The objective of this Proposal is to relate the mechanism of Ca2+ transport in cardiac sarcoplasmic reticulum [SR] to the physical structure, both of Ca2+-ATPase and its regulatory protein, phospholamban [PLB]. Two different microscopies will be used to address rather different aspects of molecular structure. To study the structure of PLB, 2-D cocrystals containing both PLB and Ca2+-ATPase will be made either from native cardiac SR or from a reconstituted system. Crystallization will be induced by vanadate, which has previously been shown to make crystals in cardiac SR and which has been used for 3-D reconstruction of skeletal Ca2+-ATPase. The presence of PLB in cocrystals will be verified by immunogold labelling and, after iodination, by elemental mapping techniques developed by Project 5. Cocrystals will then be rapidly frozen and imaged by frozen-hydrated electron microscopy. These images will be used for 3-D reconstruction at ~15Angstroms resolution, which will allow visualization (a) of the structure of cardiac Ca2+-ATPase, (b) of the oligomeric state of PLB, and (c) of their interaction. To study the dynamics of Ca2+-ATPase itself, atomic force microscopy [AFM] will be used to image Ca2+ATPase under a variety of hydrated conditions known to stabilize different conformational states. Particular conditions will stabilize five different reaction intermediates and include: (a) the presence and absence of Ca2+, (b) phosphorylation by inorganic phosphate or incubation with vanadate, and (c) phosphorylation with either CrATP or Co(NH3)4PO4. A number of specimen preparation methods, which will be developed as part of Project 2, will be used. Crystalline specimens will be imaged, first: after Fourier averaging and comparison with crystal structures from electron microscopy, these images will be used to familiarize us with the surface structure of Ca2+-ATPase. Thereafter, non-crystalline specimens will be imaged at ~10Angstroms resolution, and comparisons amongst the various non- crystalline conditions will allow us to link the reaction cycle to specific shape changes in Ca2+-ATPase, and thus, to construct a physical model for the mechanism of Ca2+ transport.
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