Fluid-fluid membrane microheterogeneity: A fluorescence resonance energy transfer study

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mi xtures were studied using fluorescence techniques (steady-state fluorescenc e intensity and anisotropy, fluorescence lifetime, and fluorescence resonan ce energy transfer (FRET)). Three compositions (cholesterol mole fraction 0 .15, 0.20, and 0.25) and two temperatures (30 and 40 degreesC) inside the c oexistence range of liquid-ordered (I,) and liquid-disordered (I,) phases w ere investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diaz ol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine(TM )-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The 1(o)/1(d) partition coefficients of the probes we re determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the I , phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRE T efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detec t very small domains (<20 nm) of I, in the cholesterol-poor end of the phas e coexistence range. In contrast, domains of I, in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleatio n being preferred in formation of I, phase from initially pure I,, and doma in growth being faster in formation of I, phase from