Localization of the hinge region of the Ca(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer.
The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum can be labelled at Cys-670 and Cys-674 with 5-[[2-[(iodoacetyl) amino]ethyl]amino]naphthalene-1-sulphonic acid (IAEDANS). Resonance energy transfer has been used to measure the distance between Cys-670/Cys-674 and Glu-439 labelled with 5-(bromomethyl)fluorescein as 40 A. The height of Cys-670/Cys-674 above the phospholipid/water interface has been measured by resonance energy transfer between IAEDANS-labelled ATPase and fluorescein-labelled phosphatidylethanolamine as 54 A. This locates the hinge region of the ATPase close to the mouth of the pore observed in the cytoplasmic region of the ATPase in electron micrographs. No significant changes in these distances can be detected by resonance energy transfer on binding Ca2+ or vanadate. The height of the IAEDANS label above the phospholipid/water interface is the same for bilayers of dimyristoleoylphosphatidylcholine and dioleoylphosphatidylcholine. Conformation changes on the Ca(2+)-ATPase appear to be localised to small regions of the ATPase.
Baker
K J
KJ
SERC Centre for Molecular Recognition, University of Southampton, UK.
East
J M
JM
Lee
A G
AG
D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009132:Muscles / Q000201:enzymology; D009282:Naphthalenesulfonates; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014867:Water
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010743:Phospholipids; D014867:Water; C081567:5-(bromomethyl)fluorescein; C006421:1,5-I-AEDANS; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate