Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer

Abstract

A fluorescent phospholipid derivative, the fluoresceinthiocarbamyl adduct of a natural phosphatidylethanolamine, has been synthesized and incorporated into sonicated single-bilayer vesicles of egg lecithin and dipalmitoyllecithin. The surface location of this probe has been confirmed by using extrinsic fluorescence quenching studies together with steady-state emission anisotropy measurements. Electronic excitation energy transfer between 1,6-diphenyl-1,3,5-hexatriene incorporated within the hydrophobic core of the bilayer and the novel derivative has been investigated to estimate the depth within the bilayer at which the former is located. Efficiencies have been measured for two different phospholipids, egg lecithin and dipalmitoyllecithin, in the latter case both above and below the phospholipid phase transition, with and without added cholesterol. The observed dependence of the transfer efficiency on the acceptor concentration was compared with that calculated according to Förster theory applied to random two-dimensional distributions of donor and acceptor molecules in parallel planes for various interplanar separations, taking into account orientational effects. The Förster R0 of about 45 A for this donor-acceptor pair is particularly well suited to such studies since it is of the order of the width of the bilayer. The experiments showed that energy-transfer spectroscopy can provide useful quantitative information as to the transverse location of diphenylhexatriene in homogeneous phospholipid bilayers and may also reflect lateral partitioning of donor or of both donor and acceptor into different phases in systems exhibiting phase separations.

Effect of cholesterol on membrane microheterogeneity: a study using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distributions

“…Kao et al [24] have shown that the presence of cholesterol reduces the apparent polarity of the hydrocarbon region of the phospholipid bilayer. In unsaturated phosphatidylcholine, Davenport et al [25] have shown that DPH is located in the central part of the bilayer. Therefore, in our experimental conditions it cannot be excluded that cholesterol favours this tendency so that the probe i~ located in a more hydrophobic and homogeneous environment.…”

Determining the Membrane Topology of Peptides by Fluorescence Quenching

“…The constraints imposed by the surface might also cause the donors and acceptors to lie at different depths in the membrane (which affects the computation of r) or not to be isotropically oriented (κ 2 * 2/3). RET quenching in membrane systems has been considered in detail by Wolber and Hudson (26) and Davenport et al (27). The effect on R 0 of various assumptions about κ 2 in membrane systems with Trp donors was examined in detail by Ladokhin et al (28) who found that R 0 was affected only moderately by the choice of κ 2 in a study of Trp quenching in cytochrome b 5 .…”

Quantification of protein–lipid selectivity using FRET

“…(1985) aswhere b  = ( R 0 / l ) 2 τ D −1/3 , σ is the acceptor density in each leaflet, and R e is now the distance between the protein axis and the second lipid shell (exclusion distance for bulk acceptors) (Fig. 3).…”