Effect of cholesterol on the formation of an interdigitated gel phase in lysophosphatidylcholine and phosphatidylcholine binary mixtures

We previously reported that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) forms an interdigitated gel phase in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine (16:0LPC) at concentrations below 30 mol%. In the present investigation, fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), X-ray diffraction, and differential scanning calorimetry (DSC) were used to investigate the effect of cholesterol on the phase behavior of 16:0LPC/DPPC binary mixtures. At 25 degrees C, 30 mol% 16:0LPC significantly decreases the DPH fluorescence intensity during the transition of DPPC from the L(beta') phase to the L(betaI) phase. However, the addition of cholesterol to 16:0LPC/DPPC mixtures results in a substantial increase in fluorescence intensity. The changes in DPH fluorescence intensity reflect the probe's redistribution from an orientation parallel to the acyl chain to the center of the bilayer, suggesting a bilayer structure transition from interdigitation to noninterdigitation. The normal repeat period of small angle X-ray diffraction patterns can be restored and a reflection appears at 0.42 nm with a broad shoulder around 0.41 nm in wide angle X-ray diffraction patterns when 10 mol% cholesterol is incorporated into 30 mol% 16:0LPC/DPPC vesicles, indicating that the mixtures are in the gel phase (L(beta')). Moreover, DSC results demonstrate that 10 mol% cholesterol is sufficient to significantly decrease the main enthalpy, cooperativity and lipid chain melting of 30 mol% 16:0LPC/DPPC binary mixtures, which are L(betaI), indicating that the transition of the interdigitated phase is more sensitive to cholesterol than that of the noninterdigitated phase. Our data imply that the interdigitated gel phase induced by 16:0LPC is prevented in the presence of 10 mol% cholesterol, but unlike ethanol, an increasing concentration of 16:0LPC is not able to restore the interdigitation structure of the lipid mixtures.     

Structure and thermodynamics of the dihexadecylphosphatidylcholine-water system

X-ray small- and wide-angle diffraction, differential scanning calorimetry (DSC), temperature scanning densitometry (TSD) and electron microscopy were used to study the lyotropic and thermotropic properties of the system 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine-water over a wide range of compositions from the dry lipids to a large excess of water, and in the temperature range between 0 degrees C and 150 degrees C. The results were used to construct a temperature-composition phase diagram. The phases have been characterized with respect to their molecular arrangements and hydrocarbon chain packing subcells. In the fully hydrated state (greater than 45 wt% H2O) four thermotropic phases were found, with readily reversible transitions at 5 degrees C, 32.5 degrees C and 43.6 degrees C, respectively. The two lower temperature phases deviate from all others in consisting of bilayers with fully interdigitated hydrocarbon chains, while above 32.5 degrees C the structures resemble closely those of the analog diester lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). At hydrations between 30 and 45 wt% H2O, and below 32 degrees C, interdigitated and non-interdigitated multilayers coexist in one coherent phase. A bilayer tilting mechanism is proposed for the formation of this coexistence of two regular structures. Below 30 wt% H2O, hydrated 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) exists in lamellar, non-interdigitated bilayers, showing very weak interbilayer swelling. There, the water molecules appear to occupy voids between the polar headgroups.     

Thermal phase behaviour and structure of hydrated mixtures between dipalmitoyl- and dihexadecylphosphatidylcholine

Mixtures of 1,2-dipalmitoyl- and 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC and DHPC) in dispersion with excess water were studied by differential scanning calorimetry (DSC) and X-ray diffraction techniques. The transition parameters of the main gel-to-liquid crystalline transition show a monotonous dependence on the composition, indicating ideal miscibility of the two lipids, in keeping with the closely similar structures of the pure, hydrated lipids in the P beta' and L alpha states. The pre-transition shows a depression to a minimum temperature of 23 degrees C occurring around equimolar mixtures. Below the pre-transition temperatures, the L beta' gel phase of DPPC maintains bimolecular structure up to DHPC admixtures of 50 mol%, with adaptations in hydrocarbon chain packing and multilayer periodicity. On the side of DHPC, the interdigitated gel structure shows full solubility for DPPC up to equimolarity without major structural changes. The crystalline Lc-phase of DPPC exhibits immiscibility with DHPC, demonstrated by the fact that the subtransition is abolished already at less than 15 mol% DHPC. DHPC, below its subtransition, can accommodate up to 50 mol% DPPC within an interdigitated layer structure with unperturbed, crystalline hydrocarbon chain packing.     

Effects of cholesterol on phospholipid membranes: inhibition of the interdigitated gel phase of F-DPPC and F-DPPC/DPPC

Unlike the parent phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the monofluorinated analog, 1-palmitoyl-2-(16-fluoropalmitoyl)sn-glycero-3-phosphocholine (F-DPPC), spontaneously forms an interdigitated gel phase (L(β)I) below the main transition temperature (T(m)). We have examined the effects of introducing cholesterol to F-DPPC and 1:1 F-DPPC/DPPC membranes using a combination of DSC, optical density, fluorescence intensity and polarization, (31)P NMR, and X-ray diffraction techniques. Cholesterol increases the fluidity of the gel phase, broadens the main transition, and decreases the main transition enthalpy. However, these results also reveal that there is an unusually large degree of phase coexistence between the L(β)I and non-interdigitated gel phases when cholesterol is added. Cholesterol encourages this phase segregation by partitioning into the thicker non-interdigitated domains. At higher cholesterol concentrations, the majority or all of the L(β)I phase of F-DPPC and 1:1 F-DPPC/DPPC is eliminated and is replaced by a non-interdigitated liquid-ordered (l(o)) phase with properties similar to DPPC/cholesterol. Consequently, cholesterol mitigates the influence the CF moiety has on the thermodynamic phase behavior of F-DPPC. Our findings demonstrate that there are multiple characteristics of cholesterol-rich membranes that disfavor interdigitation.     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Comparative study of the gel phases of ether- and ester-linked phosphatidylcholines

Calorimetric, X-ray diffraction, and 31P nuclear magnetic resonance (NMR) studies of aqueous dispersions of 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) gel phases at low temperatures (-60 to 22 degrees C) show thermal, structural, and dynamic differences when compared to aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) gel phases at corresponding temperatures. Differential scanning calorimetry of DHPC dispersions demonstrates a reversible, low-enthalpy "subtransition" at 4 degrees C in contrast to the conditionally reversible, high-enthalpy subtransition observed at 17 degrees C for annealed DPPC bilayers. X-ray diffraction studies indicate that DHPC dispersions form a lamellar gel phase with dav congruent to 46 A both above and below the "subtransition". It is suggested that the reduced dav observed for DHPC (46 A as compared to 64 A in DPPC) is due to an interdigitated lamellar gel phase which exists at all temperatures below the pretransition at 35 degrees C. 31P NMR spectra of DHPC gel-phase bilayers show an axially symmetric chemical shift anisotropy powder pattern which remains sharp down to -20 degrees C, suggesting the presence of fast axial diffusion. In contrast, 31P spectra of DPPC bilayers indicate this type of motion is frozen out at approximately 0 degrees C.     

Phase transitions and fatty acid spin label behavior in interdigitated lipid phases induced by glycerol and polymyxin

Glycerol and polymyxin have been shown by X-ray diffraction to induce interdigitated bilayers in phosphatidylcholine (PC) and phosphatidylglycerol (PG), respectively (McDaniel, R.V., et al. (1983) Biochim. Biophys. Acta 731, 97-108; Ranck, J.-L. and Tocanne, J.-F. (1982) FEBS Lett. 143, 175-178). In the present study we have investigated the phase behavior of PC and PG in the presence of glycerol and polymyxin by differential scanning calorimetry and the use of fatty acid spin labels. Interdigitation causes a large increase in the order parameter of a fatty acid spin labeled near the terminal methyl, 16-doxylstearate, so that it was similar to that of a fatty acid labeled much closer to the polar head group region, 5-doxylstearate. Thus interdigitation abolishes the fluidity gradient found in a non-interdigitated bilayer. 16-Doxylstearate may be useful in detecting interdigitation of lipid bilayers caused by other substances. The different samples all went through two transitions on heating or cooling, or both. However, use of the fatty acid spin label showed that the molecular events during these transitions varies for different samples. The results suggested that PC-glycerol freezes from the liquid-crystalline phase into a non-interdigitated gel phase. This subsequently becomes interdigitated upon lowering the temperature a few degrees, in a low enthalpy transition. PG-polymyxin shows a similar behavior except that the enthalpy of the non-interdigitated gel to interdigitated phase transition is greater and the transition is reversible on heating. Thus on heating PG-polymyxin first goes through a transition from the interdigitated phase to a non-interdigitated gel phase and then, in a separate transition, to the liquid-crystalline phase. This occurs because the fatty acid chains in the presence of polymyxin become too disordered with increase in temperature to maintain the interdigitated state. PG-glycerol goes into the interdigitated state less readily than the other mixtures. If cooled rapidly, PG-glycerol freezes into a metastable phase which is more disordered than the interdigitated phase. It goes into the interdigitated phase in an exothermic transition on heating. An increase in fatty acid chain length causes greater steric hindrance to interdigitation but also increases the stabilizing energy gained by interdigitation.     

The phase behavior of mixed aqueous dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phases and transition sequences for aqueous dispersions of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) have been studied by differential scanning calorimetry, dynamic x-ray diffraction, freeze-fracture electron microscopy, 31P-nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The results have been used to construct a dynamic phase diagram of the binary mixture as a function of temperature over the range 20 degrees-90 degrees C. It is concluded that DPPC and 1,2-DPG form two complexes in the gel phase, the first one with a DPPC/1,2-DPG molar ratio of 55:45 and the second one at a molar ratio of approximately 1:2, defining three different regions in the phase diagram. Two eutectic points are postulated to occur: one at a very low 1,2-DPG concentration and the other at a 1,2-DPG concentration slightly higher than 66 mol%. At temperatures higher than the transition temperature, lamellar phases were predominant at low 1,2-DPG concentrations, but nonlamellar phases were found to be predominant at high proportions of 1,2-DPG. A very important aspect of these DPPC/1,2-DPG mixtures was that, in the gel phase, they showed a ripple structure, as seen by freeze-fracture electron microscopy and consistent with the high lamellar repeat spacings seen by x-ray diffraction. Ripple phase characteristics were also found in the fluid lamellar phases occurring at concentrations up to 35.6 mol% of 1,2-DPG. Evidence was obtained by Fourier transform infrared spectroscopy of the dehydration of the lipid-water interface induced by the presence of 1,2-DPG. The biological significance of the presence of diacylglycerol in membrane lipid domains is discussed.     

Effect of alcohols on the phase transitions of dihexadecylphosphatidylcholine

We have systematically investigated the effect of short chain alcohols (methanol to n-propanol) on the phase transitions of 1,2-dihexadecylphosphatidylcholine (DHPC), a lipid that forms a stable interdigitated gel phase (L beta I) in aqueous solution. The temperature of the low-temperature L beta I to P beta' phase transition of DHPC was found to increase with alcohol concentration, showing that alcohol interacts preferentially with the interdigitated phase relative to the non-interdigitated gel. The main transition of DHPC exhibited a biphasic effect of alcohol concentration similar to that previously observed with DPPC (Rowe, E.S. (1983) Biochemistry 22,3299-3305). As alcohol concentration is increased the lower L beta I to P beta' and main P beta' to L alpha transitions of DHPC merge at the threshold concentration of the biphasic effect, so that above this concentration there is one phase transition from L beta I directly to L alpha. This is analogous to DPPC above its biphasic threshold. Similar to DPPC, the transition between L beta I and L alpha exhibits marked hysteresis.     

Studies of the structure and organization of cationic lipid bilayer membranes: calorimetric, spectroscopic, and x-ray diffraction studies of linear saturated P-O-ethyl phosphatidylcholines

Differential scanning calorimetry, x-ray diffraction, and infrared and (31)P-nuclear magnetic resonance ((31)P-NMR) spectroscopy were used to examine the thermotropic phase behavior and organization of cationic model membranes composed of the P-O-ethyl esters of a homologous series of n-saturated 1,2-diacyl phosphatidylcholines (Et-PCs). Differential scanning calorimetry studies indicate that on heating, these lipids exhibit single highly energetic and cooperative endothermic transitions whose temperatures and enthalpies are higher than those of the corresponding phosphatidylcholines (PCs). Upon cooling, these Et-PCs exhibit two exothermic transitions at temperatures slightly below the single endotherm observed upon heating. These cooling exotherms have both been assigned to transitions between the liquid-crystalline and gel phases of these lipids by x-ray diffraction. The x-ray diffraction data also show that unlike the parent PCs, the chain-melting phase transition of these Et-PCs involves a direct transformation of a chain-interdigitated gel phase to the lamellar liquid-crystalline phase for the homologous series of n > or = 14. Our (31)P-NMR spectroscopic studies indicate that the rates of phosphate headgroup reorientation in both gel and liquid-crystalline phases of these lipids are comparable to those of the corresponding PC bilayers. However, the shape of the (31)P-NMR spectra observed in the interdigitated gel phase indicates that phosphate headgroup reorientation is subject to constraints that are not encountered in the non-interdigitated gel phases of parent PCs. The infrared spectroscopic data indicate that the Et-PCs adopt a very compact form of hydrocarbon chain packing in the interdigitated gel phase and that the polar/apolar interfacial regions of these bilayers are less hydrated than those of corresponding PC bilayers in both the gel and liquid-crystalline phases. Our results indicate that esterification of PC phosphate headgroups results in many alterations of bilayer physical properties aside from the endowment of a positively charged surface. This fact should be considered in assessing the interactions of these compounds with naturally occurring lipids and with other biological materials.     

The distribution of alpha-tocopherol in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine

The effect of alpha-tocopherol on the structure and phase behaviour of mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine has been examined by synchrotron X-ray diffraction. Equimolar mixtures of dioleoylphosphatidylethanolamine:dioleoylphosphatidylcholine and dimyristoylphosphatidylcholine:dioleoylphosphatidylethanolamine did not show evidence of phase separation of an inverted hexagonal structure typical of alpha-tocopherol and phosphatidylethanolamine from lamellar phase. Mixed dispersions of dioleoyl derivatives of phosphatidylethanolamine:phosphatidylcholine (3:1) form a typical miscible gel phase at low temperatures but which phase separates into lamellar liquid-crystal and inverted hexagonal phases at temperatures greater than 65 degrees C. The presence of 1, 2 or 5 mol% alpha-tocopherol caused a decrease in the temperature at which the inverted hexagonal phase appears. Phase separation of non-lamellar phase from lamellar gel phase can be detected in the presence of 7.5 and 10 mol% alpha-tocopherol, indicating a limited capacity of the phosphatidylcholine to incorporate alpha-tocopherol into the lamellar domain. A partial phase diagram of the ternary mixture has been constructed from the X-ray scattering data. It was concluded that there is no preferential interaction of alpha-tocopherol with phosphatidylethanolamine in mixed aqueous dispersions containing phosphatidylcholines.     

Cubic phase is induced by cholesterol in the dispersion of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine

The effect of cholesterol, a major constituent of eukaryotic cell membranes, on the structure and thermotropic phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) dispersed in excess water was examined by synchrotron X-ray diffraction methods. Temperature scans over the range 10-75 degrees C showed that the gel to liquid-crystalline phase transition decreased from 25 to 10 degrees C in the presence of 20 mol% cholesterol, and no gel phase could be detected in the wide-angle X-ray scattering (WAXS) intensity profile of mixtures containing 35 mol% cholesterol. The small-angle X-ray scattering (SAXS) intensity profiles showed that the lamellar to nonlamellar phase transition temperature was also decreased in mixtures containing up to 30 mol% cholesterol but the trend was reversed in mixtures containing a higher proportion of cholesterol. There was evidence that the transition of the lamellar liquid-crystal phase is to cubic phases in mixtures containing less than 30 mol% cholesterol. The space group of one of these cubic phases was assigned as Pn3m. This effect of cholesterol on non-bilayer-forming phospholipids is considered in the context of the role of cholesterol in membrane organization and function.     

Use of spin labels to determine the percentage of interdigitated lipid in complexes with polymyxin B and polymyxin B nonapeptide

Long chain spin labels with the nitroxide group located near the terminal methyl of the chain were used to determine the percentage interdigitated lipid in complexes of polymyxin B (PMB) and polymyxin B nonapeptide (PMBN) with the acidic lipids dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidic acid (DPPA) at varying mole ratios of drug to lipid and at different pH values. These spin labels are more motionally restricted in the interdigitated than in the non-interdigitated gel phase bilayer. This allows determination of the percentage interdigitated lipid by resolution of the spectrum into motionally restricted and more mobile components. At nonsaturating concentrations of PMB, significantly more DPPG than that which can be maximally PMB-bound, becomes interdigitated. As the temperature approaches the gel to liquid crystalline phase transition temperature, the bilayer becomes progressively non-interdigitated. The ESR spectrum indicates that PMB also causes interdigitation of DPPA. However, in contrast to DPPG, the amount of DPPA which is interdigitated at pH 6, is less than the amount which is expected to be PMB-bound. This is attributed to the ability of DPPA to participate in lateral interlipid hydrogen bonding interactions. Such lateral interactions would be abolished in the interdigitated bilayer and thus they are expected to inhibit its formation. At pH 9, where the interlipid interactions of DPPA are weakened, PMB induces even more lipid than that which is PMB-bound to become interdigitated. Indeed, the percentage interdigitated lipid is even greater than found for DPPG. This may be partly a result of the greater negative charge of DPPA at this pH. A greater repulsive negative charge is expected to favor interdigitation. PMBN is less effective than PMB at inducing interdigitation of DPPG and causes little or no interdigitation of DPPA at pH 6, even at saturating concentrations. PMBN also does not lower the phase transition temperature of DPPA at pH 6 as much as PMB. At pH 9, the effect of PMBN on DPPA is more similar to the effect of PMB. However, even for DPPG, and DPPA at pH 9, PMBN does not maintain interdigitation of the lipids at higher temperatures as effectively as PMB. PMBN's smaller perturbing effect and greatly decreased ability to cause interdigitation of DPPA at pH values below 9 may be related to a decreased ability to cause lateral separation of the lipid molecules, which is necessary in order to weaken the interlipid interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of chain-linkage on the structure of phosphatidyl choline bilayers. Hydration studies of 1-hexadecyl 2-palmitoyl-sn-glycero-3-phosphocholine.

While hydrated dipalmitoyl phosphatidylcholine (DPPC) forms tilted chain L beta' bilayers in the gel phase, the ether-linked analogue dihexadecyl phosphatidylcholine (DHPC) exhibits gel phase polymorphism. At low hydration DHPC forms L beta' phases but at greater than 30% H2O a chain-interdigitated gel phase is observed (Ruocco, M. J., D. S. Siminovitch, and R. G. Griffin. 1985. Biochemistry. 24:2406-2411; Kim, J.T., J. Mattai, and G.G. Shipley. 1987. Biochemistry. 26:6599-6603). In this study we report the behavior of a phosphatidylcholine (PC) with both types of chain linkage, 1-hexadecyl-2-palmitoyl-sn-glycero-3-phosphocholine (HPPC). HPPC has been investigated as a function of hydration using differential scanning calorimetry (DSC) and x-ray diffraction. By DSC, over the hydration range 5. 1-70.3 wt% H2O, HPPC exhibits two reversible transitions. The reversible main chain-melting transition decreases from 69 degrees C, reaching a limiting value of 40 degrees C at full hydration. X-ray diffraction patterns of hydrated HPPC have been recorded as a function of hydration at 20 degrees and 50 degrees C. At 50 degrees C, melted-chain L alpha bilayer phases are observed at all hydrations. At 20 degrees C, at low hydrations (less than 34 wt% H2O) HPPC exhibits diffraction patterns characteristic of bilayer gel phases similar to those of the gel phase of DPPC. In contrast, at greater than or equal to 34 wt% H2O, HPPC shows a much reduced bilayer periodicity, d = 47 A, and a single sharp reflection at 4.0 A in the wide angle region. This diffraction pattern is identical to that exhibited by the interdigitated phase of DHPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-drug interaction: a structural analysis of pindolol effects on model membranes.

The ternary system constituted by distearoylphosphatidylcholine, pindolol (a vasodilator drug) and water has been investigated by using X-ray diffraction and calorimetric techniques. The structural modifications induced by the drug have been determined and a possible interaction model has been derived. In particular, the pindolol content-temperature dependent phase diagram shows the occurrence of two new phases: the first is an interdigitated gel, and the second is a lamellar structure presenting an unusual mixed disordered-ordered conformation of the hydrocarbon chains (L alpha beta). The comparative analysis of electron density profiles relative to the L alpha beta phase, reveals significant modifications in the paraffinic region of the lipid layer. In agreement with thermodynamic results, the structural data suggest that the drug induces a stiffening and a tightening of the hydrocarbon chains. Moreover, the hydrophilic properties of the membrane (particularly in P beta, and L alpha beta phases) present an evident dependence with the drug concentration.     

Trehalose-induced destabilization of interdigitated gel phase in dihexadecylphosphatidylcholine

Trehalose is believed to have the ability to protect some organisms against low temperatures. To clarify the cryoprotective mechanism of trehalose, the structure and the phase behavior of fully hydrated dihexadecylphosphatidylcholine (DHPC) membranes in the presence of various concentrations of trehalose were studied by means of differential scanning calorimetry (DSC), static x-ray diffraction, and simultaneous x-ray diffraction and DSC measurements. The temperature of the interdigitated gel (Lbeta(i))-to-ripple (Pbeta') phase transition of DHPC decreases with a rise in trehalose concentration up to approximately 1.0 M. Above a trehalose concentration of approximately 1.0 M, no Lbeta(i) phase is observed. In this connection, the electron density profile calculated from the lamellar diffraction data in the presence of 1.6 M trehalose indicates that DHPC forms noninterdigitated bilayers below the P beta' phase. It was concluded that trehalose destabilizes the Lbeta(i) phase of DHPC bilayers. This suggests that trehalose reduces the area at the interface between the lipid and water. The relation between this effect of trehalose and a low temperature tolerance was discussed from the viewpoint of cold-induced denaturation of proteins.     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

Elasticity and phase behavior of DPPC membrane modulated by cholesterol, ergosterol, and ethanol

Giant vesicles formed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and sterols (cholesterol or ergosterol) in water and water/ethanol solutions have been used to examine the effect of sterol composition and ethanol concentration on the area compressibility modulus (K(a)), overall mechanical behavior, vesicle morphology, and induction of lipid alkyl chain interdigitation. Our results from micropipette aspiration suggest that cholesterol and ergosterol impact the order and microstructure of the gel (L(beta)') phase DPPC membrane. At low concentration (10-15 mol%) these sterols disrupt the long-range lateral order and fluidize the membrane (K(a) approximately 300 mN/m). Then at 18 mol%, these sterols participate in the formation of a continuous cohesive liquid-ordered (L(o)) phase with a sterol-dependent membrane density (K(a) approximately 750 for DPPC/ergosterol and K(a) approximately 1100 mN/m for DPPC/cholesterol). Finally at approximately 40 mol% both cholesterol and ergosterol impart similar condensation to the membrane (K(a) approximately 1200 mN/m). Introduction of ethanol (5-25 vol%) results in drops in the magnitude of K(a), which can be substantial, and sometimes individual vesicles with lowered K(a) reveal two slopes of tension versus apparent area strain. We postulate that this behavior represents disruption of lipid-sterol intermolecular interactions and therefore the membrane becomes interdigitation prone. We find that for DPPC vesicles with sterol concentrations of 20-25 mol%, significantly more ethanol is required to induce interdigitation compared to pure DPPC vesicles; approximately 7 vol% more for ergosterol and approximately 10 vol% more for cholesterol. For lower sterol concentrations (10-15 mol%), interdigitation is offset, but by <5 vol%. These data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L(beta)I) phase bilayer is a major factor in the disruption of cellular activity, which typically occurs between approximately 12 and approximately 16 vol% ethanol in yeast fermentations. We summarize our findings by producing, for the first time, "elasticity/phase diagrams" over a wide range of sterol (cholesterol and ergosterol) and ethanol concentrations.     

Structure and phase behavior of hydrated mixtures of L-dipalmitoylphosphatidylcholine and palmitic acid. Correlations between structural rearrangements, specific volume changes and endothermic events

Several new features of the phase diagram of L-dipalmitoylphosphatidylcholine (DPPC)/palmitic acid mixtures in excess water were established by means of static and time-resolved X-ray diffraction, densitometry and differential scanning calorimetry (DSC). At low temperatures, palmitic acid has a biphasic effect on the lamellar subgel phases: at concentrations below 5-6 mol%, it prevents formation of the DPPC subgel phase (Lc), while at higher contents (between about 40 and 90 mol%) another subgel phase (Lccom) is formed as a result of lipid co-crystallization at 1 DPPC: 2 palmitic acid stoichiometry. A crystalline palmitic acid phase separates from Lccom above 70-80 mol% of fatty acid. The Lccomphase transforms into a lamellar gel phase (L beta) in an endothermic transition centered at 38 degrees C. At high temperatures, the mixtures form hexagonal liquid-crystalline phase (HII) in the region of 60-70 mol% and an isotropic phase (I) at 90-100 mol% of palmitic acid. No coexistence of HII phase with the fluid lamellar phase of DPPC was observed at intermediate compositions (20 and 50 mol% of palmitic acid) but rather formation of a complex phase with non-periodic geometry characterized by molten chains and a broad, continuous small-angle scattering band. No evidence for fluid phase coexistence was found also at compositions between HII and I phases. The L beta--HII transition at 60-70 mol% of palmitic acids is readily reversible and two-state in both heating and cooling modes. It is characterized by the coexistence of initial and final phases with no detectable intermediates by time-resolved and static X-ray diffraction. The crystalline-isotropic transition in palmitic acid is two-state only in heating direction. On cooling, it is characterized by strong undercooling and gradually relaxing lamellar crystalline structures. The slowly reversible Lccom--L beta transition proceeds continuously through intermediate states. Although clearly discernible by both DSC and X-ray diffraction, it is not accompanied by specific volume changes.     

Cubic phase is induced by cholesterol in the dispersion of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine

The effect of cholesterol, a major constituent of eukaryotic cell membranes, on the structure and thermotropic phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) dispersed in excess water was examined by synchrotron X-ray diffraction methods. Temperature scans over the range 10-75 °C showed that the gel to liquid-crystalline phase transition decreased from 25 to 10 °C in the presence of 20 mol% cholesterol, and no gel phase could be detected in the wide-angle X-ray scattering (WAXS) intensity profile of mixtures containing 35 mol% cholesterol. The small-angle X-ray scattering (SAXS) intensity profiles showed that the lamellar to nonlamellar phase transition temperature was also decreased in mixtures containing up to 30 mol% cholesterol but the trend was reversed in mixtures containing a higher proportion of cholesterol. There was evidence that the transition of the lamellar liquid-crystal phase is to cubic phases in mixtures containing less than 30 mol% cholesterol. The space group of one of these cubic phases was assigned as Pn3m. This effect of cholesterol on non-bilayer-forming phospholipids is considered in the context of the role of cholesterol in membrane organization and function.