G. Lindblom

Hydrophobic molecules in lecithin-water systems. I. Formation of reversed hexagonal phases at high and low water contents

The system dioleoylphosphatidylcholine (DOPC)-n-dodecane-2H2O was investigated with different nuclear magnetic resonance (NMR) techniques: (a) a tentative phase diagram was determined by 2H- and 31P-NMR, (b) translational diffusion coefficients were determined for the three components with the pulsed magnetic field gradient NMR technique, and (c) order parameters for perdeuterated n-dodecane were obtained by 2H-NMR. n-Dodecane induces the formation of reversed hexagonal (HII) phases at low and high water concentrations, and cubic phases at low water contents. The translational diffusion coefficients of n-dodecane in a cubic phase with 6 mol water per mol DOPC, and in an HII phase with 48 mol water per mol DOPC, were just approximately 2.5 times lower than in pure dodecane. Perdeuterated dodecane gave large quadrupole splittings in a lamellar phase, much smaller in an HII phase at low water contents, and a narrow single peak in an HII phase at high water contents. This latter observation indicates that a large fraction of the dodecane molecules is located in separate regions between the water cylinders. Our results support the model given by Gruner concerning the aggregation of membrane lipids in the presence of hydrophobic molecules.

Lipid and water diffusion in bicontinuous cubic phases measured by NMR

Lipid and water diffusion coefficients in bicontinuous cubic liquid crystalline phases have been determined with the NMR pulsed magnetic field gradient technique. In the monoolein-water system, a discontinuity in the variation of the water diffusion coefficient with water content is observed, which coincides with the two-phase region between the two cubic phases in this system. The degree of water association to the lipid has been determined, considering the obstruction factor for diffusion in the cubic phases. The lipid diffusion coefficient increases with increased unsaturation of the lipid, and decreases when larger amphiphile molecules like cholesterol, gramicidin-A, and lyso-oleoyl-phosphatidylcholine are solubilized in the cubic phase. In a cubic liquid crystal of monoolein (MO), dioleoylphosphatidylcholine (DOPC), and water, the individual lipid diffusion coefficients have been determined simultaneously in the same sample. The diffusion coefficients of MO and DOPC differ by a factor of two, and both decrease with increasing DOPC content. The results are discussed in relation to probe techniques for measurements of lipid diffusion.

Lipid extracts from membranes of Acholeplasma laidlawii A grown with different fatty acids have a nearly constant spontaneous curvature

X-ray diffraction methods were used to explore the variation in the spontaneous curvature of lipid extracts from Acholeplasma laidlawii strain A-EF22 grown with different mixtures of palmitic acid and oleic acid. It was shown that the cells respond to the different growing conditions by altering the polar head group compositions in order to keep the phase transition between lamellar and nonlamellar structures within a narrow temperature range. This has been interpreted to mean that the membrane lipids are adjusted toward an optimal packing (Lindblom et al. (1986) Biochemistry 25, 7502). Here it is shown that for these extracts, the membrane curvature is kept within a narrow range (58-73 A), compared to the range in curvatures exhibited by pure lipids extracts from the membrane (17-123 A). These observations support the hypothesis (Gruner (1989) J. Phys. Chem. 93, 7562) that the spontaneous curvature is a functionally important membrane parameter which is regulated by the organism and is likely to be one of the constraints controlling the lipid composition of the bilayer.

Influence of membrane-spanning alpha-helical peptides on the phase behavior of the dioleoylphosphatidylcholine/water system.

The effect of solubilized hydrophobic peptides on the phase behavior of dioleoylphosphatidylcholine (DOPC)/water system was studied by 2H- and 31P-NMR spectroscopy and by x-ray diffraction, and partial phase diagrams were constructed. The utilized peptides were HCO-AWW(LA)5WWA-NHCH2CH2OH (WALP16), which is an artificial peptide designed to resemble a transmembrane part of a membrane protein; and VEYAGIALFFVAAVLTLWSMLQYLSAAR (Pgs peptide E), a peptide that is identical to one of the putative transmembrane segments of the membrane-associated protein phosphatidylglycerophosphate synthase (Pgs) in Escherichia coli. Circular dichroism spectroscopy suggests that both peptides are mostly alpha-helical in DOPC vesicles. The most striking features in the phase diagram of the WALP16/DOPC/water system are 1) a single lamellar liquid crystalline (L alpha) phase forms only at very low peptide concentrations. 2) At low water content and above a peptide/lipid molar ratio of approximately 1:75 a reversed hexagonal liquid crystalline (H[II]) phase coexists with an L alpha phase, while in excess water this phase forms at a peptide/lipid molar ratio of approximately 1:25. 3) At peptide/lipid ratios > or =1:6 a single H(II) phase is stable. Also, the Pgs peptide E strongly affects the phase behavior, and a single L alpha phase is only found at low peptide concentrations (peptide/lipid molar ratios <1:50), and water concentrations <45% (w/w). Higher peptide content results in coexistence of L alpha and isotropic phases. Generally, the fraction of the isotropic phase increases with increasing temperature and water concentration, and at 80% (w/w) water content only a single isotropic phase is stable at 55 degrees C. Thus, both peptides were found to be able to induce nonlamellar phases, although different in structure, in the DOPC/water system. The phase transitions, the extensions of the one-phase regions, and the phase structures observed for the two systems are discussed in terms of the molecular structure of the two peptides and the matching between the hydrophobic lengths of the peptides and the bilayer thickness of DOPC.

LECITHIN TRANSLATIONAL DIFFUSION STUDIED BY PULSED NUCLEAR MAGNETIC RESONANCE

The translational diffusion coefficient of egg yolk and dilauroyl lecithin in optically isotropic phases containing sodium cholate has been measured using the pulsed NMR magnetic field gradient method. After a correction for geometrical factors the measured diffusion coefficient is found to agree well with previous determinations in phospholipid systems. The experimental data imply that the cubic mesophase of the lecithin-sodium cholate-water system contains continuous lipid aggregates. A possible model of the arrangement of the different amphiphile molecules in the cubic phase is discussed.

31P and 2H NMR Studies of Phase Equilibria in the Three Component System: Monoolein-Dioleoylphosphatidylcholine

2H and 31P NMR have been used in the determination of the phase behaviour at 28°C of the ternary system monoolein/dioleoylphosphatidylcholine/heavy water. The isothermal phase diagram shows the existence of four one-phase areas containing homogeneous liquid crystalline matter and one area with isotropic solution. Two of the liquid crystalline phases have a lamellar structure, one has a cubic and the fourth a reversed hexagonal structure. A non-aqueous mixture of 3 to 5 moles of monoolein per mole of dioleoyl-phosphatidylcholine is an isotropic oily fluid.

The effects of hydration and divalent cations on lamellar-nonlamellar phase transitions in membranes and total lipid extracts from Acholeplasma laidlawii A-EF22 - a 2H NMR study.

Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with 75 µmα-deuterated palmitic acid (16:0-d2) and 75 µmα-deuterated oleic acid (18:1c-d2), or with 150 µm 18:1c-d2. The fatty acids were incorporated into the membrane lipids and 2H NMR spectra were recorded from intact membranes, total lipid extracts, and the combined glucolipid and neutral lipid fractions of a total lipid extract. The lipids in intact membranes form a bilayer structure up to at least 70 °C. The same result was obtained with membranes digested with pronase, which removes a large fraction of the membrane proteins. A reversed hexagonal liquid crystalline (HII) phase was formed below 70 °C by the total lipid extracts hydrated with 20 and 30% (w/w) water; in the presence of 40% (w/w) water only one of the extracts formed an HII phase below 70 °C. The HII phase was formed at higher temperatures with an increasing water content. However, only a lamellar liquid crystalline (Lα) phase was formed up to 70 °C by the total lipid extracts when the water concentrations were 50% (w/w) or higher. The temperature (TLH) for the Lα to HII phase transition in the combined glucolipid and neutral lipid fractions was only 2–3 °C lower than for the total lipids, and the phospholipids thus have a very modest influence on the TLH value. Physiologically relevant concentrations of Ca2+ and Mg2+ ions did not affect the phase equilibria of total lipid extracts significantly. It is concluded from comparison with published data that the membrane lipids of the cell wall-less bacterium A. laidlawii have a smaller tendency to form reversed nonlamellar phases than the membrane lipids of three bacterial species surrounded by a cell wall.

Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol.

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.

Spectroscopic Studies on Structure and Dynamics of Lyotropic Liquid Crystals: Cubic and Reversed Hexagonal Phases and Lipid Vesicles

An overview of the formation and structure of lyotropic liquid crystals is given, in particular considering the influence of molecular shapes and packing constraints on the phase equilibria. Investigations of phase equilibria in phospholipid/hydrocarbon/water systems are found to be in good agreement with theoretical predictions. Aggregate structure, lipid head group hydration and counterion binding, have been studied by using nuclear magnetic resonance (NMR), electron spin resonance (ESR) and fluorescence light spectroscopy. The discussion is focused mainly on non-lamellar and cubic phases. NMR measurements of diffusion, spin relaxation and lineshape analysis can give detailed information about the structure and dynamics of cubic phases, information which is difficult to obtain by X-ray diffraction. ESR studies of micellar aggregates and vesicles are described. The ESR lineshape for some systems gives information about the size and shape of micellar aggregates. Time-resolved fluorescence spectroscopy is used to determine the aggregation number of micelles. The order and dynamics of fluorescence probes through the measurements of the fluorescence anisotropy decay are described. Finally, a discussion of electronic energy transfer in lamellar phases and lipid vesicles is given.