HYDROPHOBIC MISMATCH AND LONG-RANGE PROTEIN-LIPID INTERACTIONS IN BACTERIORHODOPSIN PHOSPHATIDYLCHOLINE VESICLES

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 Halobacterium halobium, was used and reconstituted with dilauroyl-(Lau2GroPCho), dimyristoyl- (Myr2GroPCho), dipalmitoyl- (Pam2GroPCho) and distearoyl- (Ste2GroPCho) 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-1,3,5-hexatriene probe. Data obtained with 1-acyl-2-[8-(2-anthroyl)-octanoyl]-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 (bacteriorhodopsin/phosphatidylcholine molar ratios almost-equal-to 1:50) and despite the presence of the endogenous lipids, bacteriorhodopsin (hydrophobic length d(p) almost-equal-to 3.0-3.1 nm) brought about a large upward shift in the phase-transition temperature of Lau2GroPCho (DELTAT almost-equal-to 40 K, mean hydrophobic thickness dBAR almost-equal-to 2.4 nm), and to a lesser extent of Myr2GroPCho (AT almost-equal-to 23 K, dBAR almost-equal-to 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 Pam2GroPCho (DELTA almost-equal-to 0 K, dBAR almost-equal-to 3.2 nm), a lipid whose mean hydrophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste2GroPCho was decreased (DELTAT almost-equal-to - 13 K, dBAR almost-equal-to 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 (bacteriorhodopsin/phosphatidylcholine molar ratios < 1:500). In this region and for Lau2GroPCho, 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 Lau2GroPCho and Ste2GroPCho. On account of microscopic theoretical models and of the relative values of d(p) and dBAR, these diagrams indicate a preference of the protein for those lipid molecules which are in the gel-ordered state in Lau2GroPCho but in the liquid disordered state in Ste2GroPCho. The phase diagram of Lau2GroPCho was also analyzed using another theoretical approach based upon elastic models within the Landau-de Gennes theory. This allowed for the estimation of the coherence length zeta which characterizes the distance over which the hydrophobic thickness of the lipid bilayer is perturbed by the protein. A value of 1.2 nm was found, agreeing relatively well with theoretical predictions.