Influence of the lamellar phase unbinding energy on the relative stability of lamellar and inverted cubic phases

Based on curvature energy considerations, nonbilayer phase-forming phospholipids in excess water should form stable bicontinuous inverted cubic (Q_II) phases at temperatures between the lamellar (L_α) and inverted hexagonal (H_II) phase regions. However, the phosphatidylethanolamines (PEs), which are a common class of biomembrane phospholipids, typically display direct L_α/H_II phase transitions and may form intermediate Q_II phases only after the temperature is cycled repeatedly across the L_α/H_II phase transition temperature, T_H, or when the H_II phases are cooled from T > T_H. This raises the question of whether models of inverted phase stability, which are based on curvature energy alone, accurately predict the relative free energy of these phases. Here we demonstrate the important role of a noncurvature energy contribution, the unbinding energy of the L_α phase bilayers, g_u, that serves to stabilize the L_α phase relative to the nonlamellar phases. The planar L_α phase bilayers must separate for a Q_II phase to form and it turns out that the work of their unbinding can be larger than the curvature energy reduction on formation of Q_II phase from L_α at temperatures near the L_α/Q_II transition temperature (T_Q). Using g_u and elastic constant values typical of unsaturated PEs, we show that g_u is sufficient to make T_Q > T_H for the latter lipids. Such systems would display direct L_α → H_II transitions, and a Q_II phase might only form as a metastable phase upon cooling of the H_II phase. The g_u values for methylated PEs and PE/phosphatidylcholine mixtures are significantly smaller than those for PEs and increase T_Q by only a few degrees, consistent with observations of these systems. This influence of g_u also rationalizes the effect of some aqueous solutes to increase the rate of Q_II formation during temperature cycling of lipid dispersions. Finally, the results are relevant to protocols for determining the Gaussian curvature modulus, which substantially affects the energy of intermediates in membrane fusion and fission. Recently, two such methods were proposed based on measuring T_Q and on measuring Q_II phase unit cell dimensions, respectively. In view of the effect of g_u on T_Q that we describe here, the latter method, which does not depend on the value of g_u, is preferable.     

The Kinetics of Non-Lamellar Phase Formation in DOPE-Me: Relevance to Biomembrane Fusion

The mechanism of the lamellar/inverted cubic (Q_II) phase transition is related to that of membrane fusion in lipid systems. N-Monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) exhibits this transition and is commonly used to investigate the effects of exogenous substances, such as viral fusion peptides, on the mechanism of membrane fusion. We studied DOPE-Me phase behavior as a first step in evaluating the effects of membrane-spanning peptides on inverted phase formation and membrane fusion. These measurements show that: a) the onset temperatures for Q_II and inverted hexagonal (H_II) phase formation both are temperature scan rate-dependent; b) longer pre-incubation times at low temperature and lower temperature scan rates favor formation of the Q_II phase; and c) in temperature-jump experiments between 61 and 65°C, the meta-stable H_II phase forms initially, and disappears slowly while the Q_II phase develops. These observations are rationalized in the context of a mechanism for both the lamellar/non-lamellar phase transition and the related process of membrane fusion. Current address for D.P.S.: Givaudan, Cincinnati, OH 45216 Data Deposition: Relevant transition temperatures in this paper have been deposited in the LIPIDAT ( )     

Screening a Peptide Library by DSC and SAXD: Comparison with the Biological Function of the Parent Proteins

We have recently identified the membranotropic regions of the hepatitis C virus proteins E1, E2, core and p7 proteins by observing the effect of protein-derived peptide libraries on model membrane integrity. We have studied in this work the ability of selected sequences of these proteins to modulate the L_β-L_α and L_α-H_II phospholipid phase transitions as well as check the viability of using both DSC and SAXD to screen a protein-derived peptide library. We demonstrate that it is feasible to screen a library of peptides corresponding to one or several proteins by both SAXD and DSC. This methodological combination should allow the identification of essential regions of membrane-interacting proteins which might be implicated in the molecular mechanism of membrane fusion and/or budding.     

Sugars favour formation of hexagonal (HII) phase at the expense of lamellar liquid-crystalline phase in hydrated phosphatidylethanolamines

The disaccharides, sucrose and trehalose, markedly decreased (up to 17-13C°) the temperature of the lamellar to hexagonal (L_α →H_II) phase transition and simultaneously increase by 2–4 C° the temperature of the lamellar gel to lamellar liquid-crystal (L_β →L_α) phase transition in hydrated dihexadecylphosphatidylethanolamine and distearoylphosphatidylethanolamine. These two transitions merge and convert into a single L_β-H_II phase transition when dispersed in 2.4 M sucrose. These results are inconsistent with recent reports by (8) and (9)) which suggest that trehalose stabilizes the L_α phase relative to the H_II phase and shifts upwards beyond detectability the L_α-H_II transition. The present results are considered as a manifestation of the Hofmeister effect in which the sugars act as kosmotropic reagents stabilizing the structure of bulk water. This tends to decrease the area of contact between the lipid and the aqueous phases and favours the H_II and L_β phases relative to L_α phase. This hypothesis is consistent with the effects of chaotropic reagents on the L_α-H_II phase transition (Yeagle and Sen (1986) Biochemistry 25, 7518–7522) and on the stability of the lamellar phase of dipalmitoylphosphatidylcholine (Oku and MacDonald (1983) J. Biol. Chem. 258, 8733–8738).     

Pivotal surfaces in inverse hexagonal and cubic phases of phospholipids and glycolipids

Data on the location and dimensions of the pivotal surfaces in inverse hexagonal (H_II) and inverse cubic (Q_II) phases of phospholipids and glycolipids are reviewed. This includes the H_II phases of dioleoyl phosphatidylethanolamine, 2:1 mol/mol mixtures of saturated fatty acids with the corresponding diacyl phosphatidylcholine, and glucosyl didodecylglycerol, and also the Q_II^230/G gyroid inverse cubic phases of monooleoylglycerol and glucosyl didodecylglycerol. Data from the inverse cubic phases are largely compatible with those from inverse hexagonal H_II-phases. The pivotal plane is located in the hydrophobic region, relatively close to the polar–apolar interface. The area per lipid at the pivotal plane is similar in size to lipid cross-sectional areas found in the fluid lamellar phase (L_α) of lipid bilayers.     

The Gaussian curvature elastic energy of intermediates in membrane fusion

The Gaussian curvature elastic energy contribution to the energy of membrane fusion intermediates has usually been neglected because the Gaussian curvature elastic modulus, κ, was unknown. It is now possible to measure κ for phospholipids that form bicontinuous inverted cubic (Q_II) phases. Here, it is shown that one can estimate κ for lipids that do not form Q_II phases by studying the phase behavior of lipid mixtures. The method is used to estimate κ for several lipid compositions in excess water. The values of κ are used to compute the curvature elastic energies of stalks and catenoidal fusion pores according to recent models. The Gaussian curvature elastic contribution is positive and similar in magnitude to the bending energy contribution: it increases the total curvature energy of all the fusion intermediates by 100 units of k_BT or more. It is important to note that this contribution makes the predicted intermediate energies compatible with observed lipid phase behavior in excess water. An order-of-magnitude fusion rate equation is used to estimate whether the predicted stalk energies are consistent with the observed rates of stalk-mediated processes in pure lipid systems. The current theory predicts a stalk energy that is slightly too large, by ∼30 k_BT, to rationalize the observed rates of stalk-mediated processes in phosphatidylethanolamine or N-monomethylated dioleoylphosphatidylethanolamine systems. Despite this discrepancy, the results show that models of fusion intermediate energy are accurate enough to make semiquantitative predictions about how proteins mediate biomembrane fusion. The same rate model shows that for proteins to drive biomembrane fusion at observed rates, they have to perform mediating functions corresponding to a reduction in the energy of a purely lipidic stalk by several tens of k_BT. By binding particular peptide sequences to the monolayer surface, proteins could lower fusion intermediate energies by altering the elastic constants of the patches of lipid monolayer that form the stalk. Here, it is shown that if peptide binding changes κ or some other combinations of local elastic constants by only tens of percents, the stalk energy and the energy of catenoidal fusion pores would decrease by tens of k_BT relative to the pure lipid value. This is comparable to the required mediating effect. The curvature energies of stalks and catenoidal fusion pores have almost the same dependence on monolayer elastic constants as the curvature energies of the rhombohedral and Q_II phases; respectively. The effects of isolated fusion-relevant peptides on the energies of these intermediates can be determined by studying the effects of the peptides on the stability of rhombohedral and Q_II phases.     

Neutron Diffraction with an Excess-Water Cell

As part of a study of the molecular basis of membrane fusion by enveloped viruses, we have used neutron diffraction to study the lamellar (L_α) to inverse hexagonal (H_II) phase transition in the phospholipid N-methylated dioleoylphosphatidylethanolamine. This lipid was chosen because its phase transitions are particularly sensitive to the presence of agents that have been demonstrated to promote or inhibit membrane fusion. Two different geometries of neutron diffraction were used: small angle scattering (SANS) and a membrane diffractometer. The SANS measurements were carried out on the SWAN instrument at KEK, Japan, using dispersions of multilamellar vesicles (MLVs). The diffractometer measurements used the V1 instrument at BeNSC-HMI, Germany, with a specially-constructed cell that holds a stack of lipid bilayers in an excess-water state. The two approaches are compared and discussed. Although the diffractometer takes considerably longer to collect the data, it records much higher resolution than the SANS instrument. The samples recorded in the excess-water cell were shown to be well aligned, despite the lipids being fully hydrated, allowing for the production of high-resolution data. Trial measurements performed have demonstrated that sample alignment is preserved throughout the L_α to H_II phase transition, thereby opening up possibilities for obtaining high-resolution data from non-lamellar phases.     

Calcium Triggered Lα-H2 Phase Transition Monitored by Combined Rapid Mixing and Time-Resolved Synchrotron SAXS

Background

Awad et al. [1] reported on the Ca²⁺-induced transitions of dioleoyl-phosphatidylglycerol (DOPG)/monoolein (MO) vesicles to bicontinuous cubic phases at equilibrium conditions. In the present study, the combination of rapid mixing and time-resolved synchrotron small-angle X-ray scattering (SAXS) was applied for the in-situ investigations of fast structural transitions of diluted DOPG/MO vesicles into well-ordered nanostructures by the addition of low concentrated Ca²⁺ solutions.

Methodology/Principal Findings

Under static conditions and the in absence of the divalent cations, the DOPG/MO system forms large vesicles composed of weakly correlated bilayers with a d-spacing of ∼140 Å (L_α-phase). The utilization of a stopped-flow apparatus allowed mixing these DOPG/MO vesicles with a solution of Ca²⁺ ions within 10 milliseconds (ms). In such a way the dynamics of negatively charged PG to divalent cation interactions, and the kinetics of the induced structural transitions were studied. Ca²⁺ ions have a very strong impact on the lipidic nanostructures. Intriguingly, already at low salt concentrations (DOPG/Ca²⁺>2), Ca²⁺ ions trigger the transformation from bilayers to monolayer nanotubes (inverted hexagonal phase, H₂). Our results reveal that a binding ratio of 1 Ca²⁺ per 8 DOPG is sufficient for the formation of the H₂ phase. At 50°C a direct transition from the vesicles to the H₂ phase was observed, whereas at ambient temperature (20°C) a short lived intermediate phase (possibly the cubic Pn3m phase) coexisting with the H₂ phase was detected.

Conclusions/Significance

The strong binding of the divalent cations to the negatively charged DOPG molecules enhances the negative spontaneous curvature of the monolayers and causes a rapid collapsing of the vesicles. The rapid loss of the bilayer stability and the reorganization of the lipid molecules within ms support the argument that the transition mechanism is based on a leaky fusion of the vesicles.     

Frequency-resolved intramolecular excimer fluorescence study of lipid bilayer and nonbilayer phases

Frequency-domain fluorescence intensity decays of the intramolecular excimer forming (DipyPE) in a fully hydrated dioleoyl-phosphatidylethanolamine (DOPE) suspension have been measured at the monomer (395 nm) and excimer (475 nm) emissions and at different temperatures (0-30°C). A classical Birks (two-state) and a new three-state kinetics models were used to analyze the frequency-domain data. The three-state model allowed us to resolve various intramolecular dynamics parameters of DipyPE in the host DOPE suspension. Those parameters are the excimer association (K_dm) and dissociation (K_md) rate constants, effective concentration (C), and lateral diffusion rate (f) of the pyrene moieties in the DipyPE. In contrast, only CK_dm and K_md were determined based on the two-state model. We observed that K_dm declined while C increased abruptly at ∼12°C, the known thermotropic lamellar liquid crystalline-to-inverted hexagonal (L_α-H_II) phase transition temperature of DOPE. No abrupt changes in K_md and f were observed at all temperatures. We concluded that the rotation of the lipid acyl chains is hindered and the free volume available for the lipid terminal methyl ends is reduced as the lipid membrane enters the highly curved H_II phase from the planar L_α phase.     

Bilayer structural destabilization by low amounts of chlorophyll a

The present study shows that small admixtures of one chlorophyll a (Chla) molecule per several hundred lipid molecules have strong destabilizing effect on lipid bilayers. This effect is clearly displayed in the properties of the L_α-H_II transformations and results from a Chla preference for the H_II relative to the L_α phase. Chla disfavors the lamellar liquid crystalline phase L_α and induces its replacement with inverted hexagonal phase H_II, as is consistently demonstrated by DSC and X-ray diffraction measurements on phosphatidylethanolamine (PE) dispersions. Chla lowers the L_α-H_II transition temperature (42 °C) of the fully hydrated dipalmitoleoyl PE (DPoPE) by ∼ 8 °C and ∼ 17 °C at Chla/DPoPE molar ratios of 1:500 and 1:100, respectively. Similar Chla effect was recorded also for dielaidoyl PE dispersions. The lowering of the transition temperature and the accompanying significant loss of transition cooperativity reflect the Chla repartitioning and preference for the H_II phase. The reduction of the H_II phase lattice constant in the presence of Chla is an indication that Chla favors H_II phase formation by decreasing the radius of spontaneous monolayer curvature, and not by filling up the interstitial spaces between the H_II phase cylinders. The observed Chla preference for H_II phase and the substantial bilayer destabilization in the vicinity of a bilayer-to-nonbilayer phase transformation caused by low Chla concentrations can be of interest as a potential regulatory or membrane-damaging factor.     

Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α → H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α → H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α → H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.     

Material Studies of Lipid Vesicles in the Lα and Lα-Gel Coexistence Regimes

In this work, we utilize micropipette aspiration and fluorescence imaging to examine the material properties of lipid vesicles made from mixtures of palmitoyloleoylphosphocholine (POPC) and dipalmitoylphosphatidylcholine (DPPC). At elevated temperatures/low DPPC fractions, these lipids are in a miscible liquid crystalline (L_α) state, whereas at lower temperatures/higher DPPC fractions they phase-separate into L_α and gel phases. We show that the elastic modulus, K, and critical tension, τ_c, of L_α vesicles are independent of DPPC fraction. However, as the sample temperature is increased from 15°C to 45°C, we measure decreases in both K and τ_c of 20% and 50%, respectively. The elasticity change is likely driven by a change in interfacial tension. We describe the reduction in critical tension using a simple model of thermally activated membrane pores. Vesicles with two-phase coexistence exhibit material properties that differ from L_α vesicles including critical tensions that are 20–40% lower. Fluorescence imaging of phase coexistent POPC/DPPC vesicles shows that the DPPC-rich domains exist in an extended network structure that exhibits characteristics of a solid. This gel network explains many of the unusual material properties of two-phase membranes.     

Structural and dielectric properties of synthetic glycolipids in mixtures with water

Three synthetically produced glycolipids, N-(β-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(β-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(β-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70°C. X-Ray diffraction revealed a direct L_β' → H transition at 20°C, 60°C, and 45°C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of ≈20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H phase the binding of water is stronger than in the L_β' phase. In the temperature range below 0°C, OSGA and OOGA show a “subzero transition” due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal → distorted hexagonal at 60°C.     

Bicontinuous inverted cubic phase stabilization as an index of antimicrobial and membrane fusion peptide activity

Some antimicrobial peptides (AMPs) and membrane fusion-catalyzing peptides (FPs) stabilize bicontinuous inverted cubic (Q_II) phases. Previous authors proposed a topological rationale: since AMP-induced pores, fusion intermediates, and Q_II phases all have negative Gaussian curvature (NGC), peptides which produce NGC in one structure also do it in another. This assumes that peptides change the curvature energy of the lipid membranes. Here I test this with a Helfrich curvature energy model. First, experimentally, I show that lipid systems often used to study peptide NGC have NGC without peptides at higher temperatures. To determine the net effect of an AMP on NGC, the equilibrium phase behavior of the host lipids must be determined. Second, the model shows that AMPs must make large changes in the curvature energy to stabilize AMP-induced pores. Peptide-induced changes in elastic constants affect pores and Q_II phase differently. Changes in spontaneous curvature affect them in opposite ways. The observed correlation between Q_II phase stabilization and AMP activity doesn't show that AMPs act by lowering pore curvature energy. A different rationale is proposed. In theory, AMPs could simultaneously stabilize Q_II phase and pores by drastically changing two particular elastic constants. This could be tested by measuring AMP effects on the individual constants. I propose experiments to do that. Unlike AMPs, FPs must make only small changes in the curvature energy to catalyze fusion. It they act in this way, their fusion activity should correlate with their ability to stabilize Q_II phases.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: II. On the fine structure of the phase transitions in hydrated dipalmitoylphosphatidylethanolamine

The phase transitions of dipalmitoylphosphatidylethanolamine (DPPE) in excess water have been examined by low-angle time-resolved x-ray diffraction and calorimetry at low scan rates. The lamellar subgel/lamellar liquid-crystalline (L_c → L_α), lamellar gel/lamellar liquid-crystalline (L_β → L_α), and lamellar liquid-crystalline/lamellar gel (L_α → L_β) phase transitions proceed via coexistence of the initial and final phases with no detectable intermediates at scan rates 0.1 and 0.5°C/min. At constant temperature within the region of the L_β → L_α transition the ratio of the two coexisting phases was found to be stable for over 30 min. The state of stable phase coexistence was preceded by a 150-s relaxation taking place at constant temperature after termination of the heating scan in the transition region. While no intermediate structures were present in the coexistence region, a well reproducible multipeak pattern, with at least four prominent heat capacity peaks separated in temperature by 0.4-0.5°C, has been observed in the cooling transition (L_α → L_β) by calorimetry. The multipeak pattern became distinct with an increase of incubation time in the liquid-crystalline phase. It was also clearly resolved in the x-ray diffraction intensity versus temperature plots recorded at slow cooling rates. These data suggest that the equilibrium state of the L_α phase of hydrated DPPE is represented by a mixture of domains that differ in thermal behavior, but cannot be distinguished structurally by x-ray scattering.     

A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein,an intrinsically disordered protein

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H^N–H^α and H^N–H^N NOEs, values for ³J_HNHα, ¹J_HαCα, ²J_CαN, and ¹J_CαN, as well as chemical shifts of ¹⁵N, ¹³C^α, and ¹³C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PP_II region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the α_R region (10–20%) is found. Analysis of ¹H–¹H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases

The phase transitions in fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases have been studied by lowangle time-resolved x-ray diffraction under conditions similar to those employed in calorimetry (scan rates 0.05-0.5°C/min and uniform temperature throughout the samples). This approach provides more adequate characterization of the equilibrium transition pathways and allows for close correlations between structural and thermodynamic data. No coexistence of the rippled gel (P_β') and liquid-crystalline (L_α) phases was found in the main transition of DPPC; rather, a loss of correlation in the lamellar structure, observed as broadening of the lamellar reflections, takes place in a narrow temperature range of ~100 mK at the transition midpoint. Formation of a long-living metastable phase, denoted by P_β'(mst), differing from the initial P_β' was observed in cooling direction by both x-ray diffraction and calorimetry. No direct conversion of P_β'(mst) into P_β' occurs for over 24 h but only by way of the phase sequence P_β'(mst) → L_β' → P_β'. According to differential scanning calorimetry (DSC), the enthalpy of the P_β'(mst)-L_α transition is by ~5% lower than that of the P_β'-L_α transition. The effects of ethanol (Rowe, E. S. 1983. Biochemistry. 22:3299-3305; Simon, S. A., and T. J. McIntosh. 1984. Biochim. Biophys. Acta 773:169-172) on the mechanism and reversibility of the DPPC main transition were clearly visualized. At ethanol concentrations inducing formation of interdigitated gel phase, the main transition proceeds through a coexistence of the initial and final phases over a finite temperature range. During the subtransition in DPPC recorded at scan rate 0.3°C/min, a smooth monotonic increase of the lamellar spacing from its subgel (L_c) to its gel (L_β') phase value takes place. The width of the lamellar reflections remains unchanged during this transformation. This provides grounds to propose a “sequential” relaxation mechanism for the subgel-gel transition which is not accompanied by growth of domains of the final phase within the initial one.     

Influence of surfactant protein C on the interfacial behavior of phosphatidylethanolamine monolayers

In the current work we study with monolayer tensiometry and Brewster angle microscopy (BAM) the surface properties of Dipalmitoleoylphosphatidylethanolamine (DPoPE) films at the air/water interface in presence and absence of specific surfactant protein C (SP-C). DPoPE is used, as it readily forms both lamellar (L_α) and non-lamellar inverted hexagonal (H_II) phases and appears as a suitable model phospholipid for probing the interfacial properties of distinct lipid phases. At pure air/water interface L_α shows faster adsorption and better surface disintegration than H_II phase. The interaction of DPoPE molecules with SP-C (predeposited at the interface) results in equalizing of the interfacial disintegration of the both phases (reaching approximately the same equilibrium surface tension) although the adsorption kinetics of the lamellar phase remains much faster. Monolayer compression/decompression cycling revealed that the effect of SP-C on dynamic surface tensions (γ _max and γ _min) of mixed films is remarkably different for the two phases. If γ _max for L_α decreased from the first to the third cycle, the opposite effect is registered for H_II where γ _max increases during cycling. Also the significant decrease of γ _min for L_α in SP-C presence is not observed for H_II phase. BAM studies reveal the formation of more uniform and homogeneously packed DPoPE monolayers in the presence of SP-C.     

Cubic topology in surfactant and lipid mixtures

Ternary systems of palmitoyl-oleoyl-phosphatidylcholine (POPC) and the non-ionic surfactant C₁₂EO₂ (di-ethylene-oxide-mono-dodecyl-ether) in water have been studied with optical microscopy, NMR, DSC and X-rays from ambient temperatures to 45 °C. Below 29 °C the system is in the lamellar liquid crystalline state. Between 30 and 32 °C it transforms into a cubic Ia3d structure which converts into the cubic Pn3m phase at 39 °C. The transitions are fully reversible. An epitaxial relationship between all three phases was found, which is an elegant and convenient way to rearrange molecules from lamellar bilayers to a network of curved surfaces. The la3d (Q²³⁰) to Pn3m (Q²²⁴) transition occurs without measurable enthalpy change. This, together with the metric relation of 1.60 between the cubic lattice constants is strong evidence for a Bonnet transformation, where the structural changes occur without change in curvature. The potential significance of the cubic phases as intermediate structures for biological processes, e. g. transport across a bilayer or fusion of membranes, are discussed.     

Structural properties of phosphatidylcholine in a monolayer at the air/water interface: Neutron reflection study and reexamination of x-ray reflection measurements

Neutron reflectivities of phosphatidylcholine monolayers in the liquid condensed (LC) phase on ultrapure H₂O and D₂O subphases have been measured on a Langmuir film balance. Using a dedicated liquid surface reflectometer, reflectivities down to R = 10^-6 in the momentum transfer range Q_z = 0-0.4 Å^-1 were accessed.

In a new approach, by refining neutron reflectivity data from chain-perdeuterated DPPC-d₆₂ in combination with x-ray measurements on the same monolayer under similar conditions it is shown that the two techniques mutually complement one another. This analysis leads to a detailed conception of the interface structure. It is found that in the LC phase (which is analogous to the L_β, phase in vesicle dispersions) the head group is interpenetrated with subphase water (4 ± 2.5 molecules per lipid) and the average tilt angle of the hydrophobic chains from the surface normal is 33 ± 3 degrees.