Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers

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Abstract
The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37.degree. C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores. Photobleaching of the bacteriorhodopsin and trypsinization of the cytochrome b5 effectively removed energy transfer, as demonstrated by a return of the lifetime centers to the values obtained for phospholipid vesicles alone. However, the value of the widths remained broad for both proteins (about 3.2 ns). From these results we would conclude that there is a distinct heterogeneity in the environment of the DPH in the protein-lipid interfacial region in lipid bilayers. Although the exact basis for the environment heterogeneity caused by membrane proteins has yet to be ascertained, since the kinetics of the fluorescence decay process is of general importance in time-resolved fluorescence spectroscopy, these results have important implications for studies of lipid bilayers containing protein.