Localization of Cys-344 on the (Ca(2+)-Mg(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer.
4-Bromomethyl-6,7-dimethoxy-coumarin labels the (Ca(2+)-Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum at Cys-344. Resonance energy transfer has been used to measure the distance between this site and Lys-515 labelled with fluorescein isothiocyanate as about 37 A. The height of Cys-344 above the phospholipid/water interface has been measured by resonance energy transfer for the ATPase reconstituted into bilayers containing fluorescein-labelled phosphatidylethanolamine; the height was found to be about 45 A. None of these distances was found to alter on changing pH, or on addition of Mg2+, Ca2+ or vanadate. Quenching of the fluorescence of the coumarin-labelled ATPase with KI suggested that the fluorophore is not fully exposed on the ATPase.
Mata
A M
AM
SERC Centre for Molecular Recognition, University of Southampton, UK.
Stefanova
H I
HI
Gore
M G
MG
Khan
Y M
YM
East
J M
JM
Lee
A G
AG
D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry*; D003374:Coumarins; D003545:Cysteine / Q000737:chemistry*; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D003374:Coumarins; C053769:4-bromomethyl-6,7-dimethoxycoumarin; D017301:Ca(2+) Mg(2+)-ATPase; D016650:Fluorescein-5-isothiocyanate; D003545:Cysteine