X-ray diffraction study of bilayer to non-bilayer phase transitions in aqueous dispersions of di-polyenoic phosphatidylethanolamines.

The low temperature phase properties of aqueous dispersions of di-18:2 and di-18:3 phosphatidylethanolamine are strongly influenced by the presence of ice. In the presence of cryoprotectants to inhibit ice formation, these lipids persist in the H(II) phase down to at least -50 degrees C. Ice formation, however, leads to a drastic reduction in the amount of available free water and a rapid reduction in the diameter of the inverted cylindrical micelles of the H(II) phase. The resulting increase in surface curvature of the micelles induces an imbalance in the forces acting in the lipid surface and the hydrophobic core which is relieved by formation of the L(alpha) phase. On reheating the lipid samples undergo an abrupt L(alpha) --> H(II) phase transition at about -20 degrees C. The radius of the water core of the inverted micelles at their point of formation is estimated to be 0.9 nm. This increases with temperature as more unfrozen water becomes available until the normal equilibrium radius of about 2.3 nm is reached at 0 degrees C when the bulk water in the sample finally melts. A small proportion of the H(II) phase lipid enters an as yet unidentified cubic phase on freezing. The spacings of the (10) planes of the H(II) phase, the (111) planes of the cubic phase and the d-spacing of the L(alpha) phase were found to be almost identical at the phase transition temperature. The cubic phase appears to disappear at low temperature but to reform on heating. Freeze-fracture studies revealed no unequivocal evidence for cubic phase lipid but the presence of residual non-bilayer lipid structures was observed even at temperatures as low as -80 degrees C. The presence of intersecting stacks of lamellar sheets in the replicas strongly suggest the existence of an epitaxial relationship between the L(alpha) and H(II) phases in these systems.

The effect of ice on membrane lipid phase behaviour.

The effects of ice on the lipid phase behaviour of di-18:1 PE and di-18:2 PE were studied by comparing the behaviour of these lipids in supercooled and frozen dispersions. The presence of ice raised the onset temperature of the L alpha--> L beta phase transition of di-18:1 PE from -10 degrees C to -6.5 degrees C and increased its molar enthalpy from 6.1 to 8.5 kcal/mol but had little effect on the co-operativity of the transition. Real-time X-ray diffraction measurements of the HII--> L alpha phase transition of di-18:2 PE suggested that this transition could take place in the presence of ice but that the corresponding L alpha--> HII phase transition could not take place until the ice melted. Measurements of the temperature dependence of the d-spacing of di-18:1 PE and di-18:2 PC in frozen dispersions indicated that the amounts of unfrozen water in such dispersions changes significantly with temperature. It was concluded that the increases in onset temperature and molar enthalpy seen for the L alpha--> L beta transition of di-18:1 PE probably reflected the effects of osmotic dehydration. The main effect of ice in the case of the HII--> L alpha phase transition, however, appeared to be to limit the ability of the lipid to undergo structural reorganisation.

Phase behaviour of membrane lipids containing polyenoic acyl chains.

The low-temperature thermal behaviour of di-18:2 phosphatidylethanolamine (di-18:2 PE) is shown to be characterized by similar broad low-enthalpy transitions to those previously reported for polyenoic samples of phosphatidylcholines (Keough and Kariel (1987) Biochim. Biophys. Acta 902, 11-18), and monogalactosyldiacylglycerol (Sanderson and Williams (1992) Biochim. Biophys. Acta. 1107, 77-85). Real-time X-ray diffraction measurements indicate that these transitions correspond to transitions between the gel (L beta) and liquid-crystal (L alpha) phases of the lipids. The gel phase of these lipids is, however, much more loosely packed than the corresponding phases of membrane lipids containing monoenoic or fully-saturated acyl chains. The low enthalpy and reduced co-operativity of the L alpha--> L beta phase transitions of the polyenoic lipids is attributed to the reduced contribution of van der Waals interactions between their acyl chains in the gel-state of these lipids. Comparison with the earlier results obtained for MGDG suggest that the acyl chains of polyenoic lipids can form well-ordered lattices but require the additional energy input associated with the formation of a hydrogen bond network between the lipid headgroups in order to do so.

A combined SAXS/WAXS investigation of the phase behaviour of di-polyenoic membrane lipids.

Real-time measurements of the SAXS/WAXS diffraction patterns of aqueous dispersions (1:1 wt/wt) of the di-polyenoic lipids di-18:2 PC, di-18:3 PC, di-18:2 PE and di-18:3 PE were made over the temperature range 10 degrees to about -80 degrees C. The results of these measurements were compared to similar measurements performed on the corresponding di-18:0 and di-18:1 derivatives. SAXS measurements of the temperature dependence of lamellar repeat distances show that the di-polyenoic lipids undergo broad second-order transitions between their gel and liquid-crystal lamellar phases spanning 30-40 degrees C. The di-18:1 and di-18:0 derivatives, in contrast, undergo abrupt first-order transitions. The gel phases of the di-18:0 derivatives are characterised by two-component WAXS patterns with a sharp component close to 0.42 nm and a broader component at narrower spacings. On cooling, these lipids appear to undergo an initial transition to an L beta, phase followed by a conversion to an Lc phase. The gel phases of the di-18:1 derivatives also show two-component patterns but with the sharp component centred closer to 0.44 nm. The di-polyenoic lipids, in contrast, are characterised by a single broad peak centred at a spacing of about 0.42 nm, close to that of conventional L beta phases. The changes in lamellar repeat distance accompanying the transitions in the di-monoenoic and di-polyenoic lipids, all of which occur in the frozen state, are very similar, indicating that the acyl chains of the polyenoic lipids are close to their maximum extension in the gel state. The WAXS patterns of the polyenoic lipids suggest that the saturated upper parts of the acyl chains are packed on a regular hexagonal lattice while their polyunsaturated termini remain relatively disordered.

Real-time X-ray diffraction study at different scan rates of phase transitions for dipalmitoylphosphatidylcholine in KSCN.

Multibilayer arrays of dipalmitoylphosphatidylcholine (DPPC) in 1 M KSCN were characterized using real-time X-ray diffraction and differential scanning calorimetry. A phase transition sequence was observed as a function of increasing temperature which involved changes from the interdigitated subgel (Lc(inter)) to interdigitated gel (L beta(inter)) to disordered (L alpha) bilayer states. The phase transition mechanisms were unambiguously determined by comparison of results from fast and slow scans. The Lc(inter)-->L beta(inter) phase transition was shown to involve a continuous change in acyl chain spacing between the rectangular subgel acyl chain unit cell into an hexagonal gel acyl chain unit cell. The mechanism is similar to that for subgel to gel state transitions involving non-interdigitated DPPC bilayers.

Sterols stabilize the ripple phase structure in dihexadecylphosphatidylcholine.

The presence of various sterols in mixtures with dihexadecylphosphatidylcholine (DHPC) was studied using static X-ray diffraction of temperature equilibrated samples, and real-time X-ray diffraction of samples undergoing temperature scans. It was found that these sterols eliminate the interdigitation of the alkyl chains in the DHPC sub-gel and gel-state bilayers while stabilizing the ripple gel-state at the expense of the gel-state bilayer phase. The ripple-ripple phase transition previously observed for dipalmitoylphosphatidylcholine in the presence of low molar concentrations of sterols (Wolfe et al. (1992) Phys. Rev. Lett. 68, 1085-1088) was also observed for similar DHPC-sterol mixtures. In addition, we show the first evidence that the presence of 5 alpha-cholestane-3 beta,5,6 beta-triol will cause the lipid mixtures to continue to adopt a ripple mesophase structure even after the DHPC alkyl chain becomes disordered.

Regulation of the surface pressure of lipid monolayers and bilayers by the activity of water: derivation and application of an equation of state.

A quasi-two-dimensional equation of state for liquid-type lipid monolayers has been derived and successfully applied to surface pressure-area isotherms obtained with a variety of lipids. For lipids with acyl moieties of similar length, the surface pressure and area at monolayer collapse can be accurately predicted from data obtained at lower surface pressure. Consideration of the rationalized activity coefficient as a linear scaler in an expression for surface pressure as a function of depth in the surface phase permits comparison of surface pressure-area data for monolayers with force-distance data for bilayers. This analysis shows the thermodynamic equivalence of monolayers at collapse and fully hydrated bilayers. It also supports the interpretation of the activity coefficient as a scaler and allows its determination solely from bilayer-derived data. Overall, the results show the common assumption that partial specific volume of water equals its bulk value to be inappropriate for the analysis of surface structure.