Nuclear magnetic resonance studies of lipid hydration in monomethyldioleoylphosphatidylethanolamine dispersions.

Solid-state proton nuclear magnetic resonance has been used to examine surface hydration in suspensions of monomethyldioleoylphosphatidylethanolamine (MeDOPE). The magic-angle spinning (MAS) 1H spectra for aqueous suspensions of MeDOPE in the L alpha phase exhibited two resonances of roughly equal intensity that could be ascribed to water protons, but both their spin-lattice relaxation times and chemical shifts converged upon conversion to the hexagonal phase. Only a single water peak was observed for analogous samples of dioleoylphosphatidylcholine (DOPC). MAS-assisted two-dimensional nuclear Overhauser effect spectroscopy (NOESY) was conducted for multibilayers of both MeDOPE and DOPC. Through-space interactions were identified between pairs of lipid protons, as expected from their chemical structure. For lamellar suspensions of MeDOPE, positive NOESY cross-peaks were observed between the downfield-shifted water resonance (only) and both CH2N and NH2CH3+ protons of the lipid headgroup. These cross-peaks were not observed in the NOESY spectra of MeDOPE in its hexagonal or cubic phases or for lamellar DOPC reference samples. Taken together, the observation of two water peaks, spin-lattice relaxation behavior, and NOESY connectivities in MeDOPE suspensions support the interpretation that the low-field water peak corresponds to hydrogen-bonded interlamellar water interacting strongly with the lipid. Such a population of water molecules exists in association with MeDOPE in the lamellar phase but not for its inverted phases or for lamellar dispersions of DOPC.

Ganglioside GD1a generates domains of high curvature in phosphatidylethanolamine liposomes as determined by solid state 31P-NMR spectroscopy.

We have studied the effects of gangliosides on the polymorphic behaviour of phosphatidylethanolamines. The ganglioside GD1a promotes the formation of phases which give rise to isotropic 31P-NMR resonance lines, particularly in the temperature range of the L alpha to HII transition. In addition, higher mol fractions of ganglioside raise the L alpha to HII phase transition temperature. Our results demonstrate that small mol fractions of gangliosides can have profound effects on the molecular organization of phosphatidylethanolamines.

Cross-polarization dynamics in 2,6-dimethylbicyclo[3.3.1]nonane-exo-2-exo-6-diol inclusion compounds as studied by 13C magic-angle spinning nuclear magnetic resonance spectroscopy.

Solid-state 13C nuclear magnetic resonance (NMR) spectra of a number of inclusion compounds of 2,6-dimethyl-bicyclo[3.3.1]nonane-exo-2-exo-6-diol (host) with small organic small molecules (guests) have been studied. With 3,4-dichloro-1,2,5-thiadiazole and tetrachloroethylene as guests, line splittings of the host resonances were observed due to the location of the guest in the host lattice. The cross-polarization (CP) dynamics of these inclusion compounds have been studied and shown to be indicative of weakly coupled systems. As expected, the proton spin lattice relaxation times in the rotating frame (T1pH) of the host are increased by the presence of rapidly moving guest because the efficiency of homonuclear dipolar relaxation in the rotating frame is reduced. However, strong transient oscillations were also observed for the guest molecules during the Hartmann-Hahn transfer of magnetisation from the more abundant 1H spins to the 13C spins during spin lattice rotating frame relaxation. These oscillations were found to be greatest for carbons with largest chemical shift anisotropies.

Calcium-independent activation of protein kinase C by the dianionic form of phosphatidic acid.

Phosphatidic acid in the form of small unilamellar vesicles has a dissociation constant of about 8.3 as determined by 31P nuclear magnetic resonance (NMR) spectroscopy. The activation of protein kinase C (PKC) by monovalent phosphatidic acid or phosphatidylserine occurs only in the presence of Ca2+. However, PKC activity on membranes of divalent anionic phosphatidic acid is independent of Ca2+ concentration.

A comparison of membrane properties and composition between cell lines selected and transfected for multi-drug resistance.

Cell lines selected (CHRC5) and transfected (LR-73-1A) for multi-drug resistance have total lipid compositions which are indistinguishable between resistant and parental cells. Lipid composition was evaluated by 1H NMR and the total fatty acid content by GLC. No change in surface hydrophobicity, as measured with the fluorescent probe dansyl-PE, was observed as a result of transfection of CHO cells with the mdr1 gene. However, the selected cell line, CHRC5, showed a decreased surface hydrophobicity. This decreased surface hydrophobicity was indicated by an 8 nm increase in the fluorescence emission of dansyl-PE in the CHRC5 cell line compared with the AB1. Both resistant cell lines showed an increase in the polarisation of the fluorescent probe, TMA-DPH in the plasma membranes corresponding to a 14% and a 24% change in fluorescence polarisation for the selected and transfected cell lines, respectively. This is indicative of reduced mobility of the acyl chains in the resistant cell lines. Both the CHRC5 and the transfected cell lines showed almost a 2-fold increase in the initial rate of membrane cycling. The membrane cycling could be inhibited by a known bilayer stabiliser, the N-carbobenzoxy-D-Phe-L-Phe-Gly. These results indicate that the properties of the plasma membrane from resistant cells are altered compared with their parental cell line.

Hydrophobic lipid additives affect membrane stability and phase behavior of N-monomethyldioleoylphosphatidylethanolamine.

The rate of formation of high-curvature intermediates or disordered cubic phases in N-methyldioleoylphosphatidylethanolamine (N-methyl-DOPE) dispersions with or without additives was studied by 31P NMR spectroscopy. In N-methyl-DOPE dispersions, both the L alpha liquid-crystalline phase and the hexagonal HII phase convert into phases of high curvature giving rise to isotropic 31P NMR resonances. Addition of the bilayer destabilizers 1,2-diolein, 1,3-diolein, or eicosane lowers the threshold temperature of the isotropic phase. The isotropic threshold temperature is strongly correlated with the L alpha-HII phase transition temperature (TH). The addition of hexagonal phase promoters does not change the rate of formation of the isotropic phase at a temperature shifted by a fixed amount below TH. However, the formation of "isotropic" phases from the additive-stabilized hexagonal phase is slow compared to that observed in pure N-methyl-DOPE lipid dispersions. Membrane leakage and fusion are promoted by the dioleins and well as by eicosane, but changes in the rates of these processes do not correlate well with the extent of formation of isotropic phases. All three additives have similar effects on phase behavior and on vesicle leakage and fusion. These similarities occur despite the fact that eicosane is believed to partition differently into the membrane than diolein. In addition to the general similarities in the effects of the two diolein isomers, 1,2-diolein is somewhat more potent in promoting the hexagonal phase and in increasing rates of leakage and fusion than is 1,3-diolein.

Partitioning behaviour of 1-hexanol into lipid membranes as studied by deuterium NMR spectroscopy.

Deuterium nuclear magnetic resonance (NMR) spectroscopy was used to study the partitioning behaviour of 1-hexanol specifically deuterated in the alpha-position into model lipid bilayers. In all systems studied, the observed deuterium NMR lineshapes were time-dependent. Initially, 1-hexanol-d2 gave rise to an isotropic deuterium resonance with a different chemical shift from that of aqueous 1-hexanol-d2. After equilibration over a period of days, a broader spectral component characteristic of a spherically-averaged powder-pattern was observed. The quadrupole anisotropy of the 1-hexanol-d2 giving rise to the broad spectrum depended upon the cholesterol content of the membrane. From quantitation of the anisotropic to isotropic deuterium NMR spectra, the partition coefficients of 1-hexanol-d2 in a number of bilayer systems (asolectin and phosphatidylcholine bilayers (the latter with and without cholesterol] were determined. The partitioning of 1-hexanol-d2 into red blood cell membranes, and a suspension of lipids extracted from red blood cell membranes, was also examined. It is suggested that 1-hexanol, and probably other lipophiles, can partition to either the bilayer surface or the bilayer interior in a time-dependent manner.

Identification of trapped and boundary lipid binding sites in M13 coat protein/lipid complexes by deuterium NMR spectroscopy.

The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order in protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell.