Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40°C) inside the coexistence range of liquid-ordered (l o ) and liquid-disordered (l d ) phases were investigated. Two common membrane probes, N-, which form a FRET pair, were used. The l o /l d partition coefficients of the probes were determined by individual photophysical measurements and global analysis of timeresolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l d phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (Ͻ20 nm) of l o in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l d in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l o phase from initially pure l d , and domain growth being faster in formation of l d phase from initially pure l o .

Resonance energy transfer between octadecyl rhodamine B (donor) and 1,1',3,3,3',3'-hexamethylindotricarbocyanine (acceptor) was studied in a model system of membranes (large unilamellar vesicles of dipalmitoylphosphatidylcholine), using both steady-state and time-resolved techniques. In the fluid phase (temperature = 50 degrees C) the decay law and the steady-state theoretical curve for energy transfer in two dimensions are verified. In the gel phase (temperature = 25 degrees C) an apparent reduction of dimensionality is observed, which is explained on the basis of probe segregation to the defect lines (grain boundaries). An estimation of the domain size from the model recovered linear probe concentrations is approximately 1750-2000 lipid molecules. In both phases, the existence of a fractal geometry was ruled out.

The aggregation of delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol or DHE), a fluorescent analog of cholesterol, was studied by photophysical techniques. It was concluded that the aqueous dispersions of DHE consist of strongly fluorescent microcrystals, and no evidence for self-quenching in micellar-type aggregates was found. The organization of DHE in model systems of membranes (phospholipid vesicles) is strongly dependent on the vesicle type. In small unilamellar vesicles, no evidence for aggregation is obtained, and the fluorescence anisotropy is rationalized on the basis of a random distribution of fluorophores. On the contrary, in large unilamellar vesicles (LUVs), a steeper concentration depolarization was observed. To explain this, a model that takes into account transbilayer dimer formation was derived. This was further confirmed from observation of excitonic absorption bands of 22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-23,24-bisnor- 5-cholen-3 beta-ol (NBD-cholesterol) in LUV, which disappear upon sonication. It is concluded that, in agreement with recent works, sterol aggregation is a very efficient process in large vesicles (and probably in natural membranes), even at very low concentrations (approximately 5 mol%).

Lipid bilayers composed of two phospholipids with significant acyl-chain mismatch behave as nonideal mixtures. Although many of these systems are well characterized from the equilibrium point of view, studies concerning their nonequilibrium dynamics are still rare. The kinetics of lipid demixing (phase separation) was studied in model membranes (large unilamellar vesicles of 1:1 dilauroylphosphatidylcholine (C(12) acyl chain) and distearoylphosphatidylcholine (C(18) acyl chain)). For this purpose, photophysical techniques (fluorescence intensity, anisotropy, and fluorescence resonance energy transfer) were applied using suitable probes (gel phase probe trans-parinaric acid and fluid phase probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanolamine). The nonequilibrium situation was induced by a sudden thermal quench from a one-fluid phase equilibrium situation (higher temperature) to the gel/fluid coexistence range (lower temperature). We verified that the attainment of equilibrium is a very slow process (occurs in a time scale of hours), leading to large domains at infinite time. The nonequilibrium structure stabilization is due essentially to temporarily rigidified C(12) chains in the interface between gel/fluid domains, which decrease the interfacial tension by acting as surfactants. The relaxation process becomes faster with the increase of the temperature drop. In addition, heterogeneity is already present in the supposed homogeneous fluid mixture at the higher temperature.

A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Förster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.