Studies on the interaction of cholesterol with diester- and dietherlecithin

The lamellar repeat distances of aqueous dispersions of rac-1,2-dioctadec-9′-cis-enyl-glycero-3-phosphorylcholine (dietherlecithin) and 1,2-dioctadec-9′-cis-enoyl-sn-glycero-3-phosphorylcholine (diesterlecithin) have been measured by X-ray diffraction as a function of water concentration. The point of maximum hydration was found to be 43% (w/w) and 40% (w/w) for dietherlecithin and diesterlecithin respectively; the corresponding lamellar repeat distances being 62.3 Å and 60.5 A. Incorporation of cholesterol above maximum hydration results in the initial increase in the lamellar repeat distance with a maximum around cholesterol concentrations of 25 and 33 mol % for dietherlecithin and die diesterlecithin respectively.The apparent partial specific volumes of the two lecithins and for lecithin-cholesterol mixtures in sonicated aqueous dispersions were measured. Values of 1.024 cm³ · g^?1 and 0.987 cm³ · g^?1 were obtained for diether- and diesterlecithin, respectively, at 20°C. Diesterlecithin-cholesterol mixtures showed a very small change in partial specific volume while mixtures of dietherlecithin-cholesterol showed a very marked decrease with increasing proportions of cholesterol.From these data a series of structure parameters are derived for the two lecithins and possible implications for the nature of the lecithin-cholesterol interaction are discussed.     

Interplay of unsaturated phospholipids and cholesterol in membranes: effect of the double-bond position

The structural and dynamical properties of lipid membranes rich in phospholipids and cholesterol are known to be strongly affected by the unsaturation of lipid acyl chains. We show that not only unsaturation but also the position of a double bond has a pronounced effect on membrane properties. We consider how cholesterol interacts with phosphatidylcholines comprising two 18-carbon long monounsaturated acyl chains, where the position of the double bond is varied systematically along the acyl chains. Atomistic molecular dynamics simulations indicate that when the double bond is not in contact with the cholesterol ring, and especially with the C18 group on its rough β-side, the membrane properties are closest to those of the saturated bilayer. However, any interaction between the double bond and the ring promotes membrane disorder and fluidity. Maximal disorder is found when the double bond is located in the middle of a lipid acyl chain, the case most commonly found in monounsaturated acyl chains of phospholipids. The results suggest a cholesterol-mediated lipid selection mechanism in eukaryotic cell membranes. With saturated lipids, cholesterol promotes the formation of highly ordered raft-like membrane domains, whereas domains rich in unsaturated lipids with a double bond in the middle remain highly fluid despite the presence of cholesterol.     

Structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains

Quantitative structures are obtained at 30 degrees C for the fully hydrated fluid phases of palmitoyloleoylphosphatidylcholine (POPC), with a double bond on the sn-2 hydrocarbon chain, and for dierucoylphosphatidylcholine (di22:1PC), with a double bond on each hydrocarbon chain. The form factors F(qz) for both lipids are obtained using a combination of three methods. (1) Volumetric measurements provide F(0). (2) X-ray scattering from extruded unilamellar vesicles provides /F(qz)/ for low q(z). (3) Diffuse X-ray scattering from oriented stacks of bilayers provides /F(qz)/ for high q(z). Also, data using method (2) are added to our recent data for dioleoylphosphatidylcholine (DOPC) using methods (1) and (3); the new DOPC data agree very well with the recent data and with (4) our older data obtained using a liquid crystallographic X-ray method. We used hybrid electron density models to obtain structural results from these form factors. The result for area per lipid (A) for DOPC 72.4 +/- 0.5 A(2) agrees well with our earlier publications, and we find A = 69.3 +/- 0.5 A2 for di22:1PC and A = 68.3 +/- 1.5 A2 for POPC. We obtain the values for five different average thicknesses: hydrophobic, steric, head-head, phosphate-phosphate and Luzzati. Comparison of the results for these three lipids and for our recent dimyristoylphosphatidylcholine (DMPC) determination provides quantitative measures of the effect of unsaturation on bilayer structure. Our results suggest that lipids with one monounsaturated chain have quantitative bilayer structures closer to lipids with two monounsaturated chains than to lipids with two completely saturated chains.     

Lecithin bilayers. Density measurement and molecular interactions.

Density measurement are reported for bilayer dispersions of a series of saturated lecithins. For chain lengths with, respectively, 14, 15, 16, 17, and 18 carbons per chain, the values for the volume changes at the main transition are 0.027, 0.031, 0.037, 0.040 and 0.045 ml/g. The main transition temperature extrapolates with increasing chain length to the melting temperature of polyethylene. Volume changes at the lower transition are an order of magnitude smaller than the main transition. Single phase thermal expansion coefficients are also reported. The combination of X-ray data and density data indicated that the volume changes are predominantly due to the hydrocarbon chains, thus enabling the volume vCH2 of the methylene groups to be computed as a function of temperature. From this and knowledge of intermolecular interactions in hydrocarbon chains, the change in the interchain van der Waals energy, delta UvdW, at the main transition is computed for the lecithins and also for the alkanes and polyethylene at the melting transition. Using the experimental enthalpies of transition and delta UvdW, the energy equation is consistently balanced for all three systems. This yields estimates of the change in the number of gauche rotamers in the lecithins at the main transition. The consistency of these calculations supports the conclusion that the most important molecular energies for the main transition in lecithin bilayers are the hydrocarbon chain interactions and the rotational isomeric energies, and the conclusion that the main phase transition is analogous to the melting transition in the alkanes from the hexagonal phase to the liquid phase, but with some modifications.     

Effect of lanthanum ions on the phase transitions of lecithin bilayers.

Interaction of lanthanum ions (La3+) with 1,2 dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) causes an increase in Tc, the temperature of maximal excess heat capacity, and the width of the gel-to-liquid crystalline transition. At a mole ratio of La3+ to DPPC sufficient to remove the hydrocarbon chain tilt angle of DPPC, the changes in the thermodynamic parameters of the pretransition are minor, Tc and the width were unaltered and the enthalpy was reduced by only 10%. This suggests that the change in tilt angle is not a necessary concomitant of the pretransition.     

Differential scanning calorimetry and 31P NMR studies on sonicated and unsonicated phosphatidylcholine liposomes.

1. Phase transitions in sonicated (vesicles) and unsonicated liposomes composed of various synthetic phosphatidylcholines are monitored using differential scanning calorimetry and 31P NMR. 2. The temperature (Tc), heat content and width of the phase transition are comparable in both vesicles and liposomes prepared from 1,2-dipalmitoyl phosphatidylcholine and 1,2-dimyristoyl phosphatidylcholine. In vesicles composed of a (1 : 1) mixture of 1,2-dipalmitoyl phosphatidylcholine and 1,2-dioleoyl phosphatidylcholine phase separation occurs as in the bilayers of the unsonicated liposomes. 3. The linewidth of the 31P resonances in vesicles is not greatly dependent upon the fatty acid composition when the lipids are in the disordered liquid crystalline state (above Tc). When the lipids are in the gel state (below Tc), however, there is a marked increase in linewidth, demonstrating a reduction in motion of the phosphate group. 4. The ratio of the amounts of phosphatidylcholine present in the outside and inside monolayter of the vesicle membrane was determined with 31P NMR using Nd3+ as a non-permeating shift reagent. 5. The outside/inside ratio is dependent upon the hydrocarbon chain length. Increasing chain length gives a lower outside/inside ratio and a larger vesicle. Introduction of cis or trans double bonds in the chain influences the outside/inside ratio slightly. 6. The incorporation of cholesterol decreases the outside/inside ratio and increases the size of 1,2-dimyristoyl phosphatidylcholine vesicles. The cholesterol concentration in the outside and inside monolayer is approximately the same. The size of the 1,2-dioleoyl phosphatidylcholine vesicles is also increased by cholesterol incorporation but the outside/inside distribution is also increased, especially between 30 and 50 mol% cholesterol. In these vesicles cholesterol is asymmetrically distributed and strongly prefers the inside monolayer of the vesicle.     

Fluid phase lipid areas and bilayer thicknesses of commonly used phosphatidylcholines as a function of temperature

The structural parameters of fluid phase bilayers composed of phosphatidylcholines with fully saturated, mixed, and branched fatty acid chains, at several temperatures, have been determined by simultaneously analyzing small-angle neutron and X-ray scattering data. Bilayer parameters, such as area per lipid and overall bilayer thickness have been obtained in conjunction with intrabilayer structural parameters (e.g. hydrocarbon region thickness). The results have allowed us to assess the effect of temperature and hydrocarbon chain composition on bilayer structure. For example, we found that for all lipids there is, not surprisingly, an increase in fatty acid chain trans-gauche isomerization with increasing temperature. Moreover, this increase in trans-gauche isomerization scales with fatty acid chain length in mixed chain lipids. However, in the case of lipids with saturated fatty acid chains, trans-gauche isomerization is increasingly tempered by attractive chain-chain van der Waals interactions with increasing chain length. Finally, our results confirm a strong dependence of lipid chain dynamics as a function of double bond position along fatty acid chains.     

Trans-unsaturated lipid dynamics: modulation of dielaidoylphosphatidylcholine acyl chain motion by ethanol.

Acyl chain dynamics of the trans-unsaturated lipid, dielaidoylphosphatidylcholine (DEPC), were studied by conventional and saturation transfer electron paramagnetic resonance spectroscopy of aqueous dispersions of DEPC spin labeled with lecithins having doxyl groups at positions 5, 10, and 14 on the sn-2 chain. The gel to liquid crystalline transition is concerted with simultaneous increases in rotational motion about the long axis of the acyl chain (libration) and in gauche-trans conformational interconversions (wobble). Relative to saturated lecithins at similar reduced temperatures the double bond (a) slowed libration by an order of magnitude in both phases, while wobble motions were several times slower, and (b)-produced a pronounced stiffness of the acyl chain near the double bond. Ethanol (0-1.6 M), in addition to its well-known colligative effect on the phase transition, was found to decrease the bilayer order in a concentration-dependent manner. This effect was smaller in the gel than in the liquid crystalline phase, most pronounced next to the double bond, and weakest deep in the bilayer. Ethanol affected slow motions little in the gel phase but wobble and libration correlation times were markedly decreased in the liquid crystalline phase.     

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)     

Effect of chain length and unsaturation on elasticity of lipid bilayers

Micropipette pressurization of giant bilayer vesicles was used to measure both elastic bending k(c) and area stretch K(A) moduli of fluid-phase phosphatidylcholine (PC) membranes. Twelve diacyl PCs were chosen: eight with two 18 carbon chains and degrees of unsaturation from one double bond (C18:1/0, C18:0/1) to six double bonds per lipid (diC18:3), two with short saturated carbon chains (diC13:0, diC14:0), and two with long unsaturated carbon chains (diC20:4, diC22:1). Bending moduli were derived from measurements of apparent expansion in vesicle surface area under very low tensions (0.001-0.5 mN/m), which is dominated by smoothing of thermal bending undulations. Area stretch moduli were obtained from measurements of vesicle surface expansion under high tensions (>0.5 mN/m), which involve an increase in area per molecule and a small-but important-contribution from smoothing of residual thermal undulations. The direct stretch moduli varied little (< +/-10%) with either chain unsaturation or length about a mean of 243 mN/m. On the other hand, the bending moduli of saturated/monounsaturated chain PCs increased progressively with chain length from 0.56 x 10(-19) J for diC13:0 to 1.2 x 10(-19) J for diC22:1. However, quite unexpectedly for longer chains, the bending moduli dropped precipitously to approximately 0.4 x 10(-19) J when two or more cis double bonds were present in a chain (C18:0/2, diC18:2, diC18:3, diC20:4). Given nearly constant area stretch moduli, the variations in bending rigidity with chain length and polyunsaturation implied significant variations in thickness. To test this hypothesis, peak-to-peak headgroup thicknesses h(pp) of bilayers were obtained from x-ray diffraction of multibilayer arrays at controlled relative humidities. For saturated/monounsaturated chain bilayers, the distances h(pp) increased smoothly from diC13:0 to diC22:1 as expected. Moreover, the distances and elastic properties correlated well with a polymer brush model of the bilayer that specifies that the elastic ratio (k(c)/K(A))(1/2) = (h(pp) - h(o))/24, where h(o) approximately 1 nm accounts for separation of the headgroup peaks from the deformable hydrocarbon region. However, the elastic ratios and thicknesses for diC18:2, diC18:3, and diC20:4 fell into a distinct group below the correlation, which showed that poly-cis unsaturated chain bilayers are thinner and more flexible than saturated/monounsaturated chain bilayers.     

13C nuclear magnetic resonance spectra of 1,2-dioctadec-cis-enoyl-sn-glycero-3-phosphorylcholines.

The 13C NMR spectra of all sixteen 1,2-dioctade-cis-enoyl-sn-glycero-3-phosphorylcholines have been obtained. Resonance lines of the olefinic, methylene, methyl and carboxyl carbon nuclei are sufficiently characteristic to permit unequivocal designation of double bond position for each isomer. Two resonances of the sn-glycero-3-phosphorylcholine structure have been reassigned.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

Potentiometric estimation of charges in barnacle muscle fibers under internal perfusion

Summary A statistical mechanical treatment of the fluidity of lipid hydrocarbon chains in phospholipid bilayers is presented, which explicitly takes some account of interchain steric restrictions. With an effective energy separation of 750 cal/mole betweengauche andtrans conformations, it is found possible to account both for the chain dependence of the entropy and enthalpy change at the liquid crystalline transition of saturated lecithins, and also for intensity data in the laser raman spectra of dipalmitoyl lecithin. The method is used to calculate conformational probabilities in the lipid chains, in particular those for 2g 1 kinks. The calculated kink concentrations are found to be in agreement with the molecular permeability theory of H. Träuble (J Membrane Biol. 4:193, 1971).     

Effects of chain unsaturation on the equation of state for lipid monolayers at the air-water interface.

An equation of state for lipid monolayers at the air-water interface is presented, which takes into account the effects of the conformation and the number and position of double bonds of the hydrocarbon chains. The total Hamiltonian of the monolayer is assumed to consist of three terms. Two of them are calculated exactly within the limitations of the formulation. These are the two-dimensional entropy of mixing of the lipid and water molecules at the surface and the conformational entropy of the chains using a model available from the literature. These two terms give rise to positive surface pressure. The third term, which includes all energies that are not amenable to calculation, was obtained as the difference between the sum of the two calculated terms and experimental data and is found to represent an approximately area-independent tension. The effects of chain unsaturation on the equation of state were modeled in the present theory in two ways; the chain bend caused by cis double bonds increases the minimal molecular area, and the double bond linkage on a chain decreases the degrees of freedom of the chain. Calculations revealed that the former is highly significant whereas the latter is negligible. The deduced equation of state reproduces experimental data with appropriate values for three parameters, which represent the collapse area, the overlap of adjacent chains, and the combined effects of the intra- and intermolecular interactions other than the surface mixing entropy and the chain conformational energy.     

Influence of chain length and unsaturation on sphingomyelin bilayers

Sphingomyelins (SMs) are among the most common phospholipid components of plasma membranes, usually constituting a mixture of several molecular species with various fatty acyl chain moieties. In this work, we utilize atomistic molecular dynamics simulations to study the differences in structural and dynamical properties of bilayers comprised of the most common natural SM species. Keeping the sphingosine moiety unchanged, we vary the amide bonded acyl chain from 16 to 24 carbons in length and examine the effect of unsaturation by comparing lipids with saturated and monounsaturated chains. As for structural properties, we find a slight decrease in average area per lipid and a clear linear increase in bilayer thickness with increasing acyl chain length both in saturated and unsaturated systems. Increasing the acyl chain length is found to further the interdigitation across the bilayer center. This is related to the dynamics of SM molecules, as the lateral diffusion rates decrease slightly for an increasing acyl chain length. Interdigitation also plays a role in interleaflet friction, which is stronger for unsaturated chains. The effect of the cis double bond is most significant on the local order parameters and rotation rates of the chains, though unsaturation shows global effects on overall lipid packing and dynamics as well. Regarding hydrogen bonding or properties related to the lipid/water interface region, no significant effects were observed due to varying chain length or unsaturation. The significance of the findings presented is discussed.     

Properties of unsaturated phospholipid bilayers: Effect of cholesterol

Properties of hydrated unsaturated phosphatidylcholine (PC) lipid bilayers containing 40 mol % cholesterol and of pure PC bilayers have been studied. Various methods were applied, including molecular dynamics simulations, self-consistent field calculations, and the pulsed field gradient nuclear magnetic resonance technique. Lipid bilayers were composed of 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules. Lateral self-diffusion coefficients of the lipids in all these bilayers, mass density distributions of atoms and atom groups with respect to the bilayer normal, the C-H and C-C bond order parameter profiles of each phospholipid hydrocarbon chain with respect to the bilayer normal were calculated. It was shown that the lateral self-diffusion coefficient of PC molecules of the lipid bilayer containing 40 mol % cholesterol is smaller than that for a corresponding pure PC bilayer; the diffusion coefficients increase with increasing the degree of unsaturation of one of the PC chains in bilayers of both types (i.e., in pure bilayers or in bilayers with cholesterol). The presence of cholesterol in a bilayer promoted the extension of saturated and polyunsaturated lipid chains. The condensing effect of cholesterol on the order parameters was more pronounced for the double C=C bonds of polyunsaturated chains than for single C-C bonds of saturated chains.     

Factors affecting molecular packing in mixed lipid monolayers and bilayers

The nature of the molecular interactions and the factors determining molecular packing in mixed phospholipid/glyceride monolayers and bilayers were investigated by monolayer and nuclear magnetic resonance (NMR) techniques. Force-area curves were obtained at various temperatures for monolayers, at the air-water interface, of synthetic lecithins and a phosphatidyl ethanolamine mixed with di- and triglycerides in different molar ratios. The linewidths of peaks in the high resolution NMR spectra of lecithin/glyceride co-dispersions in excess water at different temperatures were used to obtain information about molecular mobilities.It was found that the molecular packing in mixed lipid monolayers and bilayers is determined by the following factors: (1) Whether lipid chains are above or below their melting point (T_C). (2) The difference between experimental temperature and T_C: the larger the difference, the smaller the effect of one component on the other. (3) The degree of similarity of the chains of the components; this influences the degree of cooperativity of chain motions and the degree of mixing of the components. (4) The nature, orientation, mutual interaction and degree of hydration of the polar groups.It is shown that mean molecular area does not always reflect the state of chain motions in lipid films, because of heterogeneity of motion and structure along the molecules. Cooperativity of motion may reduce steric requirements; other effects which are of particular importance for lecithins are interactions of zwitterions, and the influence of polar group hydration.     

Cyclopentanoid analogs of dipalmitoyl phosphatidic acid: effect of backbone geometry on thermotropic properties

Seven geometrical or positional isomers of dipalmitoyl cyclopentanophosphoric acid (DPCPA) have been synthesized and studied: 1,3/2-1P (I); 1,2/3-1P (II); 1,2/3-3P (III), 1,2,3/0-1P (IV); 1,2,3/0-2P (V); 1,3/2-2P (VI); 1,2/3-2P (VII). When dispersed in 0.1 M Tris-HCl at pH 7.4, I-VII gave thermal transitions (Tc) of 60.0 degrees, 59.0 degrees, 56.8 degrees, 55.3 degrees, 38.3 degrees, 36.8 degrees and 34.0 degrees C, respectively, as measured by differential scanning calorimetry (DSC). When the lipids were dispersed at pH 9.5 in 0.1 M borate, Tc of I-IV decreased, whereas Tc of V-VII increased. In contrast, at pH 1.5 in 0.1 M HCl/KCl, Tc of I-IV decreased slightly, but Tc of V-VII rose markedly. To determine the effect of head group geometry and substitution pattern on acyl chain motion, EPR spectra of 1-palmitoyl, 2-[16-doxylstearoyl]-glycero-3-phosphoric acid in bilayers of DPCPA isomers were acquired. Abrupt spectral changes occurred at temperatures closely correlating with transition temperatures observed by DSC. These results have led to the conclusions that: (i) isomers I-IV containing vicinal acyl chains form bilayers that exhibit structural transitions at temperatures higher than those at which transitions are exhibited by isomers V-VII which have a polar phosphate group interposed between the two chains; (ii) the effects of differences in backbone structure are transmitted down the entire length of the acyl chains; (iii) the orientation of the cyclopentane ring in the isomers I-IV is significantly different from that in isomers V-VII at pH values where the phosphate group is doubly negatively charged.     

Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.     

The interfacial structure of phospholipid bilayers: differential scanning calorimetry and Fourier transform infrared spectroscopic studies of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine and its dialkyl and acyl-alkyl analogs.

The thermotropic phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) and its 1,2-dialkyl, 1-acyl 2-alkyl and 1-alkyl 2-acyl analogs was examined by differential scanning calorimetry, and the organization of these molecules in those hydrated bilayers was studied by Fourier transform infrared spectroscopy. The calorimetric data indicate that substitution of either or both of the acyl chains of DPPC with the corresponding ether-linked hydrocarbon chain results in relatively small increases in the temperature (< 4 degrees C) and enthalpy (< 1 kcal/mol) of the lipid chain-melting phase transition. The spectroscopic data reveal that replacement of one or both of the ester-linked hydrocarbon chains of DPPC with its ether-linked analog causes structural changes in the bilayer assembly, which result in an increase in the polarity of the local environments of the phosphate headgroups and of the ester carbonyl groups at the bilayer polar/apolar interface. The latter observation is unexpected, given that ester linkages are considered to be intrinsically more polar that ether linkages. This finding cannot be satisfactorily rationalized unless the conformation of the glycerol backbones of the analogs containing ether-linked hydrocarbon chains differs significantly from that of diacyl glycerolipids such as DPPC. A comparison of the alpha-methylene scissoring bands and the methylene wagging band progressions of these lipids with the corresponding absorption bands of specifically chain-perdeuterated analogs of DPPC also supports the conclusion that replacement of the ester-linked hydrocarbon chains of DPPC with the corresponding ether-linked analog induces conformational changes in the lipid glycerol backbone. The suggestion that the conformation of glycerol backbones in the alkyl-acyl and dialkyl derivatives of DPPC differs from that of the naturally occurring 1,2-diacyl glycerolipid suggests that mono- and di-alkyl glycerolipids may not be good models of their diacyl analogs. These results, and previously published evidence that DPPC analogs with ether-linked hydrocarbon chains spontaneously form chain-interdigitated gel phases at low temperatures, clearly indicate that the properties of lipid bilayers can be substantially altered by small changes in the chemical structures of their polar/polar interfaces, and highlight the critical role of the interfacial region as a determinant of the structure and organization of lipid assemblies.