioenergetics at the cellular level.
