Phosphorylated Ca 2+ -ATPase Stable Enough for Structural Studies

Henao, Fernando; Delavoie, Franck; Lacapère, Jean-Jacques; McIntosh, David B; Champeil, Philippe

Phosphorylated Ca 2+ -ATPase Stable Enough for Structural Studies

Journal Article

2001

Journal Title

The Journal of Biological Chemistry

Link to Journal

https://dx.doi.org/10.1074/jbc.M103905200

Department

Division of Chemical Pathology

Faculty

Faculty of Health Sciences

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.