Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor.

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ2. From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.

Fret studies of conformational changes in heparin-binding peptides.

FRET (Förster Resonance Energy Transfer) was applied to study structural properties of heparin-binding peptides containing the sequence XBBBXXBX where 'X' represents hydropathic or uncharged and 'B' represents basic amino acids. Internally quenched fluorogenic peptides were synthesized containing the fluorescent donor oaminobenzoic acid (o-Abz) and the acceptor dinitrophenyl ethylenediamine (Eddnp) group. Using the CONTIN computational package, distance distributions were recovered from time resolved fluorescence data, associated to end-to-end distances of the peptides. The peptides containing three or four repeat units have random structure in aqueous medium, and the interaction with low molecular weight heparin stabilized short end-to end distances. Experiments in water/trifluoroethanol (TFE) mixtures showed changes in distance distributions compatible with compact conformations stabilized above 40% volume content of TFE in the mixture. Similar changes in distance distributions were also observed for the peptides in interaction with SDS micelles in aqueous suspensions and circular dichroism data revealed alpha-helix formation in the peptides in interaction with heparin, SDS micelles or the co-solvent TFE. The process is dependent on electrostatic and hydrogen bond interactions and the end-to-end distances obtained are smaller than expected for the peptides in linear α-helix conformation, indicating the occurrence of structural arrangements leading to additional decrease in the distances.