Monopolar-bipolar lipid interactions in model membrane systems

1H-NMR, dynamic light scattering and negative staining electron microscopy have been used to study the formation and physico-chemical properties of aqueous dispersions of mixtures of monopolar lipids extracted from Sulfolobus solfataricus. This microorganism is a thermophilic archaeobacterium growing optimally at about 85 degrees C and pH 3. The two hydrolytic fractions of the membrane complex lipids that have been studied are: the symmetric lipid glycerol dialkyl glycerol tetraether (GDGT) and the asymmetric lipid glycerol dialkyl nonitol tetraether (GDNT). Electron micrographs of pure and mixed GDNT and GDGT dispersions show the formation of complex structures. Only above a critical monopolar/bipolar lipid ratio, typical of the bipolar lipid, could closed structures be formed and good agreement was obtained in sizing with NMR, electron microscopy and dynamic light scattering. NMR spectra have been carried out at several temperatures from 25 degrees to 85 degrees C, to obtain information on the temperature-dependent structural, dynamic and permeability properties of the co-dispersed vesicles. The results are discussed in terms of the steric constraints and the chemico-physical interactions occurring among the different parts of the molecules and compared with previous studies performed with different physical techniques.     

ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.     

Asymmetric lipid fluidity in human erythrocyte membrane: new spin-label evidence

We have synthesized spin-labeled analogues of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine with a short beta chain (C5) bearing a doxyl group at the fourth position. When added to an erythrocyte suspension, the labels immediately incorporate in the membrane. The orientation of the spin-labels was assessed in the bilayer (i) by addition in the medium of a nonpermeant reducer (ascorbate at 5 degrees C) or (ii) by following spontaneous reduction at 37 degrees C due to the endogenous reducing agents present in the cytosol. Both techniques prove that the spin-labels are originally incorporated in the outer leaflet and redistribute differently after incubation. After a 5-h incubation at 5 degrees C, the phosphatidylcholine derivative remained in the outer layer, while the phosphatidylethanolamine and phosphatidylserine derivatives were found principally in the inner leaflet. During the incubation, a small fraction of the spin-labels is hydrolyzed, particularly the phosphatidylserine derivative, presumably by an endogenous phospholipase A2. Because the hydrolyzed spin-labeled fatty acids are rejected in the aqueous phase, the spectra of the intact membrane-bound phospholipids can be obtained by an adequate spectral subtraction. The ESR spectrum corresponding to a probe in the outer leaflet indicates a more restricted motion than that associated with probes in the inner leaflet. Additional experiments have been carried out to prove that the difference in viscosity, which is likely to be due to anisotropic cholesterol distribution, is not attributable to modification of the cell morphology.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c induced lateral phase separation in a diphosphatidylglycerol-steroid spin-label model membrane.

The extrinsic membrane protein cytochrome c binds to lipid mixtures containing negatively charged phospholipids such as diphosphatidylglycerol (DPG). In this study the effect of cytochrome c on the lipid distribution in a DPG-steroid spin-label (3-doxyl-5alpha-cholestane) model membrane system is examined. The electron spin resonance (ESR) line-shape changes indicate that cytochrome c induces lateral phase separation at room temperature. The resulting two-dimensional lipid distribution is nonrandom, consisting of clusters of phospholipids bound to cytochrome c and patches of steroid spin-label molecules. Phase separations are also observed in the three-component system: DPG, phosphatidylcholine, and 3-doxyl-5alpha-cholestane.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.     

A spin-label study of the effect of gamma radiation on erythrocyte membrane. Influence of lipid peroxidation on membrane structure.

Gamma-irradiation of bovine erythrocyte membranes (0.1-4 Mrad) resulted in a decrease in the degree of order of membrane lipids, as measured by spin-labelled fatty acid esters, at the depth of C12 but not at the depth of C5. Dose dependence of this phenomenon corresponded to dose dependence of malondialdehyde formation in the membranes. On this basis a mechanism for the effect of lipid peroxidation on the membrance structure is proposed. Membrane proteins underwent radiation-induced conformational transitions revealed by maleimide spin label which could be also connected with lipid peroxidation.     

Concentration of oxygen in lipid bilayers using a spin-label method.

The concentration of oxygen in the hydrocarbon region of lipid bilayer has been determined using a novel electron spin resonance (ESR) nitroxide-radical spin-probe method. For dimyristoylphosphatidylcholine (DMPC), the partition coefficient above the main transition temperature is approximately 3. Rapid decrease to 0.2 occurs below the pretransition temperature indicating exclusion of oxygen in the crystalline phase. The differences of molar free energy, enthalpy, and entropy of mixing between water and lipid have been determined for each phase.     

Magnetic resonance studies on membrane and model membrane systems: II. Phosphorus spectra and relaxation rates in dispersions of lecithin

We present phosphorus magnetic resonance (PhMR) spectra, relaxation rates, and chemical shifts for unsonicated and sonicated lecithins in aqueous dispersions and for egg lecithin in chloroform and methanol. Aqueous lecithin dispersions are characterized by long values for T₁ and considerably shorter values for T₂. Both of these values as well as the value of the linewidth change with sonication. Lecithin dispersions in methanol and chloroform have relaxation rates shorter than those seen for sonicated lecithin. We do not, at this time, present a detailed interpretation of these results. On an empirical level, however, since the relaxation rates are sensitive to the type of dispersion and possibly to the solvent, we are optimistic that they will be sensitive to structural changes involving the headgroup region.     

Monte Carlo studies of lipid chains and gramicidin A in a model membrane

The Monte Carlo method has been used to simulate the equilibrium properties of a planar array of 94 saturated lipid chains and one monomer of Gramicidin A. Chains are free to move laterally in the layer plane and to change conformation via gauche rotations and long axis rotations in a continuum. All non-hydrogen atoms on chains and on the Gramicidin A monomer interact via 6-12 potentials, and periodic boundary conditions are imposed. Calculated results consist of order parameter profiles for C-14 and C-16 chains. Profiles are calculated for chains which are neighbors to the Gramicidin A molecule and for chains which are not neighbors to the peptide. The main conclusion is that the average conformations of the chains neighboring the Gramicidin A monomer are very similar to those of the bulk chains.     

Electrostatic interactions at charged lipid membranes. Hydrogen bonds in lipid membrane surfaces

Hydrogen-bonded structures within lipid membrane surfaces are not disrupted by water and are of thermodynamic and therefore potential structural importance in biological systems.     

Protein-induced membrane disorder: a molecular dynamics study of melittin in a dipalmitoylphosphatidylcholine bilayer

A molecular dynamics simulation of melittin in a hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer was performed. The 19, 000-atom system included a 72-DPPC phospholipid bilayer, a 26-amino acid peptide, and more than 3000 water molecules. The N-terminus of the peptide was protonated and embedded in the membrane in a transbilayer orientation perpendicular to the surface. The simulation results show that the peptide affects the lower (intracellular) layer of the bilayer more strongly than the upper (extracellular) layer. The simulation results can be interpreted as indicating an increased level of disorder and structural deformation for lower-layer phospholipids in the immediate vicinity of the peptide. This conclusion is supported by the calculated deuterium order parameters, the observed deformation at the intracellular interface, and an increase in fractional free volume. The upper layer was less affected by the embedded peptide, except for an acquired tilt relative to the bilayer normal. The effect of melittin on the surrounding membrane is localized to its immediate vicinity, and its asymmetry with respect to the two layers may result from the fact that it is not fully transmembranal. Melittin's hydrophilic C-terminus anchors it at the extracellular interface, leaving the N-terminus "loose" in the lower layer of the membrane. In general, the simulation supports a role for local deformation and water penetration in melittin-induced lysis. As for the peptide, like other membrane-embedded polypeptides, melittin adopts a significant 25 degree tilt relative to the membrane normal. This tilt is correlated with a comparable tilt of the lipids in the upper membrane layer. The peptide itself retains an overall helical structure throughout the simulation (with the exception of the three N-terminal residues), adopting a 30 degree intrahelical bend angle.     

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.     

A new technique of depositing phospholipid bilayers on quartz surfaces: its use in membrane spin-label studies

We have developed a new improved technique termed the parallel-beam spattering (PBS) method for depositing phospholipid bilayers on quartz surfaces. This technique involves atomizing the phospholipid mixture with a stream of nitrogen gas and passing this atomized mixture through two orifices separated by a distance to achieve a parallel beam of atomized particles before deposition on the quartz plate. A static electric field can easily be applied to the quartz surface. Also a goniometer of new design has been constructed to allow precise positioning of the deposited phospholipid bilayers with reference to the magnetic field. We have utilized the PBS method to deposit phosphatidylcholine/nitroxyl labeled cholestane mixtures on quartz plates and have found that hydrated bilayers of these mixtures yield ESR spectra with essentially the same characteristics as those obtained using more conventional techniques. The distinct advantage of the new technique for depositing bilayers is that there is no spectral anomaly present which usually is present when the more conventional method of depositing bilayers is used. The spectral anomaly is apparently caused by a portion of the bilayers aligned in directions not directly parallel to the quartz surface. For precision work the spectral anomaly is unacceptable. It is not observed with the new PBS method which has yielded highly reproducible results.     

Complement-induced decrease in membrane mobility: introducing a more sensitive index of spin-label motion.

We have used spin-labeling to investigate complement-induced changes in lipid organization of antibody-sensitized sheep erythrocyte membranes. The spectrum of methyl 5-doxylstearate incorporated into the lipid component of sheep erythrocyte membranes is typical of a membrane bilayer. The membranes from complement-lysed erythrocytes have a small, but statistically significant, reduction in fluidity when compared to membranes from osmotically-lysed erythrocytes, as indicated by a small increase in T'. In theory, measurements of the widths of the outer hyperfine extrema should be more sensitive to motion than the separation of the outer hyperfine extrema (2T'). Our results indicate that the half-width at half-height of the outer hyperfine extrema show a severalfold greater percentage change than T'. The sign and magnitude of these changes are in general agreement with previous predictions. Our results imply that motional corrections to the S formalism of Hubbell, Gaffney, and McConnell are necessary because spin-label motion appears to be explicitly represented in this type of electron spin resonance spectra.     

Filipin fluorescence quenching by spin-labeled probes: studies in aqueous solution and in a membrane model system.

A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 +/- 1.0).10(3), 20 degrees C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 16-nitroxide is aggregated at concentrations higher than approximately 5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.