TIME RESOLVED X RAY INTERFEROMETRY AND SARCOPLASMIC RETICULUM CALCIUM ATPASE

Case Western Reserve University, Cleveland OH

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Abstract

The overall 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+2 ATPase is employed as the prototypical ion pump. The high-resolution 3-dimensional structure of the detergent-solubilized sarcoplasmic reticulum Ca+2 ATPase has just been determined by x-ray crystallography. We have recently developed vectorially-oriented single monolayers of the detergent-solubilized Ca+2 ATPase and the methods for the study of the so-called profile structure of the enzyme in these monolayers to a spatial resolution of approximately 7 angstroms employing both non-resonance & resonance x-ray interferometry and neutron interferometry. The detergent utilized has been shown to be optimal for maintaining enzyme functionality. As a result of this work, together with the critical developments of a doubly-focused undulator synchrotron x-ray source and a high count-rate, rapid time-framing x-ray detector, we are now poised to investigated not only the profile structures of all enzyme intermediates within the dynamic active calcium transport processes with lifetimes greater than or equal to 1 ms (as opposed to only a few selected transiently trapped intermediates) to a spatial resolution of approximately 7 angstroms, but also the positions and metal ion occupancies of the calcium binding sites themselves within the profile structures of each of these enzyme intermediates with lifetimes greater than or equal to 100 ms. The calcium transport processes will be initiated synchronously among the enzyme monolayer ensemble via the flash-photolysis of suitable caged-substrates The key time-resolved structural information can only be obtained from such studies of the Ca+2 ATPase in its fully functional form within a liquid- crystalline membrane (or non-crystalline studies of the Ca+2 ATPase in its fully functional form within a liquid crystalline membrane (or non- crystalline membrane-like) environment. This key time-solved structural information will be interpreted via detailed molecular modeling utilizing the high-resolution 3-dimensional structure for the enzyme obtained via X-ray crystallography as a critical starting point.. In addition, the effects of site-directed mutations of residues shown to be critical to the calcium transport function will be employed to facilitate this interpretation.-these studies are made possible by the utilization of single monolayers of the vectorially-oriented enzyme. As a result, these studies will undoubtedly provide deep insight into the mechanism of active calcium transport by the Ca+2ATPase, most likely highly 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 mechanisms for the formation of transmembrane ionic gradients is an important step toward understanding their regulation as central to cardiac bioenergetics at the cellular level.