Structure and thermal history dependant phase behavior of hydrated synthetic sphingomyelin analogue: 1,2-dimyristamido-1,2-deoxyphosphatidylcholine

The physical properties of hydrated multilamellar sample of 1,2-dimyristamido-1,2-deoxyphosphatidylcholine (DDPC) were investigated by means of differential scanning calorimetry (DSC), static X-ray diffraction, and simultaneous DSC and X-ray diffraction. The DDPC is a synthetic sphingomyelin analogue and has two amide bonds in its hydrophobic parts. This paper reports on metastable phase behavior of the hydrated DDPC sample. By cooling from a chain-melted state at the rates of greater than 4 °C min⁻¹, hydrated DDPC bilayers form a metastable gel phase. In the gel phase, the hydrophobic chains are tilted with respect to the bilayer normal, as like the gel phase of glycero-phosphatidylcholines. By heating, the metastable gel phase is transformed in to a stable phase associated with an exothermic heat event at 18.3 °C (ΔH = 14.6 kJ mol⁻¹) and then the stable phase is transformed into a liquid-crystalline phase at 25.6 °C (ΔH = 42 kJ mol⁻¹). The incubation at 17 °C for more than 1 h also induces the formation of the stable phase. In the stable phase, the hydrophobic chains are packed into highly ordered crystal-like structure. However, the X-ray diffraction pattern of the stable phase suggested that the entire DDPC molecules do not form a two-dimensional molecular ordered lattice, differing from normal subgel phase of glycero-phosphatidylcholines. The structure and phase behavior of DDPC revealed by the present study are discussed from the viewpoint of hydrogen bonds.     

Synthesis and characterization of 1,2-dimyristoylamido-1, 2-deoxyphosphatidylcholine as an artificial boundary lipid

The synthesis and characterization of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), are described. DDPC has two amide bonds instead of ester bonds of regular lecithins such as 1,2-dimyristoylphosphatidylcholine (DMPC). In differential scanning calorimetry (DSC) measurements, DDPC gave two endothermic peaks: one was at 18.0 degrees C (delta H = 10.74 kJ.mol-1) and the other at 23.0 degrees C (delta H = 12.91 kJ.mol-1). The former peak was sharp and considered to be the phase transition of the hydrocarbon region, while the latter was assigned to the melt of the hydrogen-belt formed by the amide groups of DDPC. Addition of DDPC to DMPC made the DMPC membrane less fluid in the region close to the surface, and significantly increased the reconstitution efficiency of glycophorin into the membrane. This effect of DDPC was much larger than that of naturally occurring lipid, sphingomyelin.     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

Comparison of the effects of NaCl on the thermotropic behaviour of sn-1' and sn-3' stereoisomers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol

The phase behaviour of liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG) and the corresponding sn-3' stereoisomer (3'-DMPG) were studied by DSC as a function of NaCl concentration. The melting of the metastable gel phase to the liquid-crystalline phase was similar for both lipids. However, in the presence of salt and at 6 degrees C (T less than Tp) the gel phase of both stereoisomers of DMPG was shown to be metastable and a new phase nominated here as the highly crystalline phase was formed as the stable state. However, significant differences in the formation and melting of the highly crystalline phase were evident between the two polar headgroup stereoisomers. For 3'-DMPG in the presence of 300 mM NaCl the melting enthalpy of this phase is approx. 82 kJ/mol and the transition temperature about 11 degrees higher (at 33.6 degrees C) than for the gel to liquid-crystalline phase transition (25 kJ/mol at 23.0 degrees C). In the presence of 0.15-1.2 M NaCl at 6 to 10 degrees C the formation of the highly crystalline phase of 3'-DMPG is complete within 2 to 5 days, increasing [NaCl] facilitates the rate. For a 1:1 mixture of 1'- and 3'-DMPG the formation of the highly crystalline phase requires several weeks and melts at about 20 degrees higher than the gel phase (at approx. 40 degrees C). For 1'-DMPG partial conversion into the highly crystalline phase requires several months. For 3'-DMPG several intermediate phases appeared as endothermic peaks between the main phase transition temperature and the melting temperature of the highly crystalline phase. In contrast, for 1'-DMPG and the 1:1 mixture the subgel phase appears to be the only metastable intermediate phase. Different monovalent cations differ in their effect on the metastable behaviour.     

Structure and thermotropic properties of phosphatidylethanolamine and its N-methyl derivatives

The structure and thermotropic properties of hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine (DMPE) and its N-monomethyl (mmDMPE) and N,N-dimethyl (dmDMPE) derivatives have been investigated by differential scanning calorimetry and X-ray diffraction. For DMPE, mmDMPE, and dmDMPE, multilamellar dispersions (approximately 50 wt % water) show chain melting bilayer gel----bilayer liquid-crystal transitions (onset) at 49.2, 42.3, and 30.7 degrees C, respectively, with the corresponding value for 1,2-dimyristoyl-sn-glycero-3-phosphocholine occurring at 23 degrees C. Thus, the bilayer chain melting transition decreases with increasing N-methylation, as originally reported for the corresponding palmitoyl series [Vaughan, D.J., & Keough, K.M. (1974) FEBS Lett. 47, 158-161]. This transition is reversible on cooling, and DMPE, mmDMPE, and dmDMPE form the original bilayer gel phase with the rotationally disordered hydrocarbon chains packed in a hexagonal lattice. Following prolonged incubation at -4 degrees C, the bilayer gel phase is shown to be metastable, and conversion to a low-temperature "crystalline" phase occurs with the hydrocarbon chains adopting a specific packing mode. For DMPE, mmDMPE, and dmDMPE, either a single or a double endothermic transition occurs as the "crystal" bilayer phase converts to the bilayer gel phase. A similar pattern of behavior is observed for the palmitoyl series. The relatively slow kinetic conversion of the metastable bilayer gel phase with hexagonally packed hydrocarbon chains to a bilayer phase in which the chains have "crystallized" appears to be a general property of membrane phospholipids and sphingolipids.     

Structure and interactions of ether- and ester-linked phosphatidylethanolamines

The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP)

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4 degrees C upon heating and two exothermic transitions between 20 and 30 degrees C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid--crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid--crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4 degrees C, this structure converts to a liquid-crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid--crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.     

Synthesis, structure, and thermal properties of 1,2-dipalmitoylgalloylglycerol (DPGG), a novel self-adhering lipid

A novel diacyl glycerol-based lipid with a polyphenolic head group has been synthesized and characterized. X-ray diffraction experiments show that this lipid, 1,2-dipalmitoylgalloylglycerol (DPGG), hydrates to form gel phase bilayers at 20 degrees C with extremely narrow interbilayer fluid separations, indicating that apposing DPGG bilayers strongly adhere to each other. Differential scanning calorimetry shows that fully hydrated DPGG exhibits a pretransition exotherm (3.7 kcal/mol) at 52 degrees C and a high enthalpy (11.3 kcal/mol) main endothermic transition at 69 degrees C. These thermal properties are similar to those of galactosylceramides with similar hydrocarbon chain compositions. The adhesive and thermal properties of DPGG are likely due to both intermolecular hydrogen-bonding and hydrophobic interactions between the aromatic rings on the gallic acids.     

Structure and properties of totally synthetic galacto- and gluco-cerebrosides

The structural and thermal properties of aqueous dispersions of the totally synthetic cerebrosides, D-erythro-N-palmitoyl galactosyl- and glucosyl-C18-sphingosine (C16:0-GalCer and C16:0-GluCer, respectively) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction. Over the temperature range 0-100 degrees C, both C16:0-GalCer and C16:0-GluCer show complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating, hydrated C16:0-GalCer undergoes an exothermic bilayer-bilayer transition at 59 degrees C to produce a stable bilayer crystal form. X-ray diffraction at 70 degrees C reveals a bilayer structure with an ordered hydrocarbon chain-packing arrangement. This ordered bilayer phase undergoes an endothermic chain-melting transition at 85 degrees C to the bilayer liquid crystalline state. Similar behavior is exhibited by hydrated C16:0-GluCer which undergoes the exothermic transition at 49 degrees C and a chain-melting transition at 87 degrees C. The exothermic transitions observed on heating hydrated C16:0-GalCer and C16:0-GluCer are irreversible and dependent upon previous chain melting, prior cooling rate, and time of incubation at low temperatures. Thus, the structure and properties of totally synthetic C16:0-GalCer and C16:0-GluCer with identical sphingosine (C18:1) and fatty acid (C16:0) chains are quite similar, suggesting that the precise isomeric structure of the linked sugar plays only a minor role in regulating the properties of hydrated cerebrosides. Further, these studies indicate that the complex thermal behavior and bilayer phase formation exhibited by these single-sugar cerebrosides are intrinsic properties and not due to the heterogeneity of the sphingosine base found in natural and partially synthetic cerebrosides.     

Refined structure of 1,2-diacyl-P-O-ethylphosphatidylcholine bilayer membranes

1,2-diacyl-P-O-ethylphosphatidylcholines are synthetic cationic lipids that show some promising properties as nonviral DNA transfection agents. To gain further insight in the effects of the additional ethyl group in the headgroup region on the bilayer structure we systematically investigated a homologous series of fully hydrated ethylphosphatidylcholines with linear saturated chains (C14:0, C16:0, and C18:0) by small- and wide-angle X-ray diffraction. Our data show that all of them form multilamellar vesicles with chain interdigitated gel phases. Paying regard to the very importance of the liquid-crystalline phase in gene transfection, we applied the novel MCG method on high resolution X-ray diffraction data of the C16:0 derivative to be able to gain structural information on this phase. Comparison of this ethylphosphatidylcholine with its parent compound, the unesterified phosphatidylcholine, revealed that the major difference in the liquid-crystalline phase is the significantly reduced water layer between the bilayers for the cationic lipid. This may be one factor that contributes to the comparatively good DNA transfection efficiency of ethylphosphatidylcholines.     

Reorientational and Conformational Ordering Processes at Elevated Pressures in 1,2-Dioleoyl Phosphatidylcholine: A Raman and Infrared Spectroscopic Study

Raman and infrared spectra of fully hydrated bilayers of 1,2-dioleoyl phosphatidylcholine (DOPC) were measured at increasing hydrostatic pressures up to -37 kbar. Under ambient conditions aqueous dispersions of DOPC are in the liquid crystalline state. The application of an external hydrostatic pressure induces conformational and dynamic ordering processes in DOPC, which trigger a first-order structural phase transition at 5 kbar from a disordered liquid crystalline state to a highly ordered gel state. In the gel phase the methylene chains of each molecule are fully extended and the two all-trans chain segments on both sides of the rigid cis double bond form a bent structure. The bent oleoyl chains in each molecule, as well as in neighboring molecules are packed parallel to each other. To achieve this parallel interchain packing, the double bonds of the sn-1 and sn-2 chains of each molecule must be aligned at the same position with respect to the bilayer interface which is achieved by a rotation of the C—C bonds in the glycerol moiety in the head group. The extremely strong interchain interactions in the gel phase of DOPC are unique for this lipid with cis dimono-unsaturated acyl chains. Our experimental results suggest that in the pressure-induced gel phase of DOPC the olefinic CH bonds are rotated out of the phase of the bent oleoyl chains and that the oleoyl chains of opposing bilayers bend towards opposite directions.     

A Fourier-transform infrared spectroscopic study of the polymorphic phase behavior of 1,2- bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine; a polymerizable lipid which forms novel microstructures

We have investigated the phase characteristics of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC23PC), a phosphatidylcholine with diacetylenic groups in the acyl chains, and its saturated analog 1,2-ditricosanoyl-sn-glycero-3-phosphocholine (DTPC), using Fourier-transform infrared spectroscopy (FTIR). Previous studies on the phase behavior of DC23PC in H2O have shown that DC23PC exhibits: (1) formation of cylindrical structures ('tubules') by cooling fluid phase multilamellar vesicles (MLVs) through Tm (43 degrees C), and 2) metastability of small unilamellar vesicles (SUVs) in the liquid-crystalline state some 40 degrees C below Tm, with subsequent formation of a gel phase comprised of multilamellar sheets at 2 degrees C. The sheets form tubules when heated and cooled through Tm. FTIR results presented here indicate that as metastable SUVs are cooled toward the transition to bilayer sheets, spectroscopic changes occur before the calorimetric transition as measured by a reduction in the CH2 symmetric stretch frequency and bandwidth. In spite of the vastly different morphologies, the sheet gel phase formed from SUVs is spectroscopically similar to the tubule gel phase. The C-H stretch region of DC23PC gel phase shows bands at 2937 and 2810 cm-1 not observed in the saturated analog of DC23PC, which may be related to perturbations in the acyl chains introduced by the diacetylenic moiety. The narrow CH2 scissoring mode at 1470 cm-1 and the prominent CH2 wagging progression indicate that DC23PC gel phase was highly ordered acyl chains with extended regions of all-trans methylene segments. In addition, the 13 cm-1 reduction in the C = O stretch frequency (1733-1720 cm-1) during the induction of DC23PC gel phase indicates that the interfacial region is dehydrated and rigid in the gel phase.     

Effect of chain unsaturation on the structure and thermotropic properties of galactocerebrosides.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the effect of increasing chain-unsaturation on the structure and properties of the hydrated cerebrosides N-stearoyl, -oleoyl, and -linoleoyl galactosylsphingosine (NSGS, NOGS, and NLnGS, respectively). DSC of hydrated (70 wt% water) NSGS shows an endothermic transition at 85 degrees C (delta H = 18.0 kcal/mol NSGS) and a broad exothermic transition at 40-60 degrees C, the latter being dependent upon the previous cooling rate. X-Ray diffraction patterns recorded at 21, 61, and 86 degrees C provide evidence for interconversions between metastable and stable crystalline NSGS bilayer phases. The properties of the unsaturated-chain cerebrosides are more complex. Hydrated NOGS shows a single endothermic transition at 44.8 degrees C (delta H = 11.5 kcal/mol NOGS). However, incubation of NOGS at 49 degrees C for 24 h results in a second transition at 55.5 degrees C. By cycling NOGS between 0 and 49 degrees C complete conversion into this higher melting phase (delta H = 12.1 kcal/mol NOGS) is achieved. X-ray diffraction confirms a bilayer phase at all temperatures and delineates the conversions between a crystalline phase at 21 degrees C (bilayer period d = 56.5A), a second crystalline phase at 47 degrees C (d = 69.9A), and a liquid crystalline phase at 59 degrees C (d = 52.0A). The more unsaturated NLnGS shows two transitions, a sharp transition at 28 degrees C (delta H = 8.0 kcal/mol NLGS) and a broad, low-enthalpy transition at 42 degrees C (delta H = 0.4 kcal/mol NLGS). Again, incubation between the two transitions leads to a single transition at 44 degrees C (delta H = 9.3 kcal/mol NLGS). X-ray diffraction demonstrates conversions between two crystalline bilayer phases (d = 55.2A and d = 68.4A), and a liquid crystalline bilayer phase (d = 51.8A). Thus, increased unsaturation in the amide-linked fatty acyl chain of cerebrosides results in decreased chain-melting temperatures (NSGS greater than NOGS greater than NLnGS) and has marked effects on their structural properties.     

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.     

Physical properties of glycosyl diacylglycerols. 1. Calorimetric studies of a homologous series of 1,2-di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

The polymorphic phase behavior of aqueous dispersions of a homologous series of 1,2-di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols was studied by differential scanning calorimetry. At fast heating rates unannealed samples of these lipids exhibit a strongly energetic transition, which has been identified as a lamellar gel/liquid crystalline (L beta/L alpha) phase transition (short- and medium-chain compounds) or a lamellar gel to inverted hexagonal (L beta/HII) phase transition (long-chain compounds) by X-ray diffraction studies (Sen et al., 1990). At still higher temperatures, some of the lipids that form lamellar liquid-crystalline phases exhibit an additional transition, which has been identified as a transition to an inverted nonbilayer phase by X-ray diffraction studies. The lamellar gel phase formed on initial cooling of these lipids is a metastable structure, which, when annealed under appropriate conditions, transforms to a more stable lamellar gel phase, which has been identified as a poorly hydrated crystal-like phase with tilted acyl chains by X-ray diffraction measurements (Sen et al., 1990). With the exception of the di-19:0 homologue, the crystalline phases of these lipids are stable to temperatures higher than those at which their L beta phases melt and, as a result, they convert directly to L alpha or HII phases on heating. Our results indicate that the length of the acyl chain affects both the kinetic and thermodynamic properties of the crystalline phases of these lipids as well as the type of nonbilayer phase that they form. Moreover, when compared with the beta-anomers, these alpha-D-glucosyl diacylglycerols are more prone to form ordered crystalline gel phases at low temperatures and are somewhat less prone to form nonbilayer phases at elevated temperatures. Thus the physical properties of glucolipids (and possibly all glycolipids) are very sensitive to the nature of the anomeric linkage between the sugar headgroup and the glycerol backbone of the lipid molecule. We suggest that this is, in part, due to a change in orientation of the glucopyranosyl ring relative to the bilayer surface, which in turn affects the way(s) in which the sugar headgroups interact with each other and with water.     

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.     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

Formation of a new stable phase of phosphatidylglycerols.

Dilauroyl and dimyristoylphosphatidylglycerol (DMPG) form a more stable gel state when aqueous suspensions are incubated several days at low temperature (0-2 degrees C), pH 7.4 with 0.15 M NaCl. This gel state is characterized by a higher transition temperature and a higher transition enthalpy. The geometry of this gel state is distinguishable from the metastable gel state that forms rapidly upon hydration on the basis of its x-ray diffraction pattern. Infrared spectra in the CH2 scissoring region indicate that the stable gel phase of DMPG is also characterized by reduced reorientational fluctuations of acyl chains and increased interchain interactions. Analysis of vibrational bands due to ester carbonyl groups of DMPG suggests that the transition to a new gel phase is initiated by changes in the interfacial and/or headgroup region of the bilayer, most likely via formation of interlipid hydrogen bonds. The melting of the stable gel phase of DMPG is accompanied by a gross morphological change resulting in vesiculation.     

An ordered metastable phase in hydrated phosphatidylethanolamine: the Y-transition

By using time-resolved X-ray diffraction, differential scanning calorimetry and scanning densitometry, we observed rapid formation at low temperature of a metastable ordered phase, termed LR1 phase, in fully hydrated dihexadecylphosphatidylethanolamine (DHPE). The LR1 phase has the same lamellar repeat period as the gel Lbeta phase but differs from the latter in its more ordered, orthorhombic hydrocarbon chain arrangement. It forms at about 12 degrees C upon cooling and manifests itself as splitting of the sharp, symmetric wide-angle X-ray peak of the DHPE gel phase into two reflections. This transition, designated the 'Y-transition', is readily reversible and proceeds with almost no hysteresis between cooling and heating scans. Calorimetrically, the LR1-->Lbeta transition is recorded as a low-enthalpy (0.2 kcal/mol) endothermic event. The formation of the LR1 phase from the gel phase is associated with a small, about 2 microl/g, decrease of the lipid partial specific volume recorded by scanning densitometry, in agreement with a volume calculation based on the X-ray data. The formation of the equilibrium Lc phase was found to take place from within the LR1 phase. This appears to be the only observable pathway for crystallisation of DHPE upon low-temperature incubation. Once formed, the Lc phase of this lipid converts directly into Lbeta phase at 50 degrees C, skipping the LR1 phase. Thus, the LR1 phase of DHPE can only be entered by cooling of the gel Lbeta phase. The data disclose certain similarities between the low-temperature polymorphism of DHPE and that of long-chain normal alkanes.     

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.