Structural elucidation of active ion transport

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The aim of this project is to elucidate the mechanism of active transport from the structural standpoint. When this project was proposed, we had merely two kinds of crystals of CaィイD12+ィエD1-ATPase useful for electron microscopy. Since then, we succeeded in generating large crystals suitable for X-ray crystallography and expected to solve the structure at an atomic resolution within two years or so. Therefore, we shifted the stress on considering on the utilization of mutants for structural studies and asked Prof. Inesi of the University of Maryland to join, because his group was the only one that-succeeded in making large quantity of this membrane protein. This collaboration went out extremely well and produced one review and one original research paper (submitted to Biochemistry). The mutational studies agreed very well with the structure revealed by x-ray crystallography (submitted to Nature) and provided insights into the conformational changes required for active transport. On the other hand, the structure was indispensable to interpret the biochemical and mutational results correctly. Altogether, this project became a very good example of international collaboration. The next step is obviously to make crystals of mutants. For this purpose, the purification procedure used for SR CaィイD12+ィエD1-ATPase has turned out to be insufficient. We are now improving the methods for cultured cells in collaboration with Prof. Inesi.