Lipid Demixing Research Articles

Lateral Distribution of Cholesterol in Dioleoylphosphatidylcholine Lipid Bilayers: Cholesterol-Phospholipid Interactions at High Cholesterol Limit

Lateral organization of cholesterol in dioleoyl-phosphatidylcholine (DOPC) lipid bilayers at high cholesterol concentration (>45 mol%) was investigated using steady-state fluorescence anisotropy and fluorescent resonance energy transfer techniques. The recently devised Low Temperature Trap method was used to prepare compositionally uniform cholesterol/DOPC liposomes to avoid the problem of lipid demixing. The fluorescence anisotropy of diphenylhexatrience chain-labeled phosphatidylcholine (DPH-PC) in these liposomes exhibited local maxima at cholesterol mol fractions of 0.50 and 0.57, and a sharp drop at 0.67. For the liposomes labeled with both dehydroergosterol and DPH-PC, the fluorescent resonance energy transfer efficiency from dehydroergosterol to DPH-PC displayed a steep jump at cholesterol mol fraction of 0.5, and dips at 0.57 and 0.68. These results indicate the presence of highly ordered cholesterol regular distribution domains at those observed critical compositions. The observed critical mol fraction at 0.67 agreed favorably with the solubility limit of cholesterol in DOPC bilayers as independently measured by light scattering and optical microscopy. The regular distribution at 0.57 was previously predicted from a Monte Carlo simulation based on the Umbrella model. The results strongly support the hypothesis that the primary requirement for cholesterol-phospholipid mixing is that the polar phospholipid headgroups need to cover the nonpolar body of cholesterol to avoid the exposure of cholesterol to water.

The Effect of Lipid Demixing on the Electrostatic Interaction of Planar Membranes across a Salt Solution

We study the effect of lipid demixing on the electrostatic interaction of two oppositely-charged membranes in solution, modeled here as an incompressible two-dimensional fluid mixture of neutral and charged mobile lipids. We calculate, within linear and nonlinear Poisson-Boltzmann theory, the membrane separation at which the net electrostatic force between the membranes vanishes, for a variety of different system parameters. According to Parsegian and Gingell, contact between oppositely-charged surfaces in an electrolyte is possible only if the two surfaces have exactly the same charge density ( σ 1 = − σ 2). If this condition is not fulfilled, the surfaces can repel each other, even though they are oppositely charged. In our model of a membrane, the lipidic charge distribution on the membrane surface is not homogeneous and frozen, but the lipids are allowed to freely move within the plane of the membrane. We show that lipid demixing allows contact between membranes even if there is a certain charge mismatch, | σ 1| ≠ | σ 2|, and that in certain limiting cases, contact is always possible, regardless of the value of σ 1/ σ 2 (if σ 1/ σ 2 < 0). We furthermore find that of the two interacting membranes, only one membrane shows a major rearrangement of lipids, whereas the other remains in exactly the same state it has in isolation and that, at zero-disjoining pressure, the electrostatic mean-field potential between the membranes follows a Gouy-Chapman potential from the more strongly charged membrane up to the point of the other, more weakly charged membrane.

The Sensitivity of Lipid Domains to Small Perturbations Demonstrated by the Effect of Triton

The hypothesis of lipid rafts describes functional domains in biological membranes. It is often assumed that rafts form by spontaneous de-mixing of certain lipids and that they can be isolated as detergent-resistant membrane particles (DRMs) using the detergent Triton X-100 (TX). Here, we present a model that describes the process of domain formation in membranes in the presence and in the absence of TX. We measure the interactions between TX and an equimolar mixture of sphingomyelin (SM), cholesterol (Cho), and 1-palmitoyl-2-oleoyl-3-sn-glycero-phosphatidylcholine (POPC) (1:1:1, mol) by means of isothermal titration calorimetry. Comparison with pure POPC membranes reveals a very unfavorable interaction between TX and SM/Cho, which causes a substantial tendency to segregate these molecules into separate, DRM-like (SM-rich) and fluid (TX-rich), domains. If rafts are indeed formed by spontaneous de-mixing of PC and SM/Cho, they must be very sensitive, and perturbations caused by techniques used to study rafts could lead to misleading results. If, however, rafts are much more stable than PC–SM–Cho domains, there must be an unknown raft stabilizer. Subtle changes of such a promoter could serve to modulate raft function.

Adsorption of charged macromolecules on mixed fluid membranes

The adsorption of charge rigid macromolecules, such as proteins from solution, on mixed (charged and neutral) lipid membranes is affected by several important factors. First, the mobile lipids in the membrane may rearrange, and demix locally to match the charge density of the apposed macromolecule, thus lowering the adsorption free energy. On the other hand, the (electrostatic) interaction between adsorbed macromolecules tends to lower the saturation coverage of the membrane. Additional factors, such as non-ideal lipid demixing or an elastic membrane response, enhanced by the presence of the charged macromolecules, may be at the base of the experimentally observed formation of high density protein domains and lateral macro-phase separation in lipid membranes.

Effects of DNA Adsorption on the Phase Cycling of a Supported Mixed Phospholipid Bilayer

In this paper we demonstrate a powerful method to visualize directly DNA on a bilayer during a lipid phase transition. Using temperature-dependent atomic force microscopy (AFM), we observed that DNA appears to induce lipid demixing during phase cycling.

Nonequilibrium Phenomena in the Phase Separation of a Two-Component Lipid Bilayer

Lipid bilayers composed of two phospholipids with significant acyl-chain mismatch behave as nonideal mixtures. Although many of these systems are well characterized from the equilibrium point of view, studies concerning their nonequilibrium dynamics are still rare. The kinetics of lipid demixing (phase separation) was studied in model membranes (large unilamellar vesicles of 1:1 dilauroylphosphatidylcholine (C 12 acyl chain) and distearoylphosphatidylcholine (C 18 acyl chain)). For this purpose, photophysical techniques (fluorescence intensity, anisotropy, and fluorescence resonance energy transfer) were applied using suitable probes (gel phase probe trans-parinaric acid and fluid phase probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanolamine). The nonequilibrium situation was induced by a sudden thermal quench from a one-fluid phase equilibrium situation (higher temperature) to the gel/fluid coexistence range (lower temperature). We verified that the attainment of equilibrium is a very slow process (occurs in a time scale of hours), leading to large domains at infinite time. The nonequilibrium structure stabilization is due essentially to temporarily rigidified C 12 chains in the interface between gel/fluid domains, which decrease the interfacial tension by acting as surfactants. The relaxation process becomes faster with the increase of the temperature drop. In addition, heterogeneity is already present in the supposed homogeneous fluid mixture at the higher temperature.

Lipid Demixing and Protein-Protein Interactions in the Adsorption of Charged Proteins on Mixed Membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is “saturated” by adsorbed proteins), the membrane charges are “overcompensated” by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.

Formation of domains of cationic or anionic lipids in binary lipid mixtures increases the electrostatic coupling strength of water-soluble proteins to supported bilayers.

The electrostatic binding strength of water-soluble proteins having either an excess positive (cytochrome c) or negative (beta-lactoglobulin) electric charge to oppositely charged supported planar bilayers (SPBs) was studied as a function of the bilayer phase state (fluid or gel phase) by IR-ATR spectroscopy. The bilayer consisted of mixtures of zwitterionic DEPC with either cationic DMTAP or anionic DMPG. We observed drastic differences in the binding strength of both proteins for the two bilayer phase states, with the gel phase exhibiting a higher binding strength than the fluid phase, under conditions where the two lipid components had different hydrophobic chain lengths resulting in a nonideal mixing behavior. In addition, for beta-lactoglobulin we observed a strong binding to a gel phase SPB comprising DEPC/DMTAP, while raising the temperature of the SPB above the chain melting transition temperature of the mixture resulted in a complete unbinding of the protein. In contrast, for DMPC/DMTAP having the same cationic charge content but no hydrophobic chain mismatch, no phase-dependent coupling strength of the protein to the SPB was observed. Our results suggest that the formation of charge-enriched domains by partial demixing of the bilayer lipids at the transition to the gel state is crucial for modulation of the protein binding strength to the SPB, while the intrinsic charge of the solid support surface is of minor importance.

Binding of Peripheral Proteins to Mixed Lipid Membranes: Effect of Lipid Demixing upon Binding

Binding isotherms have been determined for the association of horse heart cytochrome c with dioleoyl phosphatidylglycerol (DOPG)/dioleoyl phosphatidylcholine (DOPC) bilayer membranes over a range of lipid compositions and ionic strengths. In the absence of protein, the DOPG and DOPC lipids mix nearly ideally. The binding isotherms have been analyzed using double layer theory to account for the electrostatics, either the Van der Waals or scaled particle theory equation of state to describe the protein surface distribution, and a statistical thermodynamic formulation consistent with the mass-action law to describe the lipid distribution. Basic parameters governing the electrostatics and intrinsic binding are established from the binding to membranes composed of anionic lipid (DOPG) alone. Both the Van der Waals and scaled particle equations of state can describe the effects of protein distribution on the DOPG binding isotherms equally well, but with different values of the maximum binding stoichiometry (13 lipids/protein for Van der Waals and 8 lipids/protein for scaled particle theory). With these parameters set, it is then possible to derive the association constant, K r, of DOPG relative to DOPC for surface association with bound cytochrome c by using the binding isotherms obtained with the mixed lipid membranes. A value of K r (DOPG:DOPC) = 3.3–4.8, depending on the lipid stoichiometry, is determined that consistently describes the binding at different lipid compositions and different ionic strengths. Using the value of K r obtained it is possible to derive the average in-plane lipid distribution and the enhancement in protein binding induced by lipid redistribution using the statistical thermodynamic theory.

Amphipathic peptide affects the lateral domain organization of lipid bilayers

Using lipid-specific fluorescent probes, we studied the effects of amphipathic helical, membrane active peptides of the A- and L-type on membrane domain organization. In zwitterionic binary systems composed of mixtures of phosphatidylcholine and phosphatidylethanolamine, both types of peptides associated with the fluid phase. While binding with high affinity to fluid membranes, peptides were unable to penetrate into the lipid membrane in the gel state. If trapped kinetically by cooling from the fluid phase, peptides dissociated from the gel membrane on the time scale of several hours. While the geometrical shape of the α-helical peptides determines their interactions with membranes with non-bilayer phase propensity, the shape complementarity mechanism by itself is unable to induce lateral phase separation in a fluid membrane. Charge–charge interactions are capable of inducing lateral domain formation in fluid membranes. Both peptides had affinity for anionic lipids which resulted in about 30% enrichment of acidic lipids within several nanometers of the peptide's tryptophan, but there was no long-range order in peptide-induced lipid demixing. Peptide insertion in fluid acidic membranes was accompanied by only a small increase in bilayer surface and a decrease in polarity in the membrane core. Peptide–lipid charge–charge interactions were also capable of modulating existing domain composition in the course of the main phase transition in mixtures of anionic phosphatidylglycerol with zwitterionic phosphatidylcholine.

Macroripple-structures induced by different branched-chain phosphatidylcholines in bilayers of dipalmitoylphosphatidylcholine

Branched-chain phosphatidylcholines with four chains of the length C16, C18 and C20 have been synthesized by dibranching positions C2 or C4 of the acyl chains. Macroripples have been observed by freeze-fracture electron microscopy at concentrations of 5–30 mol% branched-chain phospholipids in DPPC. Macroripples differ from pretransition ripples in structure and dimension but also in their behaviour during incubation at low temperatures for a longer time. Macroripples may survive incubation at 4°C for 14 weeks. Smaller macroripples with initial periodicity of 30–50 nm may widen to twice the primary spacing during this incubation. Macroripples are commonly accompanied by parallel stripes. The stripes, which are most prominent at the crests of the macroripples, are seen on the bilayer fracture-face but not at the bilayer surface revealed by etching. Parallel stripes can also exist without tilting of the bilayer into macroripples. We suggest that phase separation or at least partial demixing of lipids may be a prerequisite for formation of macroripples. The macroripples have a tendency for a second tilting perpendicular to the main tilting. This effect was characteristic for the C2-branched phosphatidylcholines with C18- and C20-chains. A dramatic change from normal straight macroripples into those with second tilt was induced by high-pressure freezing of multilamellar vesicles with C4-branched phosphatidylcholines. The appearance of macroripples was more dependent on chain length than on the location of branching. Branched C16-chains having the same chain length as DPPC were most effective in inducing macroripples. An elongation of the chains by two C-atoms reduced macroripples more than an elongation by four C-atoms. Both types of elongation resulted in a restriction because macroripples are present only in parts of the lamellae. A speciality of the elongated branched-chain phosphatidylcholines is the predominance of macroripples with large periodicities (100 nm and more).

Macro-ripple phase formation in bilayers composed of galactosylceramide and phosphatidylcholine

As determined by freeze fracture electron microscopy, increasing levels of bovine brain galactosylceramide (GalCer) altered the surface structure of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers by inducing a striking "macro-ripple" phase in the larger, multilamellar lipid vesicles at GalCer mole fractions between 0.4 and 0.8. The term "macro-ripple" phase was used to distinguish it from the P beta' ripple phase observed in saturated, symmetric-chain length phosphatidylcholines. Whereas the P beta' ripple phase displays two types of corrugations, one with a wavelength of 12-15 nm and the other with a wavelength of 25-35 nm, the macro-ripple phase occurring in GalCer/POPC dispersions was of one type with a wavelength of 100-110 nm. Also, in contrast to the extended linear arrays of adjacent ripples observed in the P beta' ripple phase, the macro-ripple phase of GalCer/POPC dispersions was interrupted frequently by packing defects resulting from double dislocations and various disclinations and, thus, appeared to be continuously twisting and turning. Control experiments verified that the macro-ripple phase was not an artifact of incomplete lipid mixing or demixing during preparation. Three different methods of lipid mixing were compared: a spray method of rapid solvent evaporation, a sublimation method of solvent removal, and solvent removal using a rotary evaporation apparatus. Control experiments also revealed that the macro-ripple phase was observed regardless of whether lipid specimens were prepared by either ultra-rapid or manual plunge freezing methods as well as either in the presence or absence of the cryo-protectant glycerol. The macro-ripple phase was always observed in mixtures that were fully annealed by incubation above the main thermal transition of both POPC and bovine brain GalCer before rapid freezing. If the GalCer mixed with POPC contained only nonhydroxy acyl chains or only 2-hydroxy acyl chains, then the occurrence of macro-ripple phase decreased dramatically.

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on mulitlipid systems

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.

Synthesis and characterization of new fluorescent glycolipid probes. Molecular organisation of glycosphingolipids in mixed-composition lipid bilayers

The synthesis and properties of new fluorescent analogs of glycosphingolipids (galactosylcerebrosides, gangliosides G M1, G M3, G D1a and G D3 bearing a 9-anthrylvinyl or 3-perylenoyl residue in the acyl chain are described. The synthesis includes steps: (i) hydrolytic removal of the fatty acyl from the corresponding natural compound; (ii) reacylation of the sphingosine amino group with trans-12-(9-anthryl)-11-dodecenoic or 9-(3-perylenoyl)nonanoic acid; (iii) reacetylation of the neuraminic amino group (for gangliosides). Studies of the behavior of the fluorescent glycolipids in model membranes (multilayer liposomes and sonicated vesicles) revealed that the probes are largely lipid-specific, i.e. they behave in many aspects similar to their corresponding natural counterparts. Fluorescence anisotropy measurements and studies of fluorescence energy transfer from anthryvinyl- to perylenoyl-labeled probes suggest that in mixed bilayer systems containing approx. 10 mol% glycolipid, galactosylceramide is partly segregated from phospholipids even above the temperature of gel/liquid crystal transition of the phospholipid matrix. By contrast, no indication of lipid demixing was found for phospholipid/ganglioside systems containing up to 15 mol% glycolipid.