Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Deuterium NMR study of the effect of n-alkanol anesthetics on a model membrane system

The effects of 25 mol% incorporation of two anesthetics, 1-octanol and 1-decanol, on a deuterated, saturated phospholipid in 50 wt% aqueous multilamellar dispersions have been studied by 2H-NMR spectroscopy and differential scanning calorimetry (DSC). The phospholipid used is sn-2 substituted '[2H31]-palmitoylphosphatidylcholine' (PC-d31). DSC thermograms demonstrate that PC-d31 has phase behavior qualitatively similar to that of dipalmitoylphosphatidylcholine, with a pretransition at 31 degrees C and a main gel to liquid crystalline transition at 40 degrees C. Analysis of the temperature-dependent 2H-NMR spectra in terms of the first moment, which is extremely sensitive to the phospholipid phase, shows that 1-octanol and 1-decanol depress and broaden the main transition. This is confirmed by DSC, which shows that the pretransition is eliminated by the 1-alkanols. The carbon-deuterium bond order of the phospholipid deuterated acyl chains, in the presence and absence of 1-alkanols, was determined from deuterium quadrupolar splittings. Spectra were analyzed using the depaking technique. A 1-alkanol concentration of 25 mol% had no significant effect on the profile of the carbon-deuterium bond order parameter SCD along the phospholipid acyl chain at 50 degrees C. Thus, it appears that the liquid crystalline phase is able to accommodate large amounts of linear anesthetic molecules without substantial effect on molecular ordering within the membrane bilayer. Preliminary results show that the transverse relaxation rates of the acyl chain segments are significantly decreased by the presence of 1-octanol or 1-decanol.     

Structure and Dynamics of Cholesterol-Containing Polyunsaturated Lipid Membranes Studied by Neutron Diffraction and NMR

A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, (2)H-NMR and (13)C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 ? away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid-water interface. Addition of cholesterol partially redistributes DHA density from near the lipid-water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C(8)-C(18) is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin.     

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.     

Effects of cholesterol on conformational disorder in dipalmitoylphosphatidylcholine bilayers. A quantitative IR study of the depth dependence

A method originally proposed by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of trans-gauche isomerization in hydrocarbon chains and recently applied [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939] to the quantitative determination of phospholipid acyl chain conformational order is utilized to monitor the effects of cholesterol at various depths in dipalmitoylphosphatidylcholine (DPPC) bilayers. The method is based on the observation that the CD2 rocking modes from the acyl chains of specifically deuterated phospholipids occur at frequencies in the Fourier transform infrared spectrum which depend upon the local geometry (trans or gauche) of the C-C-C skeleton surrounding a central CD2 group. Three specifically deuterated derivatives of DPPC, namely, 4,4,4',4'-d4 DPPC (4-d4 DPPC), 6,6,6',6'-d4 DPPC (6-d4 DPPC), and 12,12,12',12'-d4 DPPC (12-d4 DPPC), have been synthesized, and the effects of cholesterol addition at 2:1 DPPC/cholesterol (mol:mol) on acyl chain order at various temperatures have been determined. At 48 degrees C, cholesterol inhibits gauche rotamer formation by factors of approximately 9 and approximately 6 at positions 6 and 4, respectively, of the acyl chains, thus demonstrating a strong ordering effect in regions of the bilayer where the sterol rings are presumed to insert parallel to the DPPC acyl chains. In contrast, the ability of the sterol to order the acyl chains is much reduced at the 12-position. The sterol demonstrates only a slight disordering of phospholipid gel phases. Finally, the contributions of different classes of gauche conformers to the spectra have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of cholesterol on interactions and dynamics of ibuprofen in a lipid bilayer

In this work, molecular dynamics (MD) simulations with atomistic details were performed to examine the influence of the cholesterol on the interactions and the partitioning of the hydrophobic drug ibuprofen in a fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer. Analysis of MD simulations indicated that ibuprofen molecules prefer to be located in the hydrophobic acyl chain region of DMPC/cholesterol bilayers. This distribution decreases the lateral motion of lipid molecules. The presence of ibuprofen molecules in the bilayers with 0 and 25 mol% cholesterol increases the ordering of hydrocarbon tails of lipids whereas for the bilayers with 50 mol% cholesterol, ibuprofen molecules perturb the flexible chains of DMPC lipids which leads to the reduction of the acyl chain order parameter. The potential of the mean force (PMF) method was used to calculate the free energy profile for the transferring of an ibuprofen molecule from the bulk water into the DMPC/cholesterol membranes. The PMF studies indicated that the presence of 50 mol% cholesterol in the bilayers increases the free energy barrier and slows down the permeation of the ibuprofen drug across the DMPC bilayer. This can be due to the condensing and ordering effects of the cholesterol on the bilayer.     

Deuterium magnetic resonance of selectively deuterated cholesteryl esters in dipalmitoyl phosphatidylcholine dispersions

Dispersions (50 wt% water) containing 95 mol% dipalmitoyl phosphatidylcholine/5 mol% deuterated cholesteryl palmitate (or stearate) were studied using ²H-NMR. Incorporation of ester into the phospholipid bilayer was found to be 0.5 mol% at 50°C. From the profile of ²H quadrupolar splitting vs. chain position, support for an average conformation resembling a ‘horseshoe’ within the bilayer is obtained. Quadrupolar relaxation times $\text{T}{}_{2e}$ of approx. 250 μs and approx. 850 μs are measured for cholesteryl palmitate-2,2-d₂ and cholesteryl palmitate-16,16,16-d₃, respectively, which are less than one-half those obtained for the corresponding positions in dipalmitoyl-d₆₂ phosphatidylcholine. This is ascribed to a slower rate of motion of the ester chain and/or an extra, slow motion of the molecule.     

Chain dynamics of selectively deuterated fatty acids in high-density lipoproteins studied by deuterium NMR

Deuterium order parameters have been determined for approximately 5 mol% selectively deuterated palmitic acid incorporated into the outer monolayer of high-density lipoproteins (HDL3). The values are SCD = 0.38 for [2,2-2H2]palmitic acid, 0.38 for [4,4-2H2]palmitic acid, 0.37 for [5,5,6,6-2H4]palmitic acid, 0.23 for [11,11,12,12-2H4]palmitic acid, and 0.05 for [16,16,16-2H3]palmitic acid. Comparison of the acyl chain order parameters in HDL3 with acyl chain order parameters determined recently [Parmar, Y.I., Wassall, S.R., & Cushley, R.J. (1984) J. Am. Chem. Soc. 106, 2434-2435] for approximately 5 mol% deuterated palmitic acid in sonicated unilamellar vesicles, composed of the same ratio of phosphatidylcholine/sphingomyelin (85/15 w/w) found in HDL3, shows that acyl chain order in the HDL3 monolayer is approximately 3-5 times higher than in the vesicle bilayer. The acyl chain order in the lipoprotein monolayer is approximately 1.5-2 times higher than in the bilayer of phosphatidylcholine multilamellar dispersions. Deuterium longitudinal relaxation times have been measured for deuterated palmitic acid in HDL3, and the values T1 approximately 16 ms for C2H2 and 170 ms for C2H3 groups are a factor of more than 2 times smaller than found in phospholipid bilayers.     

Bilayers of dipalmitoyl-3-sn-phosphatidylcholine: Conformational differences between the fatty acyl chains

$\text{Dipalmitoyl}\text{-3-sn-}\text{phosphatidylcholine}$ is specifically deuterated at the C-2 position of the fatty acyl chains. Using deuterium magnetic resonance it is then possible to probe the chain conformation in the vicinity the polar head groups. Three separate quadrupole splittings are observed for bilayers of 1,2[2′-²H₂] $\text{palmitoyl}\text{-3-sn-}\text{phosphatidylcholine}$, indicating that the two chains behave differently. The synthesis of phosphatidylcholines each deuterated in only one chain allows the assignment of the three resonances. It is concluded that the beginnings of the two chains have orientations parallel and perpendicular to the bilayer normal. The data further suggest the possibility of two long-lived conformations of the glycerol constituent.     

Photosensitive liposomes as 'cages' for laser-triggered solute delivery: the effect of bilayer cholesterol on kinetics of solute release

Liposomes containing acyl chains incorporating azobenzene chromophores have been investigated as potential 'caging' agents for fast solute release. On photolysis, trapped marker dye can be released from gel-phase liposomes within milliseconds. Solute release is markedly sensitive to the presence of cholesterol in the bilayer. Phospholipids bearing one saturated acyl chain and an azobenzene-substituted chain are ineffective as sensitisers unless cholesterol is present, while doubly substituted phospholipids sensitise release in its absence. Cholesterol markedly affects the temperature profile of solute release depending on the host phospholipid chain length. Solute release is not seen for lipid hosts with unsaturated acyl chains.     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

Molecular packing in steroid-lecithin monolayers,part II: Mixed films of cholesterol with dipalmitoylphosphatidylcholine and tetradecanoic acid

Mixed film studies of the systems cholesterol/tetradecanoic acid and cholesterol/dipalmitoylphosphatidylcholine have been carried out over the entire compositional range at 21°C. When compared on an acyl chain basis the condensing effects were found to be essentially independent of which host-lipid was utilized. The phase change of the host lipid was shifted to higher pressures, then broadened and eliminated. Maximal condensation occurred at just above 42 mol% for the cholesterol/DPPC system. In both systems the two components were initially found to be miscible at all proportions.The results are interpreted in terms of the molecular packing of cholesterol with acyl boundary layers, one significantly, one weakly affected. Maximum condensation is a result of packing that provides maximum cholesterol/acyl chain contact. Consideration is given to both long term stability of such mixed monolayers and the behaviour of the corresponding bilayer states.     

N-acylphosphatidylethanolamines: effect of the N-acyl chain length on its orientation

N-Acylphosphatidylethanolamines, or NAPEs, are found in tissues involved in degenerating processes, such as dehydrated endosperm of seeds, erythrocyte membranes, or cell injury. To determine the conformation and orientation of the acyl chains of these phospholipids, NAPEs with deuterated N-acyl chains of 6 and 16 carbon atoms were synthesized and studied by transmission and attenuated total reflectance (ATR) infrared spectroscopy. For N-C16d-DPPE, the ATR measurements show that the N-acyl chain has the same orientation as the two acyl chains attached to the glycerol moiety, while the N-acyl chain of N-C6d-DPPE is randomly oriented. These results demonstrate that for N-C16d-DPPE, the N-acyl chain is embedded into the hydrophobic core of the bilayer, while for the short chain derivative the N-acyl chain remains in the lipid headgroup region. The analysis of the carbonyl stretching band and of the amide I band suggests that, for the long N-acyl chain lipid, the ester C=O and the N-H groups are linked by intermolecular hydrogen bonds.     

Orientational order of cholesteryl oleate in low-density lipoprotein observed by 2H-NMR

Cholesteryl oleate, selectively deuterated at various positions along the acyl chain, has been incorporated into fresh human serum low-density lipoprotein (LDL2). Temperature-dependent 2H-NMR spectra were recorded between 15 and 45 degrees C. For deuterons at C-2' and C-5' of the acyl chain, two 2H-NMR spectral components, a broad and a narrow signal, are observed. This is interpreted as reflecting the coexistence of two cholesteryl ester regions in the LDL2 core which possess different degrees of order. The C-2H bond order parameters, SCD, are approx. 0.12-0.20 for the more ordered region and approx. 0.04-0.06 for the less ordered region. Longitudinal relaxation times, T1, of deuterated cholesteryl oleate are found to increase between C-8' and the terminal -C2H3 group, which is consistent with an increased rate of chain motion toward the free ends of the ester acyl chains.     

Structure and cohesive properties of sphingomyelin/cholesterol bilayers.

Thermal, structural, and cohesive measurements have been obtained for both bovine brain sphingomyelin (BSM) and N-tetracosanoylsphingomyelin (C24-SM) in the presence and absence of cholesterol. A goal of these experiments has been to clarify the mechanisms responsible for the strong interaction between sphingomyelin and cholesterol. Differential scanning calorimetry shows that fully hydrated bilayers of BSM and C24-SM have main endothermic phase transitions at 39 and 46 degrees C, respectively, that reflect the melting of the acyl chains from a gel to a liquid-crystalline phase. For each lipid, the addition of cholesterol monotonically reduces the enthalpy of this transition, so that at equimolar cholesterol the transition enthalpy is zero. The addition of equimolar cholesterol to either BSM or C24-SM coverts the wide-angle X-ray diffraction reflection at 4.15 A to a broad band centered at 4.5 A. Electron density profiles of gel-phase C24-SM bilayers contain two terminal methyl dips in the center of the bilayer, indicating that the lipid hydrocarbon chains partially interdigitate so that the long saturated 24-carbon acyl chains in one monolayer cross the bilayer center and appose the shorter sphingosine chains from the other monolayer. The incorporation of cholesterol adds electron density to the hydrocarbon chain region near the head group and removes the double terminal methyl dip. These wide- and low-angle X-ray data indicate that cholesterol packs into the hydrocarbon chain region near the sphingomyelin head group, fluidizes the methylene chains near the center of the bilayer compared to the gel phase, and reduces the extent of methylene chain interdigitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane thickening by the antimicrobial peptide PGLa

Using x-ray diffraction, solid-state ²H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.     

Location and effect of procaine on lecithin/cholesterol membranes using X-ray diffraction methods

X-ray diffraction studies were made on lecithin/cholesterol multilayers with very high water content and containing the local anaesthetic procaine. Narrow-angle diffraction experiments show that the procaine molecules are located with the uncharged aromatic amine group approx. 10 Å from the centre of the bilayer. The polar tertiary amine group of these molecules is almost certainly located in the polar head-group region of the membrane. Wide-angle diffraction experiments show that the incorporation of procaine molecules into such lipid membranes produces an approx. 30% increase in the spread of acyl chain separation, although the average spacing between the chains is slightly reduced.     

The influence of oxygenated sterol compounds on dipalmitoylphosphatidylcholine bilayer structure and packing

Fourier Transform Infra-red and Raman Spectroscopies indicate that 7 alpha-hydroxycholesterol and 7-ketocholesterol have a diminished capacity to condense (increase the packing order of) fluid-state dipalmitoylphosphatidylcholine (DPPC) acyl chains when compared with the effects of cholesterol and the other oxidized sterols studied. DPPC head groups were also more ordered by 7-ketocholesterol over the temperature range 10 degrees - 70 degrees C. Primary effects of these sterols appear to be associated with the hydrophillic regions of the DPPC bilayer, although packing arrangements with acyl chains are also involved. Phosphate and acyl chain ester groups were observed to possess a packing order which was invariant which indicates that these may be the target groups in the interaction with 7-ketocholesterol. A surprising observation was the synergistic amplification of the effects of 7-ketocholesterol by the presence of cholesterol in the DPPC bilayer.     

The thermotropic behavior of dipalmitoyl phosphatidylcholine bilayers. A Fourier transform infrared study of specifically labeled lipids.

Fourier transform infrared spectroscopy has been used to study the thermotropic behavior of hydrated multibilayers of specifically deuterated derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. It is shown that throughout the gel phase there is little or no conformational disorder introduced into the acyl chains. The pretransition effect is greatest in the central segments of the acyl chains, demonstrating that the interchain interactions are more pronounced in this region than in the center of the bilayer and suggesting the presence, in the gel phase, of a "plateau" in the strength of the interchain interactions. As the temperature is reduced, the rate of rotation of the terminal methyl group decreases steadily; below 0 degree C the conformation is rigid on the infrared time scale.     

Raman spectroscopy of selectively deuterated dimyristoylphosphatidylcholine: studies on dimyristoylphosphatidylcholine-cholesterol bilayers

Dimyristoylphosphatidylcholine (DMPC), selectively deuterated in the sn-2 chain at the 3, 6, and 10 positions is used to probe DMPC-cholesterol interactions in multilamellar dispersions. Using the Raman spectral linewidths of the 2100 cm-1 C2H2 stretching modes as an index of membrane disorder, we demonstrate that cholesterol tends to order, or increase the number of trans carbon-carbon bonds within the DMPC acyl chain near the headgroup region at all temperatures. At low temperatures, cholesterol disorders the acyl chains near the methyl termini by inducing gauche conformers; cholesterol orders the entire chain at higher temperatures. These determinations are qualitatively consistent with conclusions drawn from deuterium nuclear magnetic resonance studies, but specifically reflect acyl chain trans/gauche isomerization on the 10(-12)-10(-13) s vibrational time scale.