Fluid–Fluid Membrane Microheterogeneity: A Fluorescence Resonance Energy Transfer Study

Abstract

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 .

Behaviour of NBD-head group labelled phosphatidylethanolamines in POPC bilayers: a molecular dynamics study

“…NBD-diC n PE probes have been used extensively as model phospholipids in the studies of thermodynamics and kinetics of interaction with lipid bilayers and other hydrophobic aggregates, such as serum lipoproteins. [17][18][19] Used as FRET donors, NBD-diC n PE probes form, together with acceptor rhodamine B PE derivatives, a very well-known Förster pair for the studies of membrane organization [20][21][22][23][24] as well as quantification of lipid mixing and exchange. 25 Alternatively, NBD probes can be used as acceptors to chromophores including diphenylhexatriene 26,27 or trans-parinaric acid, 28 or even as donors and acceptors simultaneously in homo-FRET studies.…”

“…25 Alternatively, NBD probes can be used as acceptors to chromophores including diphenylhexatriene 26,27 or trans-parinaric acid, 28 or even as donors and acceptors simultaneously in homo-FRET studies. 20 Several fluorescence spectroscopic and microscopic studies have also indicated that, contrary to the vast majority of membrane fluorescent probes, doubly saturated long-chained NBD-diC n PE partitions favourably to liquid ordered (lo) phases in systems displaying lo/liquid disordered phase coexistence, 22,24,29,30 although the phase preference depends both on the probe acyl chain length and the composition of the underlying lipid mixture. 17,31 Despite their wide use in membrane biophysics, the behaviour of NBD-diC n PE probes has so far lacked a characterization using MD.…”

Steady-state probe-partitioning fluorescence resonance energy transfer: A simple and robust tool for the study of membrane phase behavior

“…It has long been recognized that energy transfer between fluorescent membrane probes is eminently suited to the detection and study of coexisting membrane phase domains [19], and a number of groups have adapted FRET experiments to this end during the last decade, or so [e.g., 4,[20][21][22]. Indeed, Loura, de Almeida, Fedorov and Prieto have published several excellent studies [13,[23][24][25][26][27] based on time-resolved measurements of transfer efficiency, ) (t E , and Silvius and Nabi have recently produced a comprehensive review of FRET-based studies of membrane microdomains [28].…”

Fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids in model membranes is connected not to lipid mobility but to probe location

“…[1][2][3][4] NBD-labeled lipids are commercially available for all major phospholipids and are readily incorporated into lipid membranes. The photophysical a Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry characteristics of NBD, such as its fluorescence quantum yield and lifetime, depend strongly on the solvent, 5 which makes it a sensitive fluorescent probe.…”