Fluorescence Resonance Energy Transfer (FRET) as a Spectroscopic Ruler for the Investigation of Protein Induced Lipid Membrane Curvature: Bacteriorhodopsin and Bacteriorhodopsin Analogs in Model Lipid Membranes

Bacteriorhodopsin (bR) is a light-driven proton pump existing in the purple membranes (PM) of Halobacterium salinarum. The effects associated with changes in proton distribution (proton gradient, membrane electric potential) play a key role in ATPase stimulation. However, how the bioenergetic modulus (bR-PM-ATPase) functions remains unclear. One can find indications that hydrophobic matching and the curvature of the lipid membrane may form a functional link between bR and ATPase. To verify whether an interaction between bR and lipids can lead to curvature of the lipid membrane, a spectroscopic ruler, that is, a fluorescence resonance energy transfer (FRET) tool, was used. The distances from fluorescent lipid probes [octadecyl rhodamine B chloride (RhB), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 16-(9-anthroyloxy) palmitic acid (16AP), and hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH), to the retinal chromophore of bR incorporated into phospholipid vesicles, were measured. The incorporation of retinal analogues with changed shape and/or altered electronic properties into the binding site of a bR or bR mutant were used to strengthen the feedback between the protein surrounding and chromophore. The experiments were performed with wild-type and D96N-mutated bR carrying retinal or 14-(12-,10-, 13,14-bi-) fluororetinal. As far as it is known, this is the first time that results obtained by the FRETmethod show that bR can induce a change in lipid structure interpreted as hydrophobically induced curving of the lipid membrane. Evidence was provided that the chromophore contributed to this effect. The extent of contribution was dependent on the chromophore structure in close vicinity to the place of its link with opsin. The implications of these findings for bR-PM-ATPase module functioning are also discussed.