The functional significance of ordered nanodomains (or rafts) in cholesterol rich eukaryotic cell membranes has only begun to be explored. This study exploits the correspondence of cellular rafts and liquid ordered (Lo) phases of three-component lipid bilayers to examine permeability. Molecular dynamics simulations of Lo phase dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and cholesterol show that oxygen and water transit a leaflet through the DOPC and cholesterol rich boundaries of hexagonally packed DPPC microdomains, freely diffuse along the bilayer midplane, and escape the membrane along the boundary regions. Electron paramagnetic resonance experiments provide critical validation: the measured ratio of oxygen concentrations near the midplanes of liquid disordered (Ld) and Lo bilayers of DPPC/DOPC/cholesterol is 1.75 ± 0.35, in very good agreement with 1.3 ± 0.3 obtained from simulation. The results show how cellular rafts can be structurally rigid signaling platforms while remaining nearly as permeable to small molecules as the Ld phase.

We considered the issue of whether shifts in the metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area-per-lipid molecule and the membrane hydrophobic thickness were derived from measurements of the (2)H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain.

The interaction of bovine rhodopsin with poly-and monounsaturated lipids was studied by 1 H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly-and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.Rhodopsin is the light receptor responsible for dim light vision in the rod photoreceptor cells of vertebrates. A large body of research demonstrated that efficiency of the rhodopsindependent steps of the visual process is exquisitely sensitive to membrane lipid composition, in particular to the content of -3 polyunsaturates (1-3). Retinal membranes of mammals, similar to synaptosomal membranes in brain, contain up to 50 mol % of docosahexaenoic acid (DHA, 2 22:6n3), a polyunsaturated fatty acid with 22 carbon atoms and six double bonds that are evenly distributed over the length of the chain (4).In earlier reconstitution experiments with bovine rhodopsin it was established that the equilibrium concentration of Meta-II rhodopsin increased with the concentration of DHA hydrocarbon chains in the lipid matrix (5-7). Also the headgroups of phospholipids had a significant effect on Meta-II formation (5, 7) and activation of G t (8, 9). It was observed that phosphatidylethanolamines (PE) and the negatively charged phosphatidylserines (PS) increased the amount of Meta-II.The influence of PS on the equilibrium was assigned to changes of the membrane electric surface potential (7). In contrast, the sensitivity of membranes to PE content correlated with an alteration of membrane curvature elasticity (10) as proposed for other membrane proteins by Navarro et al. (11), Jensen and Schutzbach (12), Gruner (13), Lindblom and coworkers (14), and Cantor (15). Litman and co-workers (6, 16) found a correlation between mobility and orientation of the fluorescence probe DPH in lipid bilayers, summarized as a membrane free volume parameter, and Meta-II formation. Furthermore, they observed a preference of rhodopsin to locate in domains rich in di-22:6n3-PC that formed in di-16:0-PC/ di-22:6-PC/cholesterol mixtures. Mouritsen proposed a link between hydrophobic thickness of lipid bilayers and activity of membrane proteins (18). Brown and co-workers (10) suggested that Meta-II has a greater hydrophobic thickness than Meta-I, and that the lipid bilayer...