Spatial arrangement of selected fluorescence labels in lipid bilayer

The method for the determination the orientation factor κ2, spatial arrangement and depth position of fluorescence labels located in hydrophilic layers of vesicles bilayer from resonance energy transfer (RET) data is presented. The method is based on the broadened Wolber and Hudson RET model in two dimensions (Biophys J. 1979). The vesicles were labeled with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) as the donor and N-(Lissamine rhodamine B sulfonyl) 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NRh-PE) as the acceptor. It was found that in basic environment sodium dithionite quenches fluorescence of both labels located in outer leaflet of bilayer. Therefore, RET data prior to and following dithionite treatment were compared and the donor–acceptor cis and trans distances of the closest approach as well as cis and trans Förster radii R0, and orientation factors κ2 for cis RET equal to 0.61 ± 0.06 and for trans RET equal to 0.17 ± 0.01 were assigned. Knowing the κ2 data, the spatial arrangement of NBD and NRh labels as dipoles in dipalmitoylphosphatidylcholine bilayer were described.