Determination of the fluorescence labels location in lipid bilayer based on fluorescence quenching

A comparison of Förster Resonance Energy Transfer (FRET) data of vesicles labeled with fluorescence labels on both inner and outer layers of the bilayer, with ones labeled only on the inner layer of bilayer to permit a determination of the location of fluorescence labels in relation to the bilayer center. The theoretical description of the FRET effect is based on the FRET model generalized and extended by Wolber and Hudson. A useful version of the FRET model implies location of acceptors with respect to a trigonal or tetragonal lattice of acceptors centered on donors located deeper than the acceptor layer. Several fluorescence labels were tested in order to find FRET pairs of labels for which the same quantity of quencher reduced label fluorescence to one half of the initial value before quencher treatment. 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) was used as a fluorescence quencher of both labels located in outer layer of bilayer.