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 a nd phosphatidylcholine species with a variety of acyl-chain lengths. T he purple membrane, from the bacterium Halobacterium halobium, was use d and reconstituted with dilauroyl-(Lau2GroPCho), dimyristoyl- (Myr2Gr oPCho), dipalmitoyl- (Pam2GroPCho) and distearoyl- (Ste2GroPCho) glyce rophosphocholine. The phase behaviour of the lipids was investigated a t different temperatures and different protein/lipid molar ratios, by analyzing the fluorescence excitation spectra of the (2-anthroyl)-octa noyl]-sn-glycero-3-phosphocholine probe, and by measuring the fluoresc ence depolarization of the 1,6-diphenyl-1,3,5-hexatriene probe. Data o btained with (2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine shows that bacteriorhodopsin produced positive or negative shifts in the pha se transition temperature of the host lipids depending on the strength and sign of the mismatch between the lipid and protein hydrophobic th icknesses and also on the protein concentration and aggregation state in the lipid bilayer, In the region of high protein concentration (bac teriorhodopsin/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 la rge upward shift in the phase-transition temperature of Lau2GroPCho (D ELTAT almost-equal-to 40 K, mean hydrophobic thickness dBAR almost-equ al-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 rigidif ying effect of the protein on these short-chain lipids. Bacteriorhodop sin had no influence on the phase properties of Pam2GroPCho (DELTA alm ost-equal-to 0 K, dBAR almost-equal-to 3.2 nm), a lipid whose mean hyd rophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste2GroPCho was decreased (DELTAT almost-eq ual-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 di lution (bacteriorhodopsin/phosphatidylcholine molar ratios < 1:500). I n this region and for Lau2GroPCho, both the spectroscopic data and cir cular-dichroism spectra indicated that the protein was in the monomeri c form. Phase diagrams, in temperature versus bacteriorhodopsin concen tration, were constructed for Lau2GroPCho and Ste2GroPCho. On account of microscopic theoretical models and of the relative values of d(p) a nd 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 L au2GroPCho was also analyzed using another theoretical approach based upon elastic models within the Landau-de Gennes theory. This allowed f or 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 relat ively well with theoretical predictions.