G. Klose

DYNAMIC PROPERTIES OF WATER AT PHOSPHATIDYLCHOLINE LIPID-BILAYER SURFACES AS SEEN BY DEUTERIUM AND PULSED FIELD GRADIENT PROTON NMR

The dynamic properties of water in phosphatidylcholine lipid/water dispersions have been studied, applying a combination of 2H-NMR techniques (quadrupole splitting and spin-lattice relaxation time) and self-diffusion measurements using pulsed filed gradient (PFG) 1H-NMR. The hydration properties of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) were compared with those of DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) and EYL (egg yolk phosphatidylcholine (lecithin)). A model is presented that assumes an exponentially decaying influence of the bilayer surface on water dynamics as well as on water orientation with increasing hydration. This assumption is based on an exponentially decaying hydration potential which results from direct lipid-water and water-water interactions. The model describes successfully the experimental data for a large water concentration range, especially at low hydration, where other models failed. With the exception of a small fraction of water which is significantly influenced by the surface in slowing down the mobility, the interbilayer water has isotropic, free water characteristics in terms of correlation times and molecular order. Hydration properties of POPC are comparable with those of EYL but differ from DOPC. At very low water content the correlation times of headgroup segmental reorientation and water are similar, indicating a strong coupling of this water to the lipid lattice. The hydration properties of the three lipids studied are explained in terms of slightly different headgroup conformations due to different lateral packing of the molecules by their fatty acid chain composition.

NMR investigations of the interaction of water with lecithin in benzene solutions.

Abstract NMR investigations of 1H (chemical shifts, line widths) and of 31P (relaxation times, T1) performed on the three-component system of lecithin-benzene-water show that there is an interaction of water with the phosphate group in two regions of different mobility and structure. A fast exchange of the water molecules takes place between both regions. The region of strong interaction involves about 2 and that of the weaker interaction about 5 water molecules per lecithin molecule. When the water concentration is increased a third region is formed which is assigned to the water molecules that are located beyond the two regions of interaction with the phosphate group, but within the micelle. This water has a different structure from that of the second region of interaction with the phosphate group and may also have a different mobility. Addition of water increases the motion of the head groups of the lecithin molecules. This is due to a loosening of the packing of lecithin molecules.

Small-angle X-ray scattering and electron microscopy of crude dispersions of swelling lipids and the influence of the morphology on the repeat distance

From X-ray measurements in different lipid systems it is concluded that the repeat distance measured in lipids with limited swelling in the lamellar liquid crystalline state exhibits qualitatively the same dependence on the water concentration. Four regions of water concentrations with different structural and morphological changes can be distinguished. Their existence is qualitatively explained. The morphology of samples with water concentrations near the boundary between the single lamellar Lα phase and the two phase region α + water has a drastic influence on the repeat distance measured. A procedure of how this boundary can be determined is proposed.

Surface areas and packing constraints in POPC C (12)EO (n) membranes. A time-resolved fluorescence study.

The surface area occupied by nonionic detergents of the type C(12)EO(n) (n = 1-8) in POPC C (12)EO (n) mixed membranes was studied by means of time-resolved resonance energy transfer (RET) between the fluorescent probe molecules NBD-PE and rhodamine-PE. The area data were interpreted within the frame of Israelachvilis concept of packing constraints yielding the critical packing parameter, f, as a measure of the asymmetry of the molecular shape of the membrane constituents. The asymmetry of the molecular shape of the detergent increases with the ethylene oxide chain length and correlates with the potency of the detergent to solubilize the bilayers and the reduction of the DPH order parameter. For n = 1-3, the membrane surface was found to expand by 0.25-0.30 nm(2) per incorporated C(12)EO(n) molecule. This value corresponds to the cross section of one hydrocarbon chain in liquid-crystalline phases. On increasing n from n = 4 to n = 8 the net area per detergent molecule increases from 0.43 nm(2) to 1.16 nm(2). These surface requirements are consistent with a disordered, coiled conformation of the EO-chains hydrated with up to two water molecules per ethylene oxide unit. For n > 5 the limiting mole fraction of the bilayer saturation was deduced from the f-data in the two-component bilayer. DPH and NBD-PE fluorescence lifetime data are discussed to give an indication of the accessibility of the probe environment to water molecules.

Hydration of the dienic lipid dioctadecadienoylphosphatidylcholine in the lamellar phase--an infrared linear dichroism and x-ray study on headgroup orientation, water ordering, and bilayer dimensions.

In the phospholipid 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC) in each of the fatty acid chains, a rigid diene group is inserted in a position near the polar/apolar boundary that is exceptionally sensitive for membrane stability. DODPC transforms upon gradual dehydration from the liquid-crystalline to a metastable gel state, which rearranges into two subgel phases at low and intermediate degrees of hydration. The molecular dimensions of the respective bilayers were determined by means of x-ray diffraction. Infrared linear dichroism of selected vibrations of the phosphate and trimethylammonium groups and of the nu13(OH) band of water adsorbed onto the lipid was used to study the molecular order in the polar part of the bilayers in macroscopically oriented samples. The dense packing of the tilted acyl chains in the subgel causes the in-plane orientation of the phosphatidylcholine headgroups with direct interactions between the phosphate and trimethylammonium groups, and a strong orientation of adsorbed water molecules. In the more disordered gel, the thickness of the polar part of the bilayer increases and the lateral interactions between the lipid headgroups weaken. The higher order in the headgroup region of the subgels correlates with shorter decay lengths of the repulsive forces acting between opposite membrane surfaces. This result can be understood if the work to dehydrate the lipid is determined to a certain degree by the work to break up the lipid-water interactions without compensation by adequate lipid-lipid contacts. Almost similar area compressibility moduli are found in the liquid-crystalline and solid phases. Obviously, the lipid avoids lateral stress by the structural rearrangement.

Application of isothermal titration calorimetry for detecting lipid membrane solubilization

Abstract Isothermal titration calorimetry is presented to be an excellent method of detecting lipid membrane solubilization upon the addition of detergents. As an example, the titration of the non-ionic detergent C 12 EO 6 to the lipid POPC is studied. The main contribution to the measured titration heat is the detergents enthalpy difference upon transfer between micelles and membranes. The detergent added as micelles is transferred into membranes up to the saturating detergent per lipip mol/mol ratio, R sat = 1.45 ± 0.07. Beyond R sat the membranes are solubilized to micelles. Subsequently, the titration heat changes its sign from endothermic to exothermic. After complete membrane dissolution at R sol = 5.0 ± 0.4 the titration heat vanishes nearly completely.

Sorption isotherms and swelling of POPC in H2O and 2H2O

Abstract The hydration and swelling properties of 1-palmitoyl-2-oleoyl-glycerophosphatidylcholine (POPC) in ordinary (H2O) and heavy (2H2O) water were examined to clarify the influence of the isotopically different water species on these features. Neither the swelling curves, nor the sorption isotherms and the empirical hydration forces of the two systems show measurable isotope effects.

Membrane/water partition of oligo(ethylene oxide) dodecyl ethers and its relevance for solubilization.

Mixed aqueous dispersions containing palmitoyloleoylphosphatidylcholine and oligo(ethylene oxide) dodecyl ethers C12EOn with n = 2-8 have been investigated. The aggregates composition has been determined as a function of the aqueous detergent concentration using Laurdan fluorescence spectroscopy. The partition of the detergent between the membranes and the aqueous phase has been analyzed on the basis of the regular solution model. Solubilization has been analyzed in terms of the limiting detergent fraction in the membrane, the minimal detergent fraction in micelles and the critical aqueous detergent concentration using thermodynamical coexistence conditions for the aqueous, bilayer and micellar pseudo-phases. A thermodynamic criterion for solubilization is presented. The standard chemical potential differences of the transfer of the detergents from water to the bilayer have been found to follow the empirical relation delta u o(n = 2-8) = -37.6 + 0.9 n kJ/mol. Thus, a similar conformation and localization of the oxyethylene units within the membrane are suggested.

Thermotropic phase behaviour of the pseudobinary mixture DPPC/C12E4 at excess water

The pseudobinary phase diagram of DPPC/C12E4 at excess water was investigated by DSC, 2H-, 31P-NMR and freeze fracture electron microscopy in a wide range of concentrations and temperatures. Compared with previously published papers on phase behaviour of DPPC/Triton X-100 and DMPC/Triton X-100 [13,41], we do not find any total solubilisation of the bilayer membrane within the range of molar fractions x = 0 to x = 0.95. The estimated phase diagram implies a strong non-ideal mixing behaviour in the gel phases with peritectic and eutectic behaviour and complete miscibility in the liquid crystalline Lα phase. Two mixing gaps were observed in the gel phase between x = 0.2 and x = 0.95. One peritectic point with an incongruently melting lamellar gel compound complex formation at a molar ratio of nDPPCnC12E4 = 3:4 (x = 0.57) and one eutectic point at x = 0.9 are a peculiar feature of this system. 2H-NMR measurements employing selectively deuterated surfactant and 31P-NMR experiments clearly indicate a lamellar liquid crystalline phase Lα for the high-temperature phase in the whole mixing range. At low surfactant concentrations 0 < x < 0.15 the formation of the rippled phase Pβ′ in a defined region of temperature can be observed by freeze fracture electron microscopy.

Hydration force parameters of phosphatidylcholine lipid bilayers as determined from 2H-NMR studies of deuterated water

The continuous decrease of the quadrupolar splitting of deuterated water interacting with phosphocholine lipid bilayers with growing water concentration is analyzed as a function of the water activity. From the apparent linear dependence on water activity a measure for hydration forces is obtained. The forces calculated are in the range of published data using sorption isotherms and osmotic stress technique in combination with SAXS. A simple interaction potential which includes orientational order of water adsorbed on surfaces gives a physical base for these findings. Therefore, deuterium NMR may become a powerful tool for hydration force analysis complementing well-known methods.