Minimal Effect of Lipid Charge on Membrane Miscibility Phase Behavior in Three Ternary Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.     

Miscibility of ternary mixtures of phospholipids and cholesterol in monolayers, and application to bilayer systems

We investigate miscibility transitions of two different ternary lipid mixtures, DOPC/DPPC/Chol and POPC/PSM/Chol. In vesicles, both of these mixtures of an unsaturated lipid, a saturated lipid, and cholesterol form micron-scale domains of immiscible liquid phases for only a limited range of compositions. In contrast, in monolayers, both of these mixtures produce two distinct regions of immiscible liquid phases that span all compositions studied, the alpha-region at low cholesterol and the beta-region at high cholesterol. In other words, we find only limited overlap in miscibility phase behavior of monolayers and bilayers for the lipids studied. For vesicles at 25 degrees C, the miscibility phase boundary spans portions of both the monolayer alpha-region and beta-region. Within the monolayer beta-region, domains persist to high pressures, yet within the alpha-region, miscibility phase transition pressures always fall below 15 mN/m, far below the bilayer equivalent pressure of 32 mN/m. Approximately equivalent phase behavior is observed for monolayers of DOPC/DPPC/Chol and for monolayers of POPC/PSM/Chol. As expected, pressure-area isotherms of our ternary lipid mixtures yield smaller molecular area and compressibility for monolayers containing more saturated acyl chains and cholesterol. All monolayer experiments were conducted under argon. We show that exposure of unsaturated lipids to air causes monolayer surface pressures to decrease rapidly and miscibility transition pressures to increase rapidly.     

Coupling of cholesterol-rich lipid phases in asymmetric bilayers

We showed previously that cholesterol-rich liquid-ordered domains with lipid compositions typically found in the outer leaflet of plasma membranes induce liquid-ordered domains in adjacent regions of asymmetric lipid bilayers with apposed leaflets composed of typical inner leaflet lipid mixtures [Kiessling, V., Crane, J. M., and Tamm, L. K. (2006) Biophys. J. 91, 3313-26]. To further examine the nature of transbilayer couplings in asymmetric cholesterol-rich lipid bilayers, the effects on the lipid phase behavior in asymmetric bilayers of different lipid compositions were investigated. We established systems containing several combinations of natural extracted and synthetic lipids that exhibited coexisting liquid-ordered (lo) and liquid-disordered (ld) domains in a supported bilayer format. We find that lo phase domains are induced in all quaternary inner leaflet combinations composed of PCs, PEs, PSs, and cholesterol. Ternary mixtures of PCs/PEs/Chol, PCs/PSs/Chol also exhibit lo phases adjacent to outer leaflet lo phases. However, with the exception of brain PC extracts, binary PC/Chol mixtures are not induced to form lo phases by adjacent outer leaflet lo phases. Higher melting lipid ad-mixtures of PEs and PSs are needed for lo phase induction in the inner leaflet. It appears that the phase behavior of the inner leaflet mixtures is dominated by the intrinsic chain melting temperatures of the lipid components, rather than by their specific headgroup classes. In addition, similar studies with synthetic, completely saturated lipids and cholesterol show that lipid oxidation is not a factor in the observed phase behavior.     

Vesicles with charged domains

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.     

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.     

Synthesis and characterization of betaine-like diacyl lipids: zwitterionic lipids with the cationic amine at the bilayer interface

We synthesized and characterized a series of zwitterionic, acetate-terminated, quaternized amine diacyl lipids (AQ). These lipids have an inverted headgroup orientation as compared to naturally occurring phosphatidylcholine (PC) lipids; the cationic group is anchored at the membrane interface, while the anionic group extends into the aqueous phase. AQ lipids preferentially interact with highly polarizable anions (ClO(4)(-)) over less polarizable ions (Cl(-)), in accord with the Hofmeister series, as measured by the change in zeta potential of AQ liposomes. Conversely, AQ lipids have a weaker association with calcium than do PC lipids. The transition temperatures (Tm) of the AQ lipids are similar to the Tm observed with phosphatidylethanolamine (PE) lipids of the same chain length. AQ lipids form large lipid sheets after heating and sonication; however, in the presence of cholesterol (Chol), these lipids form stable liposomes that encapsulate carboxyfluorescein. The AQ:Chol liposomes retain their contents in the presence of serum at 37°C, and when injected intravenously into mice, their organ biodistribution is similar to that observed with PC:Chol liposomes. AQ lipids demonstrate that modulating the headgroup charge orientation significantly alters the biophysical properties of liposomes. For the drug carrier field, these new materials provide a non-phosphate containing zwitterlipid for the production of lipid vesicles.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the role of anionic lipids in charged protein interactions with membranes

We investigate the role of anionic lipids in the binding to, and subsequent movement of charged protein groups in lipid membranes, to help understand the role of membrane composition in all membrane-active protein sequences. We demonstrate a small effect of phosphatidylglycerol (PG) lipids on the ability of an arginine (Arg) side chain to bind to, and cross a lipid membrane, despite possessing a neutralizing charge. We observe similar membrane deformations in lipid bilayers composed of phosphatidylcholine (PC) and PC/PG mixtures, with comparable numbers of water and lipid head groups pulled into the bilayer hydrocarbon core, and prohibitively large ~20 kcal/mol barriers for Arg transfer across each bilayer, dropping by just 2-3 kcal/mol due to the binding of PG lipids. We explore the causes of this small effect of introducing PG lipids and offer an explanation in terms of the limited membrane interaction for the choline groups of PC lipids bound to the translocating ion. Our calculations reveal a surprising lack of preference for Arg binding to PG lipids themselves, but a small increase in interfacial binding affinity for lipid bilayers containing PG lipids. These results help to explain the nature of competitive lipid binding to charged protein sequences, with implications for a wide range of membrane binding domains and cell perturbing peptides.     

Nonequilibrium behavior in supported lipid membranes containing cholesterol

We investigate lateral organization of lipid domains in vesicles versus supported membranes and monolayers. The lipid mixtures used are predominantly DOPC/DPPC/Chol and DOPC/BSM/Chol, which have been previously shown to produce coexisting liquid phases in vesicles and monolayers. In a monolayer at an air-water interface, these lipids have miscibility transition pressures of approximately 12-15 mN/m, which can rise to 32 mN/m if the monolayer is exposed to air. Lipid monolayers can be transferred by Langmuir-Sch?fer deposition onto either silanized glass or existing Langmuir-Blodgett supported monolayers. Micron-scale domains are present in the transferred lipids only if they were present in the original monolayer before deposition. This result is valid for transfers at 32 mN/m and also at lower pressures. Domains transferred to glass supports differ from liquid domains in vesicles because they are static, do not align in registration across leaflets, and do not reappear after temperature is cycled. Similar static domains are found for vesicles ruptured onto glass surfaces. Although supported membranes on glass capture some aspects of vesicles in equilibrium (e.g., gel-liquid transition temperatures and diffusion rates of individual lipids), the collective behavior of lipids in large liquid domains is poorly reproduced.     

Resonance energy transfer study of lysozyme-lipid interactions

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.     

Thermodynamics of lipid interactions in complex bilayers

The mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (ω_AB) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.     

Cholesterol as a factor regulating the influence of natural (PAF and lysoPAF) vs synthetic (ED) ether lipids on model lipid membranes

In this work we have performed a comparative study on the effect of antineoplastic ether lipid-edelfosine (ED), its natural analogs — Platelet Activating Factor (PAF) and its precursor (lyso-PAF), both lacking anticancer properties, on cholesterol/phosphatidylcholine (Chol/PC) monolayers, serving as model membranes. Since all the above ether lipids are membrane active, it can be expected that their effect on membranes may differentiate their biological activity. Our investigations were aimed at studying potential relationship of the effect of ED, PAF and lyso-PAF on model membranes, differing in condensation. We have modified molecular packing of Chol/PC model systems either by increasing the level of sterol in the system or changing the structure of PC, while keeping the same sterol content. Additionally, we have performed a detailed comparison of the miscibility of ED, PAF and lyso-PAF with various membrane lipids. The collected data evidenced that all the investigated ether lipids influence Chol/PC films in the same way; however, in a different magnitude. Moreover, the interactions of ED, PAF and lyso-PAF with model membranes were the strongest at the highest level of sterol in the system. A thorough analysis of the obtained results has proved that the effect of the investigated ether lipids on membranes is not dependent on the condensation of the system, but it is strongly determined by the concentration of cholesterol. Since ED was found to interact with model membranes stronger than PAF and lyso-PAF, we have suggested that this fact may contribute to differences in cytotoxicity of these compounds.     

Sterol structure determines miscibility versus melting transitions in lipid vesicles

Lipid bilayer membranes composed of DOPC, DPPC, and a series of sterols demix into coexisting liquid phases below a miscibility transition temperature. We use fluorescence microscopy to directly observe phase transitions in vesicles of 1:1:1 DOPC/DPPC/sterol within giant unilamellar vesicles. We show that vesicles containing the "promoter" sterols cholesterol, ergosterol, 25-hydroxycholesterol, epicholesterol, or dihydrocholesterol demix into coexisting liquid phases as temperature is lowered through the miscibility transition. In contrast, vesicles containing the "inhibitor" sterols androstenolone, coprostanol, cholestenone, or cholestane form coexisting gel (solid) and liquid phases. Vesicles containing lanosterol, a sterol found in the cholesterol and ergosterol synthesis pathways, do not exhibit coexisting phases over a wide range of temperatures and compositions. Although more detailed phase diagrams and precise distinctions between gel and liquid phases are required to fully define the phase behavior of these sterols in vesicles, we find that our classifications of promoter and inhibitor sterols are consistent with previous designations based on fluorescence quenching and detergent resistance. We find no trend in the liquid-liquid or gel-liquid transition temperatures of membranes with promoter or inhibitor sterols and measure the surface fraction of coexisting phases. We find that the vesicle phase behavior is related to the structure of the sterols. Promoter sterols have flat, fused rings, a hydroxyl headgroup, an alkyl tail, and a small molecular area, which are all attributes of "membrane active" sterols.     

Local Partition Coefficients Govern Solute Permeability of Cholesterol-Containing Membranes

The permeability of lipid membranes for metabolic molecules or drugs is routinely estimated from the solute’s oil/water partition coefficient. However, the molecular determinants that modulate the permeability in different lipid compositions have remained unclear. Here, we combine scanning electrochemical microscopy and molecular-dynamics simulations to study the effect of cholesterol on membrane permeability, because cholesterol is abundant in all animal membranes. The permeability of membranes from natural lipid mixtures to both hydrophilic and hydrophobic solutes monotonously decreases with cholesterol concentration [Chol]. The same is true for hydrophilic solutes and planar bilayers composed of dioleoyl-phosphatidylcholine or dioleoyl-phosphatidyl-ethanolamine. However, these synthetic lipids give rise to a bell-shaped dependence of membrane permeability on [Chol] for very hydrophobic solutes. The simulations indicate that cholesterol does not affect the diffusion constant inside the membrane. Instead, local partition coefficients at the lipid headgroups and at the lipid tails are modulated oppositely by cholesterol, explaining the experimental findings. Structurally, these modulations are induced by looser packing at the lipid headgroups and tighter packing at the tails upon the addition of cholesterol.     

Local Partition Coefficients Govern Solute Permeability of Cholesterol-Containing Membranes

The permeability of lipid membranes for metabolic molecules or drugs is routinely estimated from the solute’s oil/water partition coefficient. However, the molecular determinants that modulate the permeability in different lipid compositions have remained unclear. Here, we combine scanning electrochemical microscopy and molecular-dynamics simulations to study the effect of cholesterol on membrane permeability, because cholesterol is abundant in all animal membranes. The permeability of membranes from natural lipid mixtures to both hydrophilic and hydrophobic solutes monotonously decreases with cholesterol concentration [Chol]. The same is true for hydrophilic solutes and planar bilayers composed of dioleoyl-phosphatidylcholine or dioleoyl-phosphatidyl-ethanolamine. However, these synthetic lipids give rise to a bell-shaped dependence of membrane permeability on [Chol] for very hydrophobic solutes. The simulations indicate that cholesterol does not affect the diffusion constant inside the membrane. Instead, local partition coefficients at the lipid headgroups and at the lipid tails are modulated oppositely by cholesterol, explaining the experimental findings. Structurally, these modulations are induced by looser packing at the lipid headgroups and tighter packing at the tails upon the addition of cholesterol.     

Increased rates of lipid exchange between Mycoplasma capricolum membranes and vesicles in relation to the propensity of forming nonbilayer lipid structures

We have studied the effects of modification of the endogenous phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) content of the plasma membrane of Mycoplasma capricolum on the kinetics of spontaneous [14C]cholesterol and 14C-labeled phospholipid exchange between M. capricolum membranes and lipid vesicles. The PG/DPG molar ratio of M. capricolum membranes changed when cells were grown in media supplemented with 0.5 mM CaCl2 and/or egg phosphatidylcholine (PC) (10-20 micrograms/ml), increasing from 3.9 to 6.3 on supplementation with Ca2+; this ratio decreased to 1.1 in media supplemented with PC and to 1.8 in media containing both PC and Ca2+. The ratio of palmitate to oleate in both PG and DPG decreased when cells were grown with PC or with PC and Ca2+. Bilayer disruptions were seen in freeze-fracture electron micrographs of trypsin-treated M. capricolum membranes from cells grown with both Ca2+ and PC, and numerous lipidic particles and other bilayer disruptions were observed in trypsin-treated M. capricolum membranes and their lipid extracts. The rates of spontaneous exchange of 14C-labeled cholesterol and PC from membranes isolated from cells grown with PC and Ca2+ to acceptor lipid vesicles were exchanged by approximately 30%, and the rate of the rapidly exchangeable cholesterol pool in intact cells was enhanced by 64%. The enhancements in cholesterol and PC exchange rates are considered to result from structural defects expected in the M. capricolum membranes obtained from cells grown with Ca2+ supplementation. Our findings parallel previous examples of functional modifications of membranes induced by bilayer instability arising from a pretransitional state leading to the onset of a nonlamellar phase.     

Resonance energy transfer study of lysozyme-lipid interactions

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.     

Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II

Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or “rafts” in PC membranes, ³¹P and ²H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T₂ relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.     

Lipid miscibility and size increase of vesicles composed of two phosphatidylcholines

The size increase of small unilamellar vesicles composed of binary mixtures either of saturated fatty acid phosphatidylcholines with different chain lengths or of saturated and unsaturated phosphatidylcholines was found to depend on the miscibility properties of the lipid components. No size increase was detected in vesicles formed by two miscible phosphatidylcholines. In vesicles composed of two lipids which are partially immiscible in the gel state, a size increase was observed at temperatures which mainly overlapped the range of temperatures of the lipid phase transition. The rate of size increase of vesicles composed of two lipids which are immiscible in the gel state was faster than that of vesicles composed of two partially immiscible phosphatidylcholines, and the process occurred not only at the temperature ranges of the lipid phase transition, but also when both lipids were in the gel state. The vesicle size increase process occurred without the mixing of the internal content of the vesicles. A model is proposed in which the presence of 'fractures' between membrane regions of different fluidity and/or lipid composition controls the rate of this process.