Indirect evidence for lipid-domain formation in the transition region of phospholipid bilayers by two-probe fluorescence energy transfer

The fluorescence energy transfer between two lipid probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (donor) and N-(Lissamine rhodamine B sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (acceptor), incorporated into 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine unilamellar and multilamellar lipid bilayers, is studied in the temperature region of the main phase transition. The two probes display different relative solubilities in the gel and fluid lipid-bilayer phases. A distinct maximum in the fluorescence intensity of the donor is observed in the transition region, indicating that the two probes are demixing and hence increasing their average separation. The observation is interpreted in terms of dynamic segregation of the two probes into coexisting gel and fluid lipid domains that are formed dynamically in the transition region due to strong density fluctuations. The interpretation of the experimental observations is supported by a detailed theoretical calculation using computer simulation of a microscopic model that takes full account of diffusion of the two probes and the fluctuations of gel and fluid lipid domains characteristic of the main phase transition.