The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C₉₅ on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C₉₅ had no detectible effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C₉₅. Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C₉₅. These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

The interaction of alpha-tocopherol with bilayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine

The effect of alpha-tocopherol on the structure and phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylcholine was examined by real-time synchrotron X-ray diffraction and freeze-fracture electron microscopic methods. X-ray scattering intensity was recorded from mixed aqueous dispersions of phospholipid with 2.5, 5, 10 and 20 mol% alpha-tocopherol during temperature scans at 3 degrees /min between -25 and 10 degrees C. A ripple structure is induced by the presence of alpha-tocopherol that coexists with the ripple phase characteristic of the pure phospholipid in mixtures containing 2.5 mol% alpha-tocopherol but completely replaces it in mixtures containing greater proportions of alpha-tocopherol. Freeze-fracture replicas of dispersions containing 5 mol% alpha-tocopherol indicate a ripple phase with a periodicity of about 9 nm. Increasing amounts of alpha-tocopherol result in a progressive reduction in temperature of the gel to liquid-crystal phase transition and broadening of the transition. Two lamellar phases coexist in the liquid-crystal state, one with a spacing of 6.4 nm assigned to an alpha-tocopherol-enriched lamellar structure and the other with a lamellar repeat of 6.1 nm corresponding to bilayers of pure phospholipid.     

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.     

Inverted hexagonal and cubic phases induced by alpha-tocopherol in fully hydrated dispersions of dilauroylphosphatidylethanolamine

The effect of alpha-tocopherol on the thermotropic phase behaviour and structure of aqueous dispersions of 1,2-di-lauryl-sn-glycero-3-phosphoethanolamine was examined by synchrotron X-ray diffraction. The pure phospholipid exhibited a lamellar gel to liquid-crystal phase transition at 30 degrees C on heating at 3 degrees C min(-1) between 10 degrees C and 90 degrees C. The transition was reversible with a temperature hysteresis of 0.3 degrees C on cooling. At temperatures less than 10 degrees C only lamellar gel phase of the pure phospholipid was seen in co-dispersions of up to 20 mol % alpha-tocopherol. The presence of 2.5 mol % alpha-tocopherol caused the appearance of inverted hexagonal phase at temperatures just below the main phase transition temperature that co-existed with the lamellar gel phase. The intensity of scattering from the hexagonal-II phase increased with increasing proportion of alpha-tocopherol in the mixture and in proportions greater than 10 mol % it persisted at temperatures above the main transition and co-existed with the lamellar liquid-crystal phase of the pure phospholipid. At higher temperatures all co-dispersions containing up to 15 mol % alpha-tocopherol showed the presence of cubic phases. These phases indexed a Pn3m or Pn3 space grouping. When the proportion of alpha-tocopherol was increased to 20 mol % the only non-lamellar phase observed was inverted hexagonal phase. This phase co-existed with lamellar gel and liquid-crystal phases of the pure phospholipid, but was the only phase present at temperatures >60 degrees C. The X-ray diffraction data were used to construct a partial phase diagram of the lipid mixture in excess water between 10 degrees and 90 degrees C and up to 20 mol % alpha-tocopherol in phospholipid.     

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.     

The kinetics and mechanism of the formation of crystalline phase of dipalmitoylphosphatidylethanolamine dispersed in aqueous dimethyl sulfoxide solutions

The phase transition kinetics and mechanism of formation of a lamellar-crystalline phase of dipalmitoylphosphatidylethanolamine (DPPE) dispersed in different concentrations of aqueous dimethyl sulfoxide (DMSO) during cooling have been examined by differential scanning calorimetry and synchrotron X-ray diffraction techniques. In dispersions containing mole fractions of DMSO (x<0.22), the phase transition sequence of the phospholipid is from lamellar liquid-crystal phase to lamellar-gel phase. Increasing the mole fraction of DMSO to 0.220.5 resulted in a direct transition from liquid-crystal phase to lamellar crystal phase with no detectable intermediate gel phase. A temperature versus DMSO concentration phase diagram was constructed based on calorimetric data with phase assignments made using synchrotron X-ray diffraction measurements. The non-isothermal formation kinetics of the lamellar crystal phase, which is expressed as the half time of the transformation process, was found to depend on DMSO concentration. The inducement of lamellar crystal phase in DPPE by DMSO is discussed in terms of the dehydration effect of DMSO and competitive molecular interactions between DMSO, water, and the phospholipid.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

Phase stability of phosphatidylcholines in dimethylsulfoxide solutions.

The temperature dependence of the phase stability of dispersions of dimyristoyl, dipalmitoyl, and distearoyl derivatives of phosphatidylcholines in excess aqueous dimethylsulfoxide has been examined by differential scanning calorimetry and synchrotron x-ray diffraction methods. There was a close correlation between the enthalpic transitions and the structural changes associated with the pre- and main transitions of the phospholipids in the range of concentrations up to mole fractions of dimethylsulfoxide in water of 0.1333. The temperature of the pre- and main transitions of the three phospholipids were found to increase linearly with increasing mole fraction of dimethylsulfoxide. The difference in phase stability between the lamellar gel and ripple phases induced by increasing dimethylsulfoxide concentration resulted in disappearance of the ripple phase and direct transition between lamellar gel and lamellar liquid-crystal phases. The effect of changing the properties of the solvent by the addition of dimethylsulfoxide on the dimensions of dipalmitoylphosphatidylcholine and solvent layers of the bilayer repeat structure has been determined from electron density distribution calculations. The lamellar repeat spacing recorded at 25 degrees C decreased from 6.36 nm in aqueous dispersion to 6.04 nm in a dispersion containing a mole fraction of 0.1105 dimethylsulfoxide. The results indicate that dipole interactions between solvent and phospholipid and dielectric properties of the solvent are important factors in the determination of the structure of saturated phosphatidylcholines.     

The structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine codispersed with a branched-chain phosphatidylcholine

The structure and thermotropic phase behaviour of a fully hydrated binary mixture of dipalmitoylphosphatidylcholine and a branched-chain phosphatidylcholine, 1, 2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine, were examined using differential scanning calorimetry, synchrotron X-ray diffraction and freeze-fracture electron microscopy. The branched-chain lipid forms a nonlamellar phase when dispersed alone in aqueous medium. Mixed aqueous dispersions of the two phospholipids containing less than 33 mol% of the branched-chain lipid form lamellar phases over the whole temperature range were studied (4 degrees C to 60 degrees C). When present in proportions greater than 33 mol% it induces a hexagonal phase in mixed aqueous dispersions with dipalmitoylphosphatidylcholine at temperatures above the fluid phase transition. At temperatures below 35 degrees C a hexagonal phase coexists with a gel bilayer phase. The lamellar<-->nonlamellar transition can be explained satisfactorily on the basis of the shape of the molecule expressed in terms of headgroup and chain cross-sectional areas. At temperatures below 35 degrees C macroscopic phase separation of two gel phases takes place. Freeze-fracture electron microscopy revealed that one gel phase consists of bilayers with a highly regular, periodic superstructure (macro-ripples) whereas the other phase forms flat, planar bilayers. The macro-ripple phase appears to represent a relaxation structure required to adapt to the packing constraints imposed by the incorporation of the branched-chain lipid into the dipalmitoylphosphatidylcholine host bilayer. The data suggest that structural changes that take place on cooling the mixed dispersion below the lamellar<-->nonlamellar phase transition temperature cannot be adequately described using the molecular form concept. Instead it is necessary to take into account the detailed molecular form of the guest lipid as well as its physical properties.     

The polyisoprenoid chain length influences the interaction of ubiquinones with phospholipid bilayers

The interaction between ubiquinone homologues with polyisoprenoid chain lengths varying from 3 to 10 units and dipalmitoylphosphatidylcholine bilayers has been examined by differential scanning calorimetry and wide angle X-ray diffraction analysis. Decreasing the polyisoprenoid chain lengths of ubiquinone in mixed dispersions with phospholipid in mol ratios of about 10 mol% caused a decrease in the gel-liquid crystalline phase transition temperature of the phospholipid and a broadening of the transition. Enthalpy measurements showed that most of the phospholipid (greater than 92%) was involved in the transition endotherm and the formation of a gel phase was also confirmed by the presence of a sharp X-ray reflection of 0.42 nm. These results are consistent with a model in which all of the ubiquinone homologous ultimately undergo a phase separation from phospholipid molecules entering a gel phase on cooling below the phase transition temperature. Reducing the length of the polyisoprenoid chain alters the amphipathic balance of the ubiquinone molecules and is reflected in the tendency of shorter chain ubiquinones to intercalate between the phospholipid molecules upon reheating through the main phase transition.     

Lamellar gel-lamellar liquid crystal phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from aqueous trehalose solutions. A real-time X-ray diffraction study

The mechanism of the phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from fully hydrated gel phase (L beta') in the presence of trehalose has been investigated by real-time X-ray diffraction methods. Sequential diffraction patterns were recorded with an accumulation time of 3 s during heating and 1.2 s during cooling between about 20 and 80 degrees C. A transition is observed in the range 47-53 degrees C that involves structural events typical of a lamellar gel-lamellar liquid-crystal (L beta--L alpha) transformation. This transition is completely reversible with a temperature hysteresis of 2-3 degrees C and thereby resembles the main phase transition of fully hydrated dipalmitoylphosphatidylcholine multilayers. The mechanism of the transition from L beta to L alpha as seen in the wide-angle scattering profiles show that the sharp peak at about 0.41 nm, characteristic of the gel phase, broadens and shifts progressively to about 0.44 nm towards the end of the transition. A temperature jump of 6C degrees/s through the phase transition region of a freeze-dried dipalmitoylphosphatidylcholine: trehalose mixture (molar ratio 1:1) showed that the phase transition had a relaxation time of about 2 s which is similar to that of the main transition in the fully hydrated lipid. X-ray diffraction studies of the melting of dipalmitoylphosphatidylcholine freeze-dried from the lamellar-gel phase in the absence of trehalose showed a transition at above 70 degrees C. The low-angle diffraction data of phospholipid/trehalose mixtures are consistent with an arrangement of trehalose molecules in a loosely packed 'monolayer' separating bilayers of phospholipid. Trehalose appears to reduce the direct interbilayer hydrogen bond coupling thereby modifying the thermal stability and the phase transition mechanism of the bilayers.     

Effect of substitution of hydrogen oxide by deuterium oxide on thermotropic transition between the interdigitated gel phase and the ripple phase of dihexadecylphosphatidylcholine.

Thermotropic transitions of dihexadecylphosphatidylcholine (DHPC) dispersions in hydrogen oxide (1H2O) and deuterium oxide (2H2O) were investigated by differential scanning calorimetry (DSC). In DHPC dispersions, transition temperature between interdigitated gel phase (L beta I) and ripple phase (P beta') is lower in 2H2O than in 1H2O, and transition between the ripple phase (P beta') and fluid phase (L alpha) in 2H2O occurs at a temperature slightly higher than in 1H2O. In dipalmitoylphosphatidylcholine (DPPC) dispersions, on the other hand, transition temperature between lamellar gel phase (L beta') and ripple phase is higher in 2H2O than in 1H2O. These results suggest that the interdigitated gel phase is more stable in 1H2O than in 2H2O. To account for the shift of transition temperature by the water substitution, difference of interfacial energies between these aqueous environments is discussed.     

Long N-acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form liquid-ordered phase

The structure and thermotropic phase behaviour of aqueous dispersions of dipalmitoylphosphatidylcholine and glucosylceramide rich in C-24 fatty acyl residues was investigated by synchrotron X-ray diffraction methods. Binary mixtures comprised of molar ratios 2.5:100, 6.5:100, 12.6:100, 25:100, 40:100 and 50:100, glucolipid:phospholipid were examined in heating and cooling scans of 2°/min between 25 and 85 °C. Small-angle reflections indicated coexisting lamellar structures over the entire temperature range investigated. Reversible thermotropic changes were observed in one lamellar structure that is consistent with transitions between gel, ripple and fluid lamellar phases of pure phospholipid. The temperature of these transitions, however, were progressively shifted up by about 5 °C in the mixture containing the highest proportion of glucolipid and coincided with a published endothermic peak observed in this mixture. A higher-temperature endotherm was associated with molecular rearrangements on transition of the gel phase phospholipid to the fluid phase. This rearrangement was associated with the appearance of identifiable transient intermediate structures in the small-angle scattering region. The glucolipid formed stoichiometric mixtures with the phospholipid at all temperatures investigated and there was no evidence of phase separation of pure glucolipid. Analysis of the wide-angle scattering profiles during an initial heating scan of a binary mixture comprised of 40:60 glucolipid:phospholipid was consistent with a phase transition of pure phospholipid at about 43 °C coexisting with a liquid-ordered phase formed from the two lipids. This was confirmed by analysis of the small-angle scattering peaks of this mixture recorded at 25 and 65 °C which showed that a glucolipid-rich phase coexisted with almost pure bilayers of phospholipid at both temperatures. The glucolipid-rich phase consisted of 45:55 mole ratio glucolipid:phospholipid at 25 °C with pure phospholipid in gel phase and 42:58 mole ratio at 65 °C when the phospholipid was in the fluid phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.     

Modulation of the phase heterogeneity of aminoglycerophospholipid mixtures by sphingomyelin and monovalent cations: maintenance of the lamellar arrangement in the biological membranes

The phase behaviour of mixed molecular species of phosphatidylethanolamine, phosphatidylserine and sphingomyelin of biological origin were examined in aqueous co-dispersions using synchrotron X-ray diffraction. The co-dispersions of phospholipids studied were aimed to model the mixing of lipids populating the cytoplasmic and outer leaflets in the resting or scrambled activated cell membrane. Mixtures enriched with phosphatidylethanolamine and phosphatidylserine were characterized by a phase separation of non-lamellar phases (cubic and inverted hexagonal) with a lamellar gel phase comprising the most saturated molecular species. Inclusion of sphingomyelin in the mixture resulted in a suppression of the hexagonal-II phase in favour of lamellar phases at temperatures where a proportion of the phospholipid was fluid. The effect was also dependent on the total amount of sphingomyelin in ternary mixtures, and the lamellar phase dominated in mixtures containing more than 30 mol%, irrespective of the relative proportions of phosphatidylserine/sphingomyelin. A transition from gel to liquid-crystal phase was detected by wide-angle scattering during heating scans of ternary mixtures enriched in sphingomyelin and was shown by thermal cycling experiments to be coupled with a hexagonal-II phase to lamellar transition. In such samples there was evidence of a coexistence of non-lamellar phases with a lamellar gel phase. A transition of the gel phase to the fluid state on heating from 35 to 41 °C was evidenced by a progressive increase in the lamellar d-spacing. The presence of calcium enhanced the phase separation of a lamellar gel phase from a hexagonal-II phase in mixtures enriched in phosphatidylserine. This effect was counteracted by charge screening with 150 mM NaCl. The effect of sphingomyelin on stabilizing the lamellar phase is discussed in the context of an altered composition in the cytoplasmic/outer leaflets of the plasma membrane resulting from scrambling of the phospholipid distribution. The results suggest that a lamellar structure can be retained by the inward translocation of sphingomyelin in biological membranes. The presence of monovalent cations serves also to stabilize the bilayer in activated cells where a translocation of aminoglycerophospholipids and an influx of calcium occur simultaneously.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SAXS small-angle X-ray scattering - SM sphingomyelin - WAXS wide-angle X-ray scattering - XRD X-ray diffraction     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe2PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L(beta)) or ripple gel (P(beta)') phase to the liquid crystalline (L(alpha)) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa(-1) depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L(c)) phase to the lamellar gel (L(beta) or L(beta)') phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L(c)/L(beta) transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L(c)/L(alpha) transition was observed at a temperature higher than the main-transition temperature. The main (L(beta)/L(alpha)) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L(beta)', P(beta)' and pressure-induced interdigitated gel (L(beta)I) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe2PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

Persistence at low temperature of the P beta' ripple in dipalmitoylphosphatidylcholine multilamellar vesicles containing either glycosphingolipids or cholesterol

The disappearance and reappearance of the P beta' ripple in multilamellar liposomes of dipalmitoylphosphatidylcholine (DPPC) has been examined by freeze-etch electron microscopy. The presence of less than 10 mol% of various glycosphingolipids or cholesterol in the liposomes markedly increases the time required for ripple disappearance when the vesicles are cooled from 38 degrees C to 30 degrees C, as compared to the pure phospholipid. Once the ripples have begun to disappear in the two-component vesicles, they do not uniformly reappear until the system is heated above the main transition of DPPC and allowed to cool into the pretransition region. These results suggest that the long time for ripple disappearance in the two-component systems reflects a slow molecular reorganization process which occurs when the systems are forced to change from the P beta' gel to the L beta' gel by a temperature downshift.     

Structural aspects of pressure effects on infrared spectra of mixed-chain phosphatidylcholine assemblies in D2O

The barotropic behavior of D2O dispersions of 1-stearoyl-2-caproyl-sn-glycero-3-phosphocholine, C(18):C(10)PC, a highly asymmetric phospholipid in which the length of the fully extended acyl chain at the sn-1 position of the glycerol backbone is twice as long as that at the sn-2 position, has been investigated by high-pressure Fourier transform infrared spectroscopy. This asymmetric phosphatidylcholine bilayer at room temperature displays a pressure-induced phase transition corresponding to the liquid-crystalline----gel phase transition at 1.4 kbar. A conformational ordering of the lipid acyl chains is observed to take place abruptly at the transition pressure of 1.4 kbar. However, the lamellar lipid molecules and their acyl chains remain to be orientationally disordered in the gel phase until the applied pressure reaches 5.5 kbar. In the gel phase of fully hydrated C(18):C(10)PC, the asymmetric lipid molecules assemble into mixed interdigitated bilayers with perpendicular orientation of the zigzag planes among neighboring acyl chains. The role of excess water played in the interchain structure and the behavior of excess water and bound water under high pressure are also discussed.     

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.     

Calorimetric and spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylglycerol bilayer membranes

We have examined the effects of cholesterol (Chol) on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylglycerols (PGs) by high-sensitivity differential scanning calorimetry and Fourier transform infrared and ³¹P NMR spectroscopy. We find that the incorporation of increasing quantities of Chol alters the temperature and progressively reduces the enthalpy and cooperativity of the gel-to-liquid-crystalline phase transition of the host PG bilayer. With dimyristoyl-PG:Chol mixtures, cooperative chain-melting phase transitions are completely or almost completely abolished at Chol concentrations near 50 mol%, whereas with the dipalmitoyl- and distearoyl-PG:Chol mixtures, cooperative hydrocarbon chain-melting phase transitions are still discernable at Chol concentrations near 50 mol%. We are also unable to detect the presence of significant populations of separate domains of the anhydrous or monohydrate forms of Chol in our binary mixtures, in contrast to previous reports. We ascribe the previously reported large scale formation of Chol crystallites to the fractional crystallization of the Chol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. We further show that the direction and magnitude of the change in the phase transition temperature induced by Chol addition is dependent on the hydrocarbon chain length of the PG studied. This finding agrees with our previous results with phosphatidylcholine bilayers, where we found that Chol increases or decreases the phase transition temperature in a hydrophobic mismatch-dependent manner (Biochemistry 1993, 32:516-522), but is in contrast to our previous results for phosphatidylethanolamine (Biochim. Biophys. Acta 1999, 1416:119-234) and phosphatidylserine (Biophys. J. 2000, 79:2056-2065) bilayers, where no such hydrophobic mismatch-dependent effects were observed. We also show that the addition of Chol facilitates the formation of the lamellar crystalline phase in PG bilayers, as it does in phosphatidylethanolamine and phosphatidylserine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of Chol. Moreover, the formation of the lamellar crystalline phase in PG bilayers at lower temperatures excludes Chol, resulting in an apparent Chol immiscibility in gel-state PG bilayers. We suggest that the magnitude of the effect of Chol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipids dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.     

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.