Methylation effects on the microdomain structures of phosphatidylethanolamine monolayers.

Increasing methylation of the headgroup in DPPE results in an increase of minimum area per molecule in highly compressed monolayers at the air-water interface. The shape of solid domains, as observed by epifluorescence microscopy, also exhibits marked changes upon increasing headgroup methylation. Branching domains are observed in DPPE and DP(Me)PE, whereas U-shaped or round domains are observed in DP(Me)2PE and DPPC under our experimental conditions. The domain shape is determined more by the headgroup methylatin than by the corresponding shift in critical temperatures, as shown by the study of PCs of different acyl chain moieties. In mixed lipid monolayers, PC (phosphatidylcholine) and PE (phosphatidylethanolamine) do not mix ideally, as indicated by the non-linear variation of the average area per molecule with composition, and by distinct domain shapes in LE/LC (liquid expanded/liquid condensed) coexisting phases representing PE-enriched or PC-enriched domains in those mixed monolayers.     

Conformational changes of the lecithin headgroup in monolayers at the air/water interface

The headgroup conformation of the phospholipid dipalmitoyl-glycero-phosphocholine (DPPC) in monolayers at the air/water interface has been studied by neutron reflection in the fluid like liquid-expanded (LE) and in the crystal like solid (S) phase. Information on the headgroup conformation in the two phases has been obtained by scattering contrast variation of the lipid monolayer using four differently deuterated species of DPPC: perdeuterated, chain perdeuterated, choline group perdeuterated and selectively headgroup deuterated. Since the measurements were done mainly on a subphase of null reflecting water (i.e. water scattering contrast matched to the air) there is no subphase contribution to reflectivity and the simplest one layer model can be employed for the data analysis, thus minimising the number of free parameters. A remarkable change of the headgroup orientation was observed between the LE and the S phase. We found that the phosphate-nitrogen dipole of the DPPC headgroup exhibits an in-plane orientation with respect to the monolayer in the LE phase but it assumes a more parallel orientation to the surface normal at lateral pressures above 30 mN/m (S phase). Moreover, this conformational change is accompanied by a significant alteration of the headgroup hydration.Abbreviations DPPC Dipalmitoyl-Phosphatidylcholine - DMPC Dimyristoyl-Phosphatidylcholine - DPPE Dipalmitoyl-Phosphatidylethanolamine - DMPE Dimyristoyl-Phosphatidylethanolamine - DMPA Dimyristoyl-Phosphatic Acid - DMPG Dimyristoyl-Phosphatidylglycerol Correspondence to: T M. Bayed     

Molecular organization of surfactin-phospholipid monolayers: effect of phospholipid chain length and polar head

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC>DPPC>DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC>DPPE>DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.     

Molecular structure and interaction of dipalmitoyl phosphatidylcholine in multilayers. Comparative study with phosphatidylethanolamine

As a model of phospholipid bilayers in solid an oriented multilayer film (built-up film) of L-α-dipalmitoyl phosphatidylcholine (DPPC) was prepared from the monolayer by the dipping method. Structural analysis has been carried out by measuring infrared dichroism of the built-up film. The results were compared with those of the built-up film of L-α-dipalmitoyl phosphatidylethanolamine (DPPE). The tilting of the hydrocarbon chains is larger for DPPC than for DPPE. The orientation of the bisector of the two non-esterified PO bonds is closer to the film plane for DPPC than for DPPE. The strong hydrogen bonding interaction between the polar head groups was shown for DPPE, but not for DPPC. These features resemble the structural differences between dilauroyl phosphatidylethanolamine (DLPE) and dimyristoryl phosphatidylcholine (DMPC) in crystals. The hydrogen bonding interaction of DPPE found in solid remains even in the presence of water, namely, in the gel state. More closed packing of the hydrocarbon chains of solid DPPE than DPPC in solid was concluded on the basis of infrared and Raman spectra.     

Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures

Images of giant unilamellar vesicles (GUVs) formed by different phospholipid mixtures (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1, 2-dilauroyl-sn-glycero-3-phosphocholine (DPPC/DLPC) 1:1 (mol/mol), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPE/DPPC), 7:3 and 3:7 (mol/mol) at different temperatures were obtained by exploiting the sectioning capability of a two-photon excitation fluorescence microscope. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN), 6-propionyl-2-dimethylamino-naphthalene (PRODAN), and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE) were used as fluorescent probes to reveal domain coexistence in the GUVs. We report the first characterization of the morphology of lipid domains in unsupported lipid bilayers. From the LAURDAN intensity images the excitation generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domain. On the basis of the phase diagram of each lipid mixture, we found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region in all lipid mixtures. At temperatures corresponding to the phase coexistence region we observed lipid domains of different sizes and shapes, depending on the lipid sample composition. In the case of GUVs formed by DPPE/DPPC mixture, the gel DPPE domains present different shapes, such as hexagonal, rhombic, six-cornered star, dumbbell, or dendritic. At the phase coexistence region, the gel DPPE domains are moving and growing as the temperature decreases. Separated domains remain in the GUVs at temperatures corresponding to the solid region, showing solid-solid immiscibility. A different morphology was found in GUVs composed of DLPC/DPPC 1:1 (mol/mol) mixtures. At temperatures corresponding to the phase coexistence, we observed the gel domains as line defects in the GUV surface. These lines move and become thicker as the temperature decreases. As judged by the LAURDAN GP histogram, we concluded that the lipid phase characteristics at the phase coexistence region are different between the DPPE/DPPC and DLPC/DPPC mixtures. In the DPPE/DPPC mixture the coexistence is between pure gel and pure liquid domains, while in the DLPC/DPPC 1:1 (mol/mol) mixture we observed a strong influence of one phase on the other. In all cases the domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This observation is also novel for unsupported lipid bilayers.     

Molecular simulation study of structural and dynamic properties of mixed DPPC/DPPE bilayers

Molecular dynamics simulations have been used to study structural and dynamic properties of fully hydrated mixed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) bilayers at 0, 25, 50, 75, and 100 mol % DPPE. Simulations were performed for 50 ns at 350 K and 1 bar for the liquid-crystalline state of the mixtures. Results show that the average area per headgroup reduces from 0.65 +/- 0.01 nm(2) in pure DPPC to 0.52 +/- 0.01 nm(2) in pure DPPE systems. The lipid tails become more ordered with increasing DPPE concentration, resulting in a slight increase in membrane thickness (3.43 +/- 0.01 nm in pure DPPC to 4.00 +/- 0.01 nm in pure DPPE). The calculated area per headgroup and order parameter for pure DPPE deviates significantly from available experimental measurements, suggesting that the force field employed requires further refinement. In-depth analysis of the hydrogen-bond distribution in DPPE molecules shows that the amine groups strongly interact with the phosphate and carbonyl groups through inter/intramolecular hydrogen bonds. This yields a bilayer structure with DPPE headgroups preferentially located near the lipid phosphate and ester oxygens. It is observed that increasing DPPE concentrations causes competitive hydrogen bonding between the amine groups (hydrogen-donor) and the phosphate/carbonyl groups or water (hydrogen-acceptor). Due to the increasing number of hydrogen-donors from DPPE molecules with increasing concentration, DPPE becomes more hydrated. Trajectory analysis shows that DPPE molecules in the lipid mixtures move laterally and randomly around the membrane surface and the movement becomes more localized with increasing DPPE concentrations. For the conditions and simulation time considered, no aggregation or phase separation was observed between DPPC and DPPE.     

Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy

Epifluorescence microscopy was used to investigate the effect of cholesterol on monolayers of dipalmitoylphosphatidylcholine (DPPC) and 1 -palmitoyl-2-oleoyl phosphatidylcholine (POPC) at 21 +/- 2 degrees C using 1 mol% 1-palmitoyl-2-[12-[(7-nitro-2-1, 3-benzoxadizole-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) as a fluorophore. Up to 30 mol% cholesterol in DPPC monolayers decreased the amounts of probe-excluded liquid-condensed (LC) phase at all surface pressures (pi), but did not effect the monolayers of POPC, which remained in the liquid-expanded (LE) phase at all pi. At low pi (2-5 mN/m), 10 mol% or more cholesterol in DPPC induced a lateral phase separation into dark probe-excluded and light probe-rich regions. In POPC monolayers, phase separation was observed at low pi when > or =40 mol% or more cholesterol was present. The lateral phase separation observed with increased cholesterol concentrations in these lipid monolayers may be a result of the segregation of cholesterol-rich domains in ordered fluid phases that preferentially exclude the fluorescent probe. With increasing pi, monolayers could be transformed from a heterogeneous dark and light appearance into a homogeneous fluorescent phase, in a manner that was dependent on pi and cholesterol content. The packing density of the acyl chains may be a determinant in the interaction of cholesterol with phosphatidylcholine (PC), because the transformations in monolayer surface texture were observed in phospholipid (PL)/sterol mixtures having similar molecular areas. At high pi (41 mN/m), elongated crystal-like structures were observed in monolayers containing 80-100 mol% cholesterol, and these structures grew in size when the monolayers were compressed after collapse. This observation could be associated with the segregation and crystallization of cholesterol after monolayer collapse.     

Molecular organization of surfactin-phospholipid monolayers: Effect of phospholipid chain length and polar head

Mixed monolayers of the surface-active lipopeptide surfactin-C₁₅ and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC > DPPC > DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC > DPPE > DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.     

Molecular studies of the gel to liquid-crystalline phase transition for fully hydrated DPPC and DPPE bilayers

Molecular dynamics simulations were used for a comprehensive study of the structural properties of saturated lipid bilayers, DPPC and DPPE, near the main phase transition. Though the chemical structure of DPPC and DPPE are largely similar (they only differ in the choline and ethanolamine groups), their transformation process from a gel to a liquid-crystalline state is contrasting. For DPPC, three distinct structures can be identified relative to the melting temperature (Tm): below Tm with "mixed" domains consisting of lipids that are tilted with partial overlap of the lipid tails between leaflet; near Tm with a slight increase in the average area per lipid, resulting in a rearrangement of the lipid tails and an increase in the bilayer thickness; and above Tm with unhindered lipid tails in random motion resulting in an increase in %gauche formed and increase in the level of interdigitation between lipid leaflets. For DPPE, the structures identified were below Tm with "ordered" domains consisting of slightly tilted lipid tails and non-overlapping lipid tails between leaflets, near Tm with minimal rearrangement of the lipids as the bilayer thickness reduces slightly with increasing temperature, and above Tm with unhindered lipid tails as that for DPPC. For DPPE, most of the lipid tails do not overlap as observed to DPPC, which is due to the tight packing of the DPPE molecules. The non-overlapping behavior of DPPE above Tm is confirmed from the density profile of the terminal carbon atoms in each leaflet, which shows a narrow distribution near the center of the bilayer core. This study also demonstrates that atomistic simulations are capable of capturing the phase transition behavior of lipid bilayers, providing a rich set of molecular and structural information at and near the transition state.     

Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines.

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atomic force microscopy studies of ganglioside GM1 domains in phosphatidylcholine and phosphatidylcholine/cholesterol bilayers.

The distribution of ganglioside in supported lipid bilayers has been studied by atomic force microscopy. Hybrid dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylethanolamine (DPPE) and (2:1 DPPC/cholesterol)/DPPE bilayers were prepared using the Langmuir Blodgett technique. Egg PC and DPPC bilayers were prepared by vesicle fusion. Addition of ganglioside GM1 to each of the lipid bilayers resulted in the formation of heterogeneous surfaces that had numerous small raised domains (30--200 nm in diameter). Incubation of these bilayers with cholera toxin B subunit resulted in the detection of small protein aggregates, indicating specific binding of the protein to the GM1-rich microdomains. Similar results were obtained for DPPC, DPPC/cholesterol, and egg PC, demonstrating that the overall bilayer morphology was not dependent on the method of bilayer preparation or the fluidity of the lipid mixture. However, bilayers produced by vesicle fusion provided evidence for asymmetrically distributed GM1 domains that probably reflect the presence of ganglioside in both inner and outer monolayers of the initial vesicle. The results are discussed in relation to recent inconsistencies in the estimation of sizes of lipid rafts in model and natural membranes. It is hypothesized that small ganglioside-rich microdomains may exist within larger ordered domains in both natural and model membranes.     

Nuclear magnetic resonance and thermal studies on the interaction between salicylic acid and model membranes

DSC and (1H and 31P) NMR measurements are used to investigate the perturbation caused by the keratolytic drug, salicylic acid (SA) on the physicochemical properties of the model membranes. Model membranes (in unilamellar vesicular (ULV) form) in the present studies are prepared with the phospholipids, dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidic acid (DPPA) and mixed lipid DPPC-DPPE (with weight ratio, 2.5:2.2). These lipids have the same acyl (dipalmitoyl) chains but differed in the headgroup. The molar ratio of the drug to lipid (lipid mixture), is in the range 0 to 0.4. The DSC and NMR results suggest that the lipid head groups have a pivotal role in controlling (i) the behavior of the membranes and (ii) their interactions with SA. In the presence of SA, the main phase transition temperature of (a) DPPE membrane decreases, (b) DPPA membrane increases and (c) DPPC and DPPC-DPPE membranes are not significantly changed. The drug increases the transition enthalpy (i.e., acyl chain order) in DPPC, DPPA and DPPC-DPPE membranes. However, the presence of the drug in DPPC membrane formed using water (instead of buffer), shows a decrease in the transition temperature and enthalpy. In all the systems studied, the drug molecules seem to be located in the interfacial region neighboring the glycerol backbone or polar headgroup. However, in DPPC-water system, the drug seems to penetrate the acyl chain region also.     

Lipid headgroup discrimination by antimicrobial peptide LL-37: insight into mechanism of action

Interaction of the human antimicrobial peptide LL-37 with lipid monolayers has been investigated by a range of complementary techniques including pressure-area isotherms, insertion assay, epifluorescence microscopy, and synchrotron x-ray scattering, to analyze its mechanism of action. Lipid monolayers were formed at the air-liquid interface to mimic the surface of the bacterial cell wall and the outer leaflet of erythrocyte cell membrane by using phosphatidylglycerol (DPPG), phosphatidylcholine (DPPC), and phosphatidylethanolamine (DPPE) lipids. LL-37 is found to readily insert into DPPG monolayers, disrupting their structure and thus indicating bactericidal action. In contrast, DPPC and DPPE monolayers remained virtually unaffected by LL-37, demonstrating its nonhemolytic activity and lipid discrimination. Specular x-ray reflectivity data yielded considerable differences in layer thickness and electron-density profile after addition of the peptide to DPPG monolayers, but little change was seen after peptide injection when probing monolayers composed of DPPC and DPPE. Grazing incidence x-ray diffraction demonstrated significant peptide insertion and lateral packing order disruption of the DPPG monolayer by LL-37 insertion. Epifluorescence microscopy data support these findings.     

Orientation of gramicidin D incorporated into phospholipid multibilayers: a Fourier transform infrared-attenuated total reflection spectroscopic study

Polarized Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy was applied to study the orientation of the linear pentadecapeptide antibiotic gramicidin D incorporated into phospholipid multibilayers, which were cast on a germanium ATR plate from chloroform solution. In DMPC and DPPC multibilayers, the CH2 stretching bands of lipid hydrocarbon chains were slightly shifted to the higher frequency side and bandwidth was increased in the presence of gramicidin. However, in DPPE multibilayers, frequencies and bandwidths of these bands were unaltered. In each case, gramicidin produced little effect on the orientation of lipid hydrocarbon chains, suggesting that gramicidin penetrates into lipid layers without noticeable perturbations. Upon incubation of cast films in contact with water above the gel-liquid-crystalline transition temperature (Tc) of lipids, the reorientation of gramicidin in lipid multibilayers occurred, the degree thereof depending upon the fluidity of the lipid hydrocarbon chains and the amount of surrounding water. In DMPC multibilayers, the helix axis of gramicidin was oriented almost parallel to the lipid hydrocarbon chains after incubation. In DPPC multibilayers, on the other hand, the helix axis of gramicidin was tilted on average about 15 degrees from the lipid hydrocarbon chains after incubation. However, in DPPE multibilayers, which are known to have the most rigid bilayer structures, the reorientation of gramicidin could not be seen.     

The effect of methylation of phosphatidylethanolamine on the behaviour of lipid monolayers at the air-water interface

Monolayers of DPPE and its N-methylated derivatives including DPPC have been investigated at 23 and 37 degrees C using a modified Langmuir-Wilhelmy surface balance. The monolayers have been subjected to dynamic compression and expansion, and some characteristics of the surfaces have been determined. The minimum surface tension attained by surfaces containing the lipids (maximum surface pressures sustained by the films) depended on the extent of methylation of the head group. Monolayers of DPPE or N-MeDPPE collapsed at surface tensions of 12-16 mN.m-1, whereas those containing N,N-diMeDPPE and DPPC could be compressed to near zero surface tension. The areas per molecule occupied by these lipids under high compression varied slightly and not systematically with head-group methylation. Monolayers containing mixtures of DPPC and DPPE were also studied under the same conditions. The monolayers showed some deviation from the behaviour expected if they were to have characteristics of ideally mixed systems. The minimum surface tensions attained suggested that monolayers containing 50 mol% or more DPPC might be further enriched during compression by some selective exclusion of the DPPE. At high surface pressures, some positive deviations in nominal areas per molecule from that expected for ideal mixing were observed in the monolayers made with 50 mol% or more DPPC. These deviations might be caused by packing disruptions associated with the explosion of lipid from the films.     

The influence of headgroup structure and fatty acyl chain saturation of phospholipids on monolayer behavior: a comparative rheological study

This paper compares six phospholipidic monolayers at the water/chloroform interface by performing dilational rheological measurements with a drop tensiometer apparatus. The chosen lipids differ both in their headgroup structure and fatty acyl chain saturation or symmetry. The study concentrated on monolayers formed with DPPC, DPPE, DOPC, DOPE, POPC and POPE. Using a generalized Maxwell rheological model, transposed at the interface, the intimate intermolecular interactions between amphiphilic molecules are studied on and off the monolayer plane. The equilibrium and nonequilibrium phenomena are analyzed and, respectively, correlated with monolayer cohesion and with monolayer/sub-surface interactions. The purpose of this work is to gain further insights into the influences (as slight as they are) of the weak changes in phospholipid structure and on the behavior of the monolayers. The results, widely described, provide further details on nuances existing between very similar molecules, and likewise, on the synergies created between the different effects.     

Anomalous mixing of zwitterionic and anionic phospholipids with double-chain cationic amphiphiles in lipid bilayers.

High-sensitivity scanning calorimetry has been used to examine the thermotropic behavior of mixtures combining dipalmitoylphosphatidylcholine (DPPC), phosphatidylethanolamine (DPPE) and O-methylphosphatidic acid (DPPA-OMe) with the double-chain cationic amphiphiles N,N-dihexadecyl-N,N- dimethylammonium chloride (DHDAC), 1,2-dipalmitoyloxy-3-(trimethylammonio)propane (DPTAP) and the corresponding monomethylated tertiary amino compounds (DHMMA-H+ and DPDAP-H+). At physiological ionic strength, mixtures of these cationic amphiphiles with the anionic phospholipid DPPA-OMe can show gel-to-liquid-crystalline phase transitions at considerably higher temperatures than do either of the pure components. Surprisingly, binary mixtures of DPPC and these cationic amphiphiles also show strongly nonideal mixing, with phase diagrams exhibiting pronounced maxima in their solidus and liquidus curves. Similar behavior is not observed for mixtures of DPPC with DPPA-OMe, which closely resembles DPTAP and DPDAP-H+ in backbone configuration and polar headgroup size. The present results suggest that perturbation of the orientation of the phosphatidylcholine headgroup by cationic amphiphiles, as demonstrated previously by Seelig and co-workers (Biochemistry 28 [1989], 7720-7728), can significantly affect the thermotropic behavior of phospholipids such as DPPC. Such effects may exert a generally important (though not always easily recognizable) influence on the organization and thermotropic behavior of systems where zwitterionic phospholipids are combined with charged bilayer-associated molecules.     

A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).     

Landau theory of two-component phospholipid bilayers. I. Phosphatidylcholine/phosphatidylethanolamine mixtures

Priest's phenomenological model (Mol. Cryst. Liq. Cryst. 60 (1980) 167.) on one- and two-component PC bilayers is extended here. We constructed a new excess free energy term in the state function to describe the thermodynamic properties of the two-component phospholipid bilayers where the chain lengths and the polar heads of the components can be different simultaneously. By means of this generalized state function, we can calculate the phase diagrams of DPPC/DPPE, DMPC/DMPE, DMPC/DPPE, DPPC/DMPE and DSPC/DMPE mixtures. We obtained complete miscibility both in the liquid crystalline and in the gel phase if the chain lengths of the components were the same. If the chain length of the PE component was longer than that of the PC component, we obtained a peritectic system. A eutectic system was obtained in the reverse case. The results of the model were compared with the experimental data available. Applying the quasichemical approximation, we determined the molecular meaning of the phenomenological model parameters. Namely, sigma and gamma are proportional to the sublimation heat of the CH2 group in the long-chain alkanes and to the hydrogen-bonding energy between the polar heads of the ethanolamines; otherwise the model resulted in--1.94 kcal/mol per CH2 for the sublimation heat and --1.4 kcal/mol for the hydrogen-bond energy.