Orientation of specifically 13C=O labeled phosphatidylcholine multilayers from polarized attenuated total reflection FT-IR spectroscopy.

Oriented multilayers of 1-myristoyl-2(1-13C)-myristoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DMPC) and 1-palmitoyl-2(1-13C)-palmitoyl-sn-glycero-3-phosphatidylcholine (2[1-13C]DPPC) were investigated by use of attenuated total reflection infrared spectroscopy with polarized light. Experiments were performed with the aim to determine the orientation of the two ester groups in these phospholipids in the solid state and in the hydrated state at temperatures below and above the respective gel to liquid-crystalline phase transitions. Substitution of the naturally occurring 12C carbonyl carbon atom by 13C in the ester group of the sn-2 chain of DMPC and DPPC shifts the infrared absorption of the carbonyl double bond stretching vibration to lower frequency. This results in two well-resolved ester C=O bands which can be assigned unequivocally to the sn-1 and sn-2 chains as they are separated by more than 40 cm-1. The two ester CO-O single bond stretching vibrations of the molecular fragments-CH2CO-OC-are also affected and the corresponding infrared absorption band shifts by 20 cm-1 on 13C-labeling of the carbonyl carbon atom. From the dichroic ratios of the individual ester bands in 2(1-13C)DMPC and 2(1-13C)DPPC we were able to demonstrate that the sn-1 and sn-2 ester C=O groups are similarly oriented with respect to the bilayer plane, with an angle greater than or equal to 60 degrees relative to the bilayer normal. The two CO-O single bonds on the other hand have very different orientations. The CH2CO-OC fragment of the sn-1 chain is oriented along the direction of the all-trans methylene chain, whereas the same molecular segment of the sn-2 carbon chain is directed toward the bilayer plane. This orientation of the ester groups is retained in the liquid-crystalline phase. The tilt angle of the hydrocarbon all-trans chains, relative to the membrane normal, is 25 degrees in the solid state of DMPC and DPPC multibilayers. In the hydrated gel state this angle varies between 26 degrees and 30 degrees, depending on temperature. Neither the orientation of the phosphate group, nor that of the choline group varies significantly in the different physical states of these phospholipids.     

Fourier transform infrared spectroscopy of 13C = O-labeled phospholipids hydrogen bonding to carbonyl groups

Fourier transform infrared spectroscopy has been used to characterize the carbonyl stretching vibration of DMPC, DMPE, DMPG, and DMPA, all labeled with 13C at the carbonyl group of the sn-2 chain. Due to the vibrational isotope effect, the 13C = O and the 12C = O vibrational bands are separated by ca. 40-43 cm-1. This frequency difference does not change when the labeling is reversed with the 13C = O group at the sn-1 chain. For lipids in organic solvents possible conformational differences between the sn-1 and sn-2 ester groups have no effect on the vibrational frequency of the C = O groups. In aqueous dispersion unlabeled phospholipids always show a superposition of two bands for the C = O vibration located at ca. 1740 and 1727 cm-1. These two bands have previously been assigned to the sn-1 and sn-2 C = O groups. FT-IR spectra of 13C-labeled phospholipids show that the vibrational bands of both, the sn-1 as well as the sn-2 C = O group, are clearly superpositions of at least two underlying components of different frequency and intensity. Band frequencies were determined by Fourier self-deconvolution and second-derivative spectroscopy. The difference between the component bands is ca. 11-17 cm-1. Again, the conformational effect as shown by reversed labeling is negligible with only 1-2 cm-1. The splitting of the C = O vibrational bands in H2O and D2O is caused by hydrogen bonding of water molecules to both C = O groups as shown by a comparison with spectra of model ester compounds in different solvents. To extract quantitative information about changes in hydration, band profiles were stimulated with Gaussian-Lorentzian functions. The chemical nature of the head group and its electronic charge have distinctive effects on the extent of hydration of the carbonyl groups. In the gel and liquid-crystalline phase of DMPC the sn-2 C = O group is more hydrated than the sn-1 C = O. This is accord with the conformation determined by X-ray analysis. In DMPG the sn-1 C = O group seems to be more accessible to water, indicating a different conformation of the glycerol backbone.     

Restatement of order parameters in biomembranes: calculation of C-C bond order parameters from C-D quadrupolar splittings.

An expression for the C-C bond order parameter, SCC, of membrane hydrocarbon chains has been derived from the observed C-D bond order parameters. It allows calculation of the probability of each of the C-C bond rotamers and, consequently, the number of gauche defects per chain as well as their projected average length onto the bilayer normal, thus affording the calculation of accurate hydrophobic bilayer thicknesses. The effect of temperature has been studied on dilauroyl-, dimyristoyl-, and dipalmitoylphosphatidylcholine (DLPC, DMPC, DPPC) membranes, as has the effect of cholesterol on DMPC. The salient results are as follows: 1) an odd-even effect is observed for the SCC versus carbon position, k, whose amplitude increases with temperature; 2) calculation of SCC, from nonequivalent deuterons on the sn-2 chain of lipids, SCC2, leads to negative values, indicating the tendency for the C1-C2 bond to be oriented parallel to the bilayer surface; this bond becomes more parallel to the surface as the temperature increases or when cholesterol is added; 3) calculation on the sn-2 chain length can be performed from C1 to Cn, where n is the number of carbon atoms in the chain, and leads to 10.4, 12.2, and 13.8 A for DLPC, DMPC, and DPPC close to the transition temperature, TC, of each of the systems and to 9.4, 10.9, and 12.6 for T-TC = 30-40 degrees C, respectively; 4) separation of intra- and intermolecular motions allows quantitation of the number of gauche defects per chain, which is equal to 1.9, 2.7, and 3.5 for DLPC, DMPC, and DPPC near TC and to 2.7, 3.5, and 4.4 at T-TC = 30-40 degrees C, respectively. Finally, the validity of our model is discussed and compared with previously published models.     

Computation of mixed phosphatidylcholine-cholesterol bilayer structures by energy minimization.

The energetically preferred structures of dimyristoylphosphatidylcholine (DMPC)-cholesterol bilayers were determined at a 1:1 mole ratio. Crystallographic symmetry operations were used to generate planar bilayers of cholesterol and DMPC. Energy minimization was carried out with respect to bond rotations, rigid body motions, and the two-dimensional lattice constants. The lowest energy structures had a hydrogen bond between the cholesterol hydroxyl and the carbonyl oxygen of the sn-2 acyl chain, but the largest contribution to the intermolecular energy was from the nonbonded interactions between the flat alpha surface of cholesterol and the acyl chains of DMPC. Two modes of packing in the bilayer were found; in structure A (the global minimum), unlike molecules are nearest neighbors, whereas in structure B (second lowest energy) like-like intermolecular interactions predominate. Crystallographic close packing of the molecules in the bilayer was achieved, as judged from the molecular areas and the bilayer thickness. These energy-minimized structures are consistent with the available experimental data on mixed bilayers of lecithin and cholesterol, and may be used as starting points for molecular dynamics or other calculations on bilayers.     

Cholesterol-lipid interactions: An infrared and raman spectroscopic study of the carbonyl stretching mode region of 1,2-dipalmitoyl phosphatidylcholine bilayers

Infrared and Raman spectra were obtained for the 1690–1770 cm^?1 carbonyl stretching mode region for 1,2-dipalmitoyl phosphatidylcholine (DPPC) bilayers in the anhydrous, partially hydrated and completely hydrated states. Spectral features at approx. 1740 and 1721 cm^?1 are assigned to CO stretching modes associated with the 1- and 2-chain carbonyl groups, respectively. Splittings of the primary transitions at 1743, 1738, ～1731 and ～1721 cm^?1 are attributed to rotational isomers involving the entire chain. Hydrogen bond formation between the fatty acid carbonyl and 3βOH cholesterol groups was investigated for anhydrous DPPC bilayers. Examination of frequencies, intensities and half-widths of the carbonyl bands indicates that no hydrogen bonding occurs at either of the two carbonyl sites. However, the addition of cholesterol to completely hydrated DPPC dispersions reduces the conformational inequivalence between the two fatty acid carbonyl groups by specifically perturbing the 2-chain. For cholesterol containing systems the carbonyl stretching mode transitions were also used to monitor lattice effects within the interface region as water binds to the bilayer head groups. Specifically, the addition of approx. 2 molecules of water per lipid molecule orders the lipid lattice and increases the bilayer packing density, while the subsequent addition of 4 molecules of water per lipid molecule releases the packing constraints within the interface region and thereby decreases the packing density.     

Alcohols dehydrate lipid membranes: an infrared study on hydrogen bonding.

The effects of alcohols (methanol, ethanol, and n-butanol) on the hydrogen bonding of dipalmitoylphosphatidylcholine (DPPC) were studied by Fourier-transform infrared spectroscopy (FTIR) in water-in-oil (carbon tetrachloride) reversed micelles. The bound O-H stretching mode of water, bonded to DPPC, appeared as a broad band at around 3400 cm-1. The O-H bending mode of this complex appeared as a weak broad band at 1644 cm-1. No free O-H signal was observed. When alcohols were added, a part of DPPC-bound water was replaced by the alcohols. The released 'free' water appeared at 3680 cm-1. This free O-H stretching band represents water-alcohol complex. A new broad band of O-H stretching appeared at 3235 cm-1, which represents the alcohol molecules bound to the phosphate moiety of DPPC. When the alcohol concentration was increased, the intensities of the free O-H stretching and bending bands increased. The P = O- antisymmetric stretching band at 1238 cm-1 became broader and shifted to lower frequencies. This means that alcohols interacted with the phosphate moiety and replaced the bound water. In the deconvoluted spectra of the C = O stretching mode, the ratio between the free sn-2 and the hydrogen-bonded sn-2 bands increased; a part of the bound water at the sn-2 carbon in the glycerol skeleton is also released and the free sn-2 signal increased. From the change in the intensity of the P = O- stretching band, the partition coefficients of alcohols between the phosphate region of DPPC and water were estimated: methanol 7.8, ethanol 16.7 at 22.0 degrees C in mole fraction bases. In molality, these values translates into methanol 0.21 and ethanol 0.45. These results indicate that short-chain alcohols interact with lipid membranes at the phosphate moiety at the hydrophilic head, weaken the membrane-water interaction, and destabilize membranes.     

Membrane water-penetration profiles from spin labels

Spin label hyperfine splittings in mixtures of protic and aprotic solvents are used to obtain association constants K(A,h) for hydrogen bonding to oxazolidine nitroxides. With the Onsager approach to account for the variation in local dielectric constant, these results are used to determine the effective penetration profile of water into fluid phospholipid membranes, from recent electron paramagnetic resonance (EPR) studies on phospholipids spin-labelled systematically down the sn-2 chain. Water penetration is appreciable, depends on chain unsaturation, and is strongly affected by cholesterol.     

Charge pairing of headgroups in phosphatidylcholine membranes: A molecular dynamics simulation study

Molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine (DMPC) bilayer membrane in the liquid-crystalline phase was carried out for 5 ns to study the interaction among DMPC headgroups in the membrane/water interface region. The phosphatidylcholine headgroup contains a positively charged choline group and negatively charged phosphate and carbonyl groups, although it is a neutral molecule as a whole. Our previous study (Pasenkiewicz-Gierula, M., Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi. 1997. J. Phys. Chem. 101:3677-3691) showed the formation of water cross-bridges between negatively charged groups in which a water molecule is simultaneously hydrogen bonded to two DMPC molecules. Water bridges link 76% of DMPC molecules in the membrane. In the present study we show that relatively stable charge associations (charge pairs) are formed between the positively and negatively charged groups of two DMPC molecules. Charge pairs link 93% of DMPC molecules in the membrane. Water bridges and charge pairs together form an extended network of interactions among DMPC headgroups linking 98% of all membrane phospholipids. The average lifetimes of DMPC-DMPC associations via charge pairs, water bridges and both, are at least 730, 1400, and over 1500 ps, respectively. However, these associations are dynamic states and they break and re-form several times during their lifetime.     

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.     

IR spectroscopic study of phospholipid emulsions containing cholesteryl oleate

As models for the lipid organization of low density lipoproteins (LDL), protein-free aqueous emulsions are prepared from dimyristoyl phosphatidyl choline (DMPC), dipalmitoyl phosphatidyl choline (DPPC), and cholesteryl oleate (CO). Aqueous dispersions containing these lipids are sonicated and yield stable particles with diameters varying between 20 and 40 nm as measured through electron microscopy. IR spectroscopy shows that emulsions consisting of DMPC, DPPC, and CO at 3/1/1 and 1/1/1 ratios undergo specific thermal transitions, depending on their composition, that can be assigned to the phospholipids forming the surface layer of the emulsion particles and to core-located CO. However, at the 1/3/1 DMPC/DPPC/CO ratio this lipid system exhibits an order-disorder transition of the mixed phospholipids with no significant transition associated with core-located CO. Observation of the methylene C&bond;H and C&bond;D stretching modes of nondeuterated and deuterated lipids enables the packing characteristics and conformational order of each lipid to be monitored separately. The transition temperature changes compared to the temperatures for the analogous transitions in neat CO and CO-free phospholipid vesicles suggest the existence of interactions between CO and the above phospholipids in the ternary emulsion particles; these interactions are stronger at the 1/3/1 DMPC/DPPC/CO ratio. The results show that interactions between core and surface phases are dependent on the emulsion lipid composition and that these findings may be extended to native lipoproteins.     

The effect of lung surfactant apolipoproteins B/C on the chemical shift anisotropy of sn-2-[1-13C]dipalmitoylphosphatidylcholine

Nuclear magnetic resonance spectroscopy has been used to investigate the effect of the lung surfactant apolipoproteins B/C on dipalmitoylphosphatidylcholine to address the mechanism by which the adsorption rate of phospholipids from the bulk to the air/water interface is enhanced. Apolipoproteins B/C were isolated from bovine lung and separated from associated lipids by lipophilic Sephadex column chromatography. Amino acid analysis indicated the presence of both apolipoproteins B and C. The 13C chemical shift anisotropy of DPPC was determined as a function of temperature. Previous workers (Wittebort et al., Biochemistry, 20 (1981) 3487-3502) have concluded that the observed magnitude of the chemical shift anisotropy of the carbonyl group of the sn-2 acyl chain in pure DPPC is a result of rapid rotation about an axis along the length of the phospholipid both in the gel and liquid crystalline state. The orientation of the carbonyl group with respect to the axis of diffusion, however, undergoes an approximately 25-30 degrees shift in passage from the gel to liquid crystalline state, with the intermediate, rippled (P beta') state composed of an exchange between these two orientations. The presence of physiological concentrations SP-B/C reduced the width of the anisotropy of DPPC below but had no effect on lipids above the main phase transition temperature. This suggests that SP-B/C has a general effect on the entire assembly of lipids. The temperature of the onset of the orientational change is lowered indicating a portion of the lipids are affected by the lung surfactant apolipoproteins.     

Cholesterol effects on the phosphatidylcholine bilayer polar region: a molecular simulation study

A molecular dynamics (MD) simulation of a fully hydrated, liquid-crystalline dimyristoylphosphatidylcholine (DMPC)-Chol bilayer membrane containing approximately 22 mol% Chol was carried out for 4.3 ns. The bilayer reached thermal equilibrium after 2.3 ns of MD simulation. A 2.0-ns trajectory generated during 2.3-4.3 ns of MD simulation was used for analyses to determine the effects of Chol on the membrane/water interfacial region. In this region, 70% of Chol molecules are linked to DMPC molecules via short-distance interactions, where the Chol hydroxyl group (OH-Chol) is 1) charge paired to methyl groups of the DMPC choline moiety ( approximately 34%), via the hydroxyl oxygen atom (Och); 2) water bridged to carbonyl ( approximately 19%) and nonester phosphate ( approximately 14%) oxygen atoms, via both Och and the hydroxyl hydrogen atom (Hch); and 3) directly hydrogen (H) bonded to carbonyl ( approximately 11%) and nonester phosphate ( approximately 5%) oxygen atoms, via Hch ( approximately 17% of DMPC-Chol links are multiple). DMPC's gamma-chain carbonyl oxygen atom is involved in 44% of water bridges and 51% of direct H bonds formed between DMPC and Chol. On average, a Chol molecule forms 0.9 links with DMPC molecules, while a DMPC molecule forms 2.2 and 0.3 links with DMPC and Chol molecules, respectively. OH-Chol makes hydrogen bonds with 1.1 water molecules, preferentially via Hch. The average number of water molecules H bonded to the DMPC headgroup is increased by 7% in the presence of Chol. These results indicate that inclusion of Chol decreases interlipid links and increases hydration in the polar region of the membrane.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ether phosphatidylcholines: comparison of miscibility with ester phosphatidylcholines and sphingomyelin, vesicle fusion, and association with apolipoprotein A-I

Nonhydrolyzable matrices of ether-linked phosphatidylcholines (PCs) and sphingomyelin have been used to study the mechanism of action of lipolytic enzymes. Since ether PCs, sphingomyelin, and ester PCs vary in the number of hydrogen bond donors and acceptors in the carbonyl region of the bilayer, we have examined several physical properties of ether PCs and sphingomyelin in model systems to validate their suitability as nonhydrolyzable lipid matrices. The intermolecular interactions of ether PCs with ester PCs, sphingomyelin, and cholesterol were investigated by differential scanning calorimetry. Phase diagrams constructed from the temperature dependence of the gel to liquid-crystalline phase transition of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether) and 1,2-O-ditetradecyl-sn-glycero-3-phosphocholine (DMPC-ether) with both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) demonstrated complete lipid miscibility in the gel and liquid-crystalline phases. Additionally, phase diagrams of egg yolk sphingomyelin (EYSM) with DMPC or DMPC-ether and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-O-dioctadecyl-sn-glycero-3-phosphocholine (DSPC-ether) demonstrated no major differences in miscibility of EYSM in ester and ether PCs. The effect of 10 mol % cholesterol on the thermal transitions of mixtures of ester and ether PCs also indicates little preference of cholesterol for either lipid. The fusion of small single bilayer vesicles of DMPC, DMPC-ether, DPPC, and DPPC-ether to larger aggregates as determined by gel filtration indicated that the ester PC vesicles were somewhat more stable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal behavior of liposomes containing PCs with long and very long chain PUFAs isolated from retinal rod outer segment membranes

About one-fourth the phosphatidylcholines (PC) from retina photoreceptor rod outer segment (ROS) membranes contain docosahexaenoic acid (22:6n-3) at sn-2 and a very long chain polyunsaturated fatty acid (VLCPUFA) (C24 to C36) at the sn-1 position of the glycerol backbone. In order to study the thermotropic behavior of these PCs, subfractions and molecular species of PC (16:0/22:6, 18:0/22:6, 22:6/22:6, 32:5/22:6, 32:6/22:6, 34:5/22:6), were isolated from bovine ROS, and liposomes containing different proportions of these PCs and dimyristoyl-PC (DMPC) or dipalmitoyl PC (DPPC) were compared using the fluorescence probes Laurdan and 1,6-diphenyl-1,3,5-hexatriene (DPH). With both probes, the 22:6n-3 containing PCs from ROS, in all proportions tested, decreased the transition temperature (Tt) of both DMPC and DPPC. Below the transition temperature, coexistence of phases was evidenced in all cases. Liposomes formed with 100% of any of these PCs did not show phase transitions in the temperature range studied (8 degrees C to 50 degrees C). At physiological temperatures, as it is likely to be the case in ROS membranes, all of these PC species were in the liquid-crystalline state. With Laurdan, all dipolyunsaturated PCs seemed to behave similarly: despite the large number of double bonds per molecule, all of them decreased the Tt of DPPC less than did the hexaenoic PCs. With DPH, an ample difference was detected between the dipolyunsaturates, 22:6/22:6-PC and VLCPUFA/22:6-PCs, and between the latter and hexaenoic PCs throughout the temperature range studied. This difference is consistent with the interpretation that the largest "disorder" produced by PCs containing a VLCPUFA like 32:6n-3 at the sn-1 position occurs toward the center of the membrane.     

Spin-label electron spin resonance studies on the mode of anchoring and vertical location of the N-acyl chain in N-acylphosphatidylethanolamines

Electron spin resonance (ESR) studies have been performed on N-myristoyl dimyristoylphosphatidylethanolamine (N-14-DMPE) membranes using both phosphatidylcholines spin-labeled at different positions in the sn-2 acyl chain and N-acyl phosphatidylethanolamines spin-labeled in the N-acyl chain to characterize the location and mobility of the N-acyl chain in the lipid membranes. Comparison of the positional dependences of the spectral data for the two series of spin-labeled lipids suggests that the N-acyl chain is positioned at approximately the same level as the sn-2 chain of the phosphatidylcholine spin-label. Further, similar conclusions are reached when the ESR spectra of the N-acyl PE spin-labels in dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylethanolamine (DMPE) host matrixes are compared with those of phosphatidylcholine spin-labels in these two lipids. Finally, the chain ordering effect of cholesterol has also been found to be similar for the N-acyl PE spin-label and PC spin-labels, when the host matrix is either DMPC and cholesterol or N-14-DMPE and cholesterol at a 6:4 mole ratio. In both cases, the gel-to-liquid crystalline phase transition is completely abolished but cholesterol perturbs the gel-phase mobility of N-14-DMPE more readily than that of DMPC. These results demonstrate that the long N-acyl chains are anchored firmly in the hydrophobic interior of the membrane, in an orientation that is parallel to that of the O-acyl chains, and are located at nearly the same vertical position as that of the sn-2 acyl chains in the lipid bilayer. There is a high degree of dynamic compatibility between the N-acyl chains and the O-acyl chains of the lipid bilayer core, although bilayers of N-acyl phosphatidylethanolamines possess a more hydrophobic interior than phosphatidylcholine bilayers. These results provide a structural basis for rationalizing the biological properties of NAPEs.     

Comparative study of the interaction of fullerenol nanoparticles with eukaryotic and bacterial model membranes using solid-state NMR and FTIR spectroscopy

Native fullerene is notoriously insoluble in water and forms aggregates toxic to cell membranes, thus limiting its use in nanomedicine. In contrast, water-soluble fullerenol is compatible with biological systems and shows low in vivo toxicity on human cell lines. The interaction mechanism between these hydrophilic nanoparticles and biological membranes is however not well understood. Therefore, in this work, the effect of fullerenol on model eukaryotic and bacterial membranes was investigated using (31)P- and (2)H solid-state NMR as well as FTIR spectroscopy. DPPC/cholesterol and DPPC/DPPG bilayers were used to mimic eukaryotic and bacterial cell membranes, respectively. Our results show low affinity of fullerenol for DPPC/cholesterol bilayers but a clear interaction with model bacterial membranes. A preferential affinity of fullerenol for the anionic phospholipids DPPG in DPPC/DPPG membranes is also observed. Our data suggest that fullerenol remains at the water/bilayer interface of eukaryote-like membranes. They also indicate that the presence of a polar group such as DPPG's hydroxyl moiety at the bilayer surface plays a key role in the interaction of fullerenol with membranes. Hydrogen bonding of fullerenol nanoparticles with DPPGs' OH groups is most likely responsible for inducing lipid segregation in the lipid bilayer. Moreover, the location of the nanoparticles in the polar region of DPPG-rich regions appears to disturb the acyl chain packing and increase the membrane fluidity. The preferential interaction of fullerenol with lipids mostly found in bacterial membranes is of great interest for the design of new antibiotics.     

Interaction of carbohydrates with dry dipalmitoylphosphatidylcholine

Interactions of six carbohydrates (trehalose, sucrose, glucose, raffinose, inositol, and glycerol) with dry dipalmitoylphosphatidylcholine (DPPC) were studied using differential scanning calorimetry (DSC) and infrared spectroscopy (ir) in order to elucidate the mechanism by which some of these carbohydrates preserve structural and functional integrity of dry membranes. Results with DSC showed that trehalose depressed the main transition temperature (Tmid) of dry DPPC below that of fully hydrated DPPC, and raised the enthalpy of that transition more than did addition of water. Results obtained with ir spectroscopy suggested a potential mechanism for this interaction. In the presence of most of the carbohydrates the ir spectrum for DPPC showed changes similar to those seen when water was added to dry DPPC, and the asymmetric P = O stretching band was diminished in intensity. The degree to which the carbohydrates tested affected the integrated intensity of this band and the Tmid was correlated with the ability of those carbohydrates to preserve dry membranes. Also, bands assigned to -OH deformations in the trehalose and other carbohydrates were depressed in the presence of DPPC. Based on these observations, it is suggested that the mechanism of interaction between the carbohydrate and lipid involves hydrogen bonding between -OH groups on the carbohydrate and the phosphate head group of the phospholipid. The only exceptions to this pattern are glycerol, which depresses Tmid of dry DPPC, and myo-inositol, which has no effect on Tmid or the ir spectrum of DPPC; neither carbohydrate can preserve dry membranes. It is suggested, based on ir spectroscopy and previous results with monolayer preparations, that glycerol interacts with phospholipids by a mechanism different from that shown by the other carbohydrates.     

Dynamic chain conformations in dimyristoyl glycerol-dimyristoyl phosphatidylcholine mixtures. 2H-NMR studies.

The dynamic molecular lipid chain conformations in fully hydrated dimyristoyl phosphatidylcholine (DMPC)-dimyristoyl glycerol (DMG) mixtures have been investigated by 2H-NMR spectroscopy of the individual lipid components, the sn-2 chains of which were perdeuterated or, in the case of DMG, specifically deuterated at the C-2 position. Mixtures of compositions corresponding to the three different regions of the binary phase diagram in which the fluid phase is lamellar (DMPC:DMG 70:30 mol/mol), inverted hexagonal (DMPC:DMG 45:55 and 40:60 mol/mol), or isotropic (DMPC:DMG 20:80 mol/mol) were investigated. The gel phase in all three regions of the phase diagram has a lamellar structure, with the lipid chains rotating about the molecular long axis but executing only limited angular excursions. In the fluid lamellar phase of the 70:30 mol/mol DMPC-DMG mixture the profile of segmental chain flexibility is similar to that in single-component phospholipid bilayers and is characterized by an order parameter plateau for both lipid components. The chain order of the DMPC component is greater than in bilayers of DMPC alone and is also greater than that of the DMG component. In the inverted hexagonal phase of the 45:55 mol/mol DMPC-DMG mixture the chain flexibility profile is characterized by more widely spaced segmental order parameters off the plateau region. The intrinsic degree of chain order in the inverted hexagonal phase is less than in the lamellar phase of the 70:30 mol/mol mixture, and the difference in chain order between the DMPC and DMG components is reduced relative to that in the lamellar phase. The unique conformational features at the C-2 position of the sn-2 chain that characterize bilayers of diacyl phospholipids are found also for the diacylglycerol molecules in the fluid lamellar phase and most probably also in the inverted hexagonal phase. The DMG molecules are therefore integrated in the membrane (or nonlamellar lipid phase) in a configuration that is similar to that of the phospholipids and different from the crystal structure of diacylglycerols.     

Molecular structure and interaction of dipalmitoyl phosphatidylcholine in multilayers. Comparative study with phosphatidylethanolamine

As a model of phospholipid bilayers in solid an oriented multilayer film (built-up film) of L-α-dipalmitoyl phosphatidylcholine (DPPC) was prepared from the monolayer by the dipping method. Structural analysis has been carried out by measuring infrared dichroism of the built-up film. The results were compared with those of the built-up film of L-α-dipalmitoyl phosphatidylethanolamine (DPPE). The tilting of the hydrocarbon chains is larger for DPPC than for DPPE. The orientation of the bisector of the two non-esterified PO bonds is closer to the film plane for DPPC than for DPPE. The strong hydrogen bonding interaction between the polar head groups was shown for DPPE, but not for DPPC. These features resemble the structural differences between dilauroyl phosphatidylethanolamine (DLPE) and dimyristoryl phosphatidylcholine (DMPC) in crystals. The hydrogen bonding interaction of DPPE found in solid remains even in the presence of water, namely, in the gel state. More closed packing of the hydrocarbon chains of solid DPPE than DPPC in solid was concluded on the basis of infrared and Raman spectra.