Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s(o)) and liquid-disordered (l(d)) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l(o)) and l(d) phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s(o)-l(d) and l(o)-l(d) phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Small-angle neutron scattering studies of the effects of amphotericin B on phospholipid and phospholipid-sterol membrane structure

Small-angle neutron scattering (SANS) studies have been performed to study the structural changes induced in the membranes of vesicles prepared (by thin film evaporation) from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations and different aggregation states of the anti-fungal drug, amphotericin B (AmB). In the majority of the experiments reported, the lipid vesicles were prepared with the drug added directly to the lipid dispersions dissolved in solvents favouring either AmB monomers or aggregates, and the vesicles then sonicated to a mean size of ~100 nm. Experiments were also performed, however, in which micellar dispersions of the drug were added to pre-formed lipid and lipid-sterol vesicles. The vesicles were prepared using the phospholipid palmitoyloleoylphosphatidylcholine (POPC), or mixtures of this lipid with either 30 mol% cholesterol or 30 mol% ergosterol. Analyses of the SANS data show that irrespective of the AmB concentration or aggregation state, there is an increase in the membrane thickness of both the pure POPC and the mixed POPC-sterol vesicles-in all cases amounting to ~4 ?. The structural changes induced by the drug's insertion into the model fungal cell membranes (as mimicked by POPC-ergosterol vesicles) are thus the same as those resulting from its insertion into the model mammalian cell membranes (as mimicked by POPC-cholesterol vesicles). It is concluded that the specificity of AmB for fungal versus human cells does not arise because of (static) structural differences between lipid-cholesterol-AmB and lipid-ergosterol-AmB membranes, but more likely results from differences in the kinetics of their transmembrane pore formation and/or because of enthalpic differences between the two types of sterol-AmB complexes.     

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld)?+?liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld?+?Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Interaction of high density lipoprotein particles with membranes containing cholesterol

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78A diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquid-ordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.     

A new method for measuring edge tensions and stability of lipid bilayers: effect of membrane composition

We report a novel and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (eggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of DOPC with cholesterol and dioleoylphosphatidylethanolamine. Our data confirm previous results for eggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of dioleoylphosphatidylethanolamine is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. It is presumed that interlipid hydrogen bonding is the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on bilayers made of stearoyloleoylphosphatidylcholine, suggesting that cholesterol influences the membrane mechanical stability in a lipid-specific manner.     

Quantitation of cholesterol incorporation into extruded lipid bilayers

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33-75 mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

Dependence on phospholipid composition of the fraction of cholesterol undergoing spontaneous exchange between small unilamellar vesicles

Spontaneous cholesterol exchange between small unilamellar vesicles comprised of different phospholipids and their binary mixtures has been studied in order to understand the factors involved in the establishment and maintenance of intracellular cholesterol distributions. Exchange was performed from neutral donor vesicles containing different cholesterol concentrations, traces of [3H]cholesterol, and [14C]cholesteryl oleate as a nonexchangeable marker. The acceptor vesicles, in 10-fold excess, had the same composition, but 15 mol % phosphatidylglycerol was included to permit chromatographic separation. Data were best fitted by a single exponential and a base value. In donor vesicles containing only one phospholipid, the kinetic rate constants agreed with data reported previously; however, the base values were larger than the expected equilibrium value of 9.09%. The size of this nonexchangeable pool and the exchange rate were found to depend on the type of phospholipid. In binary phospholipid donor systems, well above the transition temperatures of the lipid components, the exchange parameters were preferentially closer to those of one component according to the order POPC greater than DMPC greater than DPPC greater than bovine brain SPM.     

The effect of cholesterol on the solubilization of phosphatidylcholine bilayers by the non-ionic surfactant Triton X-100

Most of the studies on the solubilization of model membranes by Triton X-100 (TR) involve one lipid. The aim of the present study was to evaluate the effect of the addition of cholesterol on the solubilization of bilayers made of palmitoyloleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC). Detailed investigation of the kinetics of solubilization of the cholesterol-containing bilayers by TR at different temperatures reveals that: (i) At 4 degrees C, solubilization of both systems is relatively slow. Hence, in order to prevent misleading conclusions from turbidity measurements it is important to monitor the solubilization after steady-state values of optical density (OD) are reached. (ii) Studies of the temperature-induced changes of the aggregates present in mixtures of TR, POPC and cholesterol indicate that the state of aggregation at all temperatures (including 4 degrees C) represents equilibrium. By contrast, for DPPC/cholesterol/TR mixtures "kinetic traps" may occur not only at 4 degrees C but at higher temperatures as well (e.g. 37 degrees C). (iii) The presence of cholesterol in POPC bilayers makes the bilayers more resistant to solubilization at low temperatures, especially at 4 degrees C. As a consequence, the temperature dependence of the TR concentration required for complete solubilization (Dt(sol)) is no longer a monotonically increasing function (as for POPC bilayers) but a bell-shaped function, with a minimum at about 25 degrees C. Inclusion of cholesterol in DPPC bilayers makes the bilayers more resistant to solubilization at all temperatures except 4 degrees C. In this system, we observe a bell-shaped dependence of Dt(sol) on temperature, with a minimum at 37 degrees C. (iv) Both the rate of vesicle size growth and the rate of the solubilization of POPC vesicles are not affected by the inclusion of cholesterol in the bilayers. Similarly, cholesterol did not affect significantly the rate of size growth of DPPC bilayers at all temperatures, but reduced the rate of solubilization at 4 degrees C.     

Docosahexaenoic acid promotes micron scale liquid-ordered domains. A comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures

The understanding of the functional role of the lipid diversity in biological membranes is a major challenge. Lipid models have been developed to address this issue by using lipid mixtures generating liquid-ordered (L_o)/liquid-disordered (L_d) immiscibility. The present study examined mixtures comprising Egg sphingomyelin (SM), cholesterol (chol) and phosphatidylcholine (PC) either containing docosahexaenoic (PDPC) or oleic acid (POPC). The mixtures were examined in terms of their capability to induce phase separation at the micron- and nano-scales. Fluorescence microscopy, electron spin resonance (ESR), X-ray diffraction (XRD) and calorimetry methods were used to analyze the lateral organization of the mixtures. Fluorescence microscopy of giant vesicles could show that the temperature of the micron-scale L_o/L_d miscibility is higher for PDPC than for POPC ternary mixtures. At 37 °C, no micron-scale L_o/L_d phase separation could be identified in the POPC containing mixtures while it was evident for PDPC. In contrast, a phase separation was distinguished for both PC mixtures by ESR and XRD, indicative that PDPC and POPC mixtures differed in micron vs nano domain organization. Compared to POPC, the higher line tension of the L_o domains observed in PDPC mixtures is assumed to result from the higher difference in L_o/L_d order parameter rather than hydrophobic mismatch.     

Thermodynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state studied by the orientational order parameter.

It is shown that good estimates of the activity of cholesterol in phosphatidylcholine-cholesterol mixed model membranes are obtained by examining the orientational order parameter S of cholestane spin probe (CSL) that is obtained from electron spin resonance by spectral simulation. By introducing thermodynamic stability conditions of liquid mixtures, the variation of activity (or S) as a function of cholesterol mole fraction is utilized to predict the concentration at which the phase separation occurs. These results for DMPC and cholesterol binary mixtures agree very well with those of Tempo-partitioning experiments. The comparison of activity coefficients and the phase boundary in DMPC/cholesterol mixtures with those of POPC/cholesterol mixtures suggests that acyl chain unsaturation leads to poorer mixing of cholesterol in phosphatidylcholine model membranes at higher temperatures (i.e., greater than 35 degrees C). In ternary solutions of DMPC, POPC, and cholesterol, it is found that cholesterol shows less deviation from ideality than in either of the two binary mixtures, and this implies that the phase separation occurs at higher cholesterol concentration than in either of the two binary mixtures. The present analysis suggests that there may not be a critical point in DMPC/cholesterol mixtures, even though phase separation does occur.     

Dynamics of phosphatidylcholine-cholesterol mixed model membranes in the liquid crystalline state.

The effects of cholesterol on the dynamics of cholestane spin probe (CSL) in various phosphatidylcholine-cholesterol mixed model membranes are examined. The lateral diffusion, D of CSL in DMPC/POPC/cholesterol ternary mixtures, is measured utilizing an improved dynamic imaging electron spin resonance method. It shows a factor of two decrease at 10 mol % and 25 degrees C, whereas it shows only a 40% decrease at 50 mol % and 50 degrees C. A comparison with results in POPC/cholesterol mixtures, which show a stronger effect of cholesterol on D, indicates that acyl chain unsaturation leads to stronger self association of cholesterol in PC model membranes. An S2CSL dependence of the activation energy for D, has been confirmed for the DMPC/POPC/cholesterol mixtures. Here SCSL is the order parameter for CSL. A similar correlation of R perpendicular, the perpendicular component of the rotational diffusion coefficient, with SCSL, which is true for all three mixtures (DMPC/cholesterol, POPC/cholesterol, and DMPC/POPC/cholesterol) we have studied, is also found. These are associated with the effects of enhanced local ordering on the free volume needed for translation and reorientation. Such correlations of dynamic properties D and R perpendicular with the thermodynamic quantity S, as well as the consistent interpretations of the effect of acyl chain unsaturation on the dynamics in terms of the activity coefficients, strongly emphasize the interrelation between the dynamic structure and the thermodynamics of the PC/cholesterol mixtures.     

Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation

Docosahexaenoic acid (DHA, 22:6) is an n−3 polyunsaturated fatty acid (n−3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state ²H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts.     

A fluorescence and radiolabel study of sterol exchange between membranes

The fluorescent sterols delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) and delta 5,7,9,(11)-cholestatrien-3 beta-ol (cholestatrienol) as well as [1,2-3H]cholesterol were utilized as cholesterol analogues to examine spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Exchange of fluorescent sterols was monitored at 24 degrees C by release from self-quenching of polarization from the time of mixing without separation of donor and acceptor vesicles. The polarization curve for 35 mol% sterol in POPC best fitted a two-exponential function, with a fast-exchange rate constant k1 = 0.0217 min-1, 1t1/2 = 32 min, size pool 1 = 12%, and a slow rate constant k2 = 2.91.10(-3) min-1, 2t1/2 = 238 min, size pool 2 = 88%. In addition to the above two exchangeable pools of sterol, the data were consistent with the presence of a slowly or nonexchangeable pool, 42% of total sterol, that was highly dependent on sterol content. These results were confirmed by simultaneous monitoring of [1,2-3H]cholesterol radioactivity and dehydroergosterol fluorescence intensity after separation of donor and acceptor vesicles by ion-exchange column chromatography. Thus, dehydroergosterol or cholestatrienol exchange as measured by fluorescence parameters (polarization and/or intensity) provides two new methods to follow cholesterol spontaneous exchange. These methods allow resolution and quantitation of a shorter exchange t1/2 near 30 min previously not reported. Thus, the cholesterol desorption rate from membranes may be faster than previously believed. In addition, the presence of a slowly non-exchangeable pool was confirmed.     

Photo-activated phase separation in giant vesicles made from different lipid mixtures

Using giant unilamellar vesicles (GUVs) made from POPC, DPPC, cholesterol and a small amount of a porphyrin-based photosensitizer that we name PE-porph, we investigated the response of the lipid bilayer under visible light, focusing in the formation of domains during the lipid oxidation induced by singlet oxygen. This reactive species is generated by light excitation of PE-porf in the vicinity of the membrane, and thus promotes formation of hydroperoxides when unsaturated lipids and cholesterol are present. Using optical microscopy we determined the lipid compositions under which GUVs initially in the homogeneous phase displayed Lo-Ld phase separation following irradiation. Such an effect is attributed to the in situ formation of both hydroperoxized POPC and cholesterol. The boundary line separating homogeneous Lo phase and phase coexistence regions in the phase diagram is displaced vertically towards the higher cholesterol content in respect to ternary diagram of POPC:DPPC:cholesterol mixtures in the absence of oxidized species. Phase separated domains emerge from sub-micrometer initial sizes to evolve over hours into large Lo-Ld domains completely separated in the lipid membrane. This study provides not only a new tool to explore the kinetics of domain formation in mixtures of lipid membranes, but may also have implications in biological signaling of redox misbalance.     

Temperature and composition dependence of the interaction of delta-lysin with ternary mixtures of sphingomyelin/cholesterol/POPC

The kinetics of carboxyfluorescein efflux induced by the amphipathic peptide delta-lysin from vesicles of porcine brain sphingomyelin (BSM), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol (Chol) were investigated as a function of temperature and composition. Sphingomyelin (SM)/Chol mixtures form a liquid-ordered (L(o)) phase whereas POPC exists in the liquid-disordered (L(d)) phase at ambient temperature. delta-Lysin binds strongly to L(d) and poorly to L(o) phase. In BSM/Chol/POPC vesicles the rate of carboxyfluorescein efflux induced by delta-lysin increases as the POPC content decreases. This is explained by the increase of delta-lysin concentration in L(d) domains, which enhances membrane perturbation by the peptide. Phase separations in the micrometer scale have been observed by fluorescence microscopy in SM/Chol/POPC mixtures for some SM, though not for BSM. Thus, delta-lysin must detect heterogeneities (domains) in BSM/Chol/POPC on a much smaller scale. Advantage was taken of the inverse variation of the efflux rate with the L(d) content of BSM/Chol/POPC vesicles to estimate the L(d) fraction in those mixtures. These results were combined with differential scanning calorimetry to obtain the BSM/Chol/POPC phase diagram as a function of temperature.     

Effect of phospholipid composition on an amphipathic peptide-mediated pore formation in bilayer vesicles

To better understand the influence of phospholipid acyl-chain composition on the formation of pores by cytotoxic amphipathic helices in biological membranes, the leakage of aqueous contents induced by the synthetic peptide GALA (WEAALAEALAE ALAEHLAEALAEALEALAA) from large unilamellar phospholipid vesicles of various compositions has been studied. Peptide-mediated leakage was examined at pH 5.0 from vesicles made of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with the following acyl-chain compositions: 1-palmitoyl-2-oleoyl (PO), 1,2-dioleoyl (DO), 1, 2-dielaidoyl (DE), and 1,2-dipetroselinoyl (DPe). A mathematical model predicts and simulates the final extents of GALA-mediated leakage of 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX) from 1-palmitoyl-2-oleoyl-phosphatidylcholine/1-palmitoyl-2-oleoyl-phospha tidylglycerol (POPC/POPG) and 1, 2-dielaidoyl-sn-glycero-3-phosphocholine/1, 2-dielaidoyl-phosphatidylglycerol (DEPC/DEPG) liposomes at pH 5.0 as a function of peptide concentration in the bilayer, by considering that GALA pores responsible for this leakage have a minimum size of 10 +/- 2 monomers and are formed by quasiirreversible aggregation of the peptide. With the phospholipid acyl-chain compositions tested, GALA-induced ANTS/DPX leakage follows the rank order POPC/POPG approximately DEPC/DEPG > DPePC/DPePG > DOPC/DOPG. Results from binding experiments reveal that this reduced leakage from DOPC/DOPG vesicles cannot be explained by a reduced binding affinity of the peptide to these membranes. As shown by monitoring the leakage of a fluorescent dextran, an increase in the minimum pore size also does not explain the reduction in ANTS/DPX leakage. The data suggest that surface-associated GALA monomers or aggregates are stabilized in bilayers composed of phospholipids containing a cis unsaturation per acyl chain (DO and DPe), while transbilayer peptide insertion is reduced. GALA-induced ANTS/DPX leakage is also decreased when the vesicles contain phosphatidylethanolamine (PE). This lends further support to the suggestion that factors stabilizing the surface state of the peptide reduce its insertion and subsequent pore formation in the bilayer.     

A calorimetry and deuterium NMR study of mixed model membranes of 1-palmitoyl-2-oleylphosphatidylcholine and saturated phosphatidylcholines

Binary phase diagrams have been constructed from differential scanning calorimetry (DSC) data for the systems 1-palmitoyl-2-oleylphosphatidylcholine (POPC)/dimyristoylphosphatidylcholine (DMPC), POPC/dipalmitoylphosphatidylcholine (DPPC) and POPC/distearoylphosphatidylcholine (DSPC). Mixtures of POPC with DMPC exhibit complete miscibility in the gel and liquid crystalline states. Mixtures of POPC with DPPC or with DSPC exhibit gel phase immiscibility over the composition range 0-75% DPPC (or DSPC). These results, when taken together with previous studies of mixtures of phosphatidylcholines, are consistent with the hypothesis that PCs whose order-disorder transition temperatures (Tm values) differ by less than 33 deg. C exhibit gel state miscibility. Those whose Tm values differ by more than 33 deg. C exhibit gel state immiscibility. 2H-NMR spectroscopy has been used to further study mixed model membranes composed of POPC and DPPC, in which either lipid has been labeled with deuterium in the 2-, 10- or 16-position of the palmitoyl chain(s) or in the N-methyls of the choline head group. POPC/DPPC mixtures in the liquid crystalline state are intermediate in order between pure POPC and DPPC at the same temperature. The POPC palmitoyl chain is always more disordered than the palmitoyl chains of DPPC in liquid crystalline POPC/DPPC mixtures. This is attributed to the fact that a POPC palmitoyl chain is constrained by direct bonding to have at least one oleyl chain among its nearest neighbors, while a DPPC palmitoyl chain must have at least one neighboring palmitoyl chain. When liquid crystalline POPC, DPPC and POPC/DPPC mixtures are compared at a reduced temperature (relative to the acyl chain order-disorder transition), POPC/DPPC mixtures are more disordered than predicted from the behavior of the pure components, in agreement with enthalpy data derived from DSC studies. Within the temperature range of the broad phase transition of 1:1 POPC/DPPC, a superposition of gel and liquid crystalline spectra is observed for 1:1 POPC/[2H]DPPC, while 1:1[2H]POPC/DPPC exhibits only a liquid crystalline spectrum. Thus, at temperatures within the phase transition region, the liquid crystalline phase is POPC-rich and the gel phase is DPPC-rich. Comparison of the liquid crystalline quadrupole splittings within the thermal phase transition range suggests that mixing of the residual liquid crystalline POPC and DPPC is highly non-ideal.     

Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed. Received: 10 March 1999/Revised: 22 June 1999