title: Phosphorylated Ca2+-ATPase stable enough for structural studies.
abstract: The atomic structure of sarcoplasmic reticulum Ca(2+)-ATPase, in a Ca(2+)-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655). Important steps for further understanding the mechanism of ion pumps will be the atomic structural characterization of different key conformational intermediates of the transport cycle, including phosphorylated intermediates. Following our previous report (Champeil, P., Henao, F., Lacapère, 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 Ca(2+)-ATPase (labeled with fluorescein isothiocyanate) with a week-long stability both in membranes and in mixed lipid-detergent micelles. We show that this phosphorylated fluorescein isothiocyanate-ATPase can form two-dimensional arrays in membranes, similar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca(2+)-free unphosphorylated ATPase. The results also provide hope that crystals of phosphorylated Ca(2+)-ATPase suitable for x-ray crystallography will be achieved.
authors.affiliation: Departamento de Bioquimica y Biologia Molecular, Universidad de Extremadura, 06080 Badajoz, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Henao
authors.forename: F
authors.initials: F
authors.lastname: Delavoie
authors.forename: J J
authors.initials: JJ
authors.lastname: Lacapère
authors.forename: D B
authors.initials: DB
authors.lastname: McIntosh
authors.forename: P
authors.initials: P
authors.lastname: Champeil
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D003460:Crystallization; D004795:Enzyme Stability; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D010766:Phosphorylation; D014638:Vanadates / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D014638:Vanadates; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate