The primary aim of this proposal is to solve by cryoelectron microscopy the structure of phospholamban (PLB), a small transmembrane protein that is central to beta-adrenergic regulation of cardiac contractility. Co-crystals containing PLB together with the calcium pump from sarcoplasmic reticulum (SR Ca-ATPase), have already been made by co-reconstitution of purified Ca-ATPase with recombinant PLB. Initially, Dr. Stokes will aim for 15 A resolution which will reveal the oligomeric state (which is thought to be pentameric) of PLB and its site of binding to Ca -ATPase. Co-crystals will also be made from single site mutants of PLB that produce monomers and tetramers and their structure will help define the role of oligomerization in this regulation. Finally, PLB will be crystallized in the absence of Ca-ATPase by reconstitution and by using monoclonal antibodies to mediate crystal contacts. Such crystals will allow Dr. Stokes to compare PLB structure before and after phosphorylation, which is the physiological switch controlling its regulation of Ca-ATPase. Ultimately, Dr. Stokes hope to improve the resolution to better than 10 A, thus revealing the arrangement of alpha-helices in both PLB and Ca-ATPase and better defining their structural interactions. Another aim of this proposal is to use methods previously developed for PLB and Ca-ATPase for reconstitution and crystallization of three related ion pumps, Na/K-ATPase, H/K-ATPase and plasma membrane Ca-ATPase, with the ultimate goal of investigating their structures by cryoelectron microscopy. Purified protein will be obtained from 3 different collaborators and initial reconstitution will be done at high lipid-to-protein ratios to assess the activity of the proteins. Thereafter, this ratio will be lowered and 2D membrane crystals will be induced in the reconstituted membranes; previous work has defined crystallization conditions which will be used as a starting point. H/K-ATPase is the target of clinical therapies for ulcers and structural information wil help us understand the mechanism of the various drugs. The family of ion pumps include those responsible for Menkes and Wilsons disease, and extrapolation of the structures will improve understanding of the molecular mechanisms for these diseases.
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