Phosphorylated Ca2+-ATPase stable enough for structural studies

The atomic structure of sarcoplasmic reticulum Ca2+-ATPase, in a Ca2+-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M, No mura, H. & Ogawa, H, (2000) Nature 405, 647-655). Important steps for furth er understanding the mechanism of ion pumps will be the atomic structural c haracterization of different key conformational intermediates of the transp ort cycle, including phosphorylated intermediates. Following our previous r eport (Champeil, P., Henao, F., Lacapere, J.-J, & McIntosh, D. B, (2000) J. Biol. Chem, 276, 5795-5803), we show here that it is possible to prepare a phosphorylated form of sarcoplasmic reticulum Ca2+-ATPase (labeled with fl uorescein isothiocyanate) with a meek-long stability both in membranes anal in mixed lipid-detergent micelles. We show that this phosphorylated fluore scein isothiocyanate-ATPase can form two-dimensional arrays in membranes, s imilar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca2+-free unphosphoryl ated ATPase. The results also provide hope that crystals of phosphorylated Ca2+- ATPase suitable for x-ray crystallography will be achieved.