Mismatch between the hydrophobic thicknesses of transmembrane proteins and the supporting lipid bilayer and its consequences on the lateral organization of lipids have been investigated with bacteriorhodopsin and phosphatidylcholine species with a variety of acyl-chain lengths. The purple membrane, from the bacterium Halobacteriuin halobium, was used and reconstituted with dilauroyl-(Lau,GroPCho), dimyristoyl-(Myr,GroPCho), dipalmitoyl-(Pam,GroPCho) and distearoyl-(Ste, GroPCho) glycerophosphocholine. The phase behaviour of the lipids was investigated at different temperatures and different protein/lipid molar ratios, by analyzing the fluorescence excitation spectra of the 1 -acyl-2-[ 8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, and by measuring the fluorescence depolarization of the 1,6-diphenyl-l,3,5-hexatriene probe. Data obtained with 1 -acyl-2-[8-(2-anthroyl)-oct~oyl]-sn-glycero-3-phosphocholine shows that bacteriorhodopsin produced positive or negative shifts in the phase transition temperature of the host lipids depending on the strength and sign of the mismatch between the lipid and protein hydrophobic thicknesses and also on the protein concentration and aggregation state in the lipid bilayer. In the region of high protein concentration (bacteriorhodopsidphosphatidylcholine molar ratios = 1 : 50) and despite the presence of the endogenous lipids, bacteriorhodopsin (hydrophobic length d, = 3.0 -3.1 nm) brought about a large upward shift in the phase-transition temperature of Lau,GroPCho (AT = 40 K, mean hydrophobic thickness d = 2.4 nm), and to a lesser extent of Myr,GroPCho (AT = 23 K, d = 2.8 nm), accounting for a strong rigidifying effect of the protein on these short-chain lipids. Bacteriorhodopsin had no influence on the phase properties of Pam,GroPCho (AT = 0 K, d 3.2 nm), a lipid whose mean hydrophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste,GroPCho was decreased (AT = -13 K, d = 3.7 nm), indicating a fluidifying effect of the protein on this long-chain lipid. Similar effects on the lipid acyl-chain order were observed in the region of high-protein dilution (bacteriorhodopsidphosphatidylcholine molar ratios < 1 : 500).In this region and for Lau,GroPCho, both the spectroscopic data and circular-dichroism spectra indicated that the protein was in the monomeric form. Phase diagrams, in temperature versus bacteriorhodopsin concentration, were constructed for Lau,GroPCho and Ste,GroPCho. On account of microscopic theoretical models and of the relative values of d, and d, these diagrams indicate a preference of the protein for those lipid molecules which are in the gel-ordered state in Lau2. GroPCho but in the liquid disordered state in Ste,GroPCho. The phase diagram of Lau,GroPCho was also analyzed using another theoretical approach based upon elastic models within the Landaude Gennes theory. This allowed for the estimation of the coherence length < which characterizes the distance over which the hydrophobic thickness of the lipid bilay...

Through steady-state and time-resolved fluorescence experiments, the polarity of the bilayers of egg phosphatidylcholine vesicles was studied by means of the solvatochromic 2-anthroyl fluorophore which we have recently introduced for investigating the environmental micropolarity of membranes and which was incorporated synthetically in phosphatidylcholine molecules (anthroyl-PC) in the form of 8-(2-anthroyl)octanoic acid. Fluorescence quenching experiments carried out with N,N-dimethylaniline and 12-doxylstearic acid as quenchers showed that the 2-anthroyl chromophore was located in depth in the hydrophobic region of the lipid bilayer corresponding to the C9-C16 segment of the acyl chains. Steady-state fluorescence spectroscopy revealed a nonstructured and red-shifted (lambda em(max) = 464 nm) spectrum for the probe in egg-PC bilayers, which greatly differed from the structured and blue (lambda em(max) = 404 nm) spectrum the fluorophore was shown to display in n-hexane. While the fluorescence decays of the fluorophore in organic solvents were monoexponential, three exponentials were required to account for the fluorescence decays of anthroyl-PC in egg-PC vesicles, with average characteristic times of 1.5 ns, 5.5 ns, and 20 ns. These lifetime values were independent of the emission wavelength used. Addition of cholesterol to the lipid did not alter these tau values. One just observed an increase in the fractional population of the 1.5-ns short-living species detrimental to the population of the 20-ns long-living ones. These observations enabled time-resolved fluorescence spectroscopy measurements to be achieved in the case of the 1/1 (mol/mol) egg-PC/cholesterol mixture. Three distinct decay associated spectra (DAS) were recorded, with maximum emission wavelengths, respectively, of 410 nm, 440 nm, and 477 nm for the 1.5-ns, 6-ns, and 20-ns lifetimes found in this system. On account of the properties and the polarity scale previously established for the 2-anthroyl chromophore in organic solvents, these data strongly suggest the occurrence of three distinct excited states for anthroyl-PC in egg-PC bilayers, corresponding to three environments for the 2-anthroyl chromophore, differing in polarity. The lifetime of 1.5 ns and the corresponding structured and blue (lambda em(max) = 410 nm) DAS account for a hydrophobic environment, with an apparent dielectric constant of 2, which is that expected for the hydrophobic core of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)

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Micrococcus luteus bacterium has been used as a tool for the preparation of a fluorescent (anthracene labeled) cardiolipin by a method involving two steps. First, 9-(2-anthryl)nonanoic acid is incorporated into the lipids of the bacterium by usual metabolic pathways; then phosphatidylglycerol, a major lipid in this bacterium, is converted into cardiolipin by endogeneous cardiolipin synthetase. It is shown that cardiolipin synthetase transforms anthracene–phosphatidylglycerol into anthracene–cardiolipin at a high rate. The method can be extended to the synthesis of cardiolipin molecules bearing other hydrophobic marker groups.Key words: cardiolipin, fluorescence, biosynthesis, Micrococcus luteus.