On the localization of ubiquinone in phosphatidylcholine bilayers

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05-0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [13C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the 13C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (P beta') phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.     

Differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes

We have examined the effects of cholesterol on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylserines by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. We find that the incorporation of increasing quantities of cholesterol progressively reduces the temperature, enthalpy, and cooperativity of the gel-to-liquid-crystalline phase transition of the host phosphatidylserine bilayer, such that a cooperative chain-melting phase transition is completely or almost completely abolished at 50 mol % cholesterol, in contrast to the results of previous studies. We are also unable to detect the presence of a separate anhydrous cholesterol or cholesterol monohydrate phase in our binary mixtures, again in contrast to previous reports. We further show that the magnitude of the reduction in the phase transition temperature induced by cholesterol addition is independent of the hydrocarbon chain length of the phosphatidylserine studied. This result contrasts with our previous results with phosphatidylcholine bilayers, where we found that cholesterol increases or decreases the phase transition temperature in a chain length-dependent manner (1993. Biochemistry, 32:516-522), but is in agreement with our previous results for phosphatidylethanolamine bilayers, where no hydrocarbon chain length-dependent effects were observed (1999. Biochim. Biophys. Acta, 1416:119-234). However, the reduction in the phase transition temperature by cholesterol is of greater magnitude in phosphatidylethanolamine as compared to phosphatidylserine bilayers. We also show that the addition of cholesterol facilitates the formation of the lamellar crystalline phase in phosphatidylserine bilayers, as it does in phosphatidylethanolamine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of cholesterol. We ascribe the limited miscibility of cholesterol in phosphatidylserine bilayers reported previously to a fractional crystallization of the cholesterol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. In general, the results of our studies to date indicate that the magnitude of the effect of cholesterol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipid dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.     

Solid-state NMR investigation of the selective disruption of lipid membranes by protegrin-1

The interaction of a beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), with various lipid membranes is investigated by (31)P, (2)H, and (13)C solid-state NMR. Mixed lipid bilayers containing anionic lipids and cholesterol are used to mimic the bacterial and mammalian cell membranes, respectively. (31)P and (2)H spectra of macroscopically oriented samples show that PG-1 induces the formation of an isotropic phase in anionic bilayers containing phosphatidylglycerol. Two-dimensional (31)P exchange experiments indicate that these isotropic lipids are significantly separate from the residual oriented lamellar bilayers, ruling out toroidal pores as the cause for the isotropic signal. (1)H spin diffusion experiments show that PG-1 is not exclusively bound to the isotropic phase but is also present in the residual oriented lamellar bilayers. This dynamic and morphological heterogeneity of the anionic membranes induced by PG-1 is supported by the fact that (13)C T(2) relaxation times measured under cross polarization and direct polarization conditions differ significantly. In contrast to the anionic membrane, the zwitterionic phosphatidylcholine (PC) membrane does not form an isotropic phase in the presence of PG-1 but shows significant orientational disorder. The addition of cholesterol to the PC bilayer significantly reduces this orientational disorder. The (13)C T(2) relaxation times of the PC lipids in the presence of both cholesterol and PG-1 suggest that the peptide may decrease the dynamic heterogeneity of the cholesterol-containing membrane. The observed selective interaction of PG-1 with different lipid membranes is consistent with its biological function and may be caused by its strong cationic and amphipathic structure.     

Calorimetric and spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylglycerol bilayer membranes

We have examined the effects of cholesterol (Chol) on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylglycerols (PGs) by high-sensitivity differential scanning calorimetry and Fourier transform infrared and ³¹P NMR spectroscopy. We find that the incorporation of increasing quantities of Chol alters the temperature and progressively reduces the enthalpy and cooperativity of the gel-to-liquid-crystalline phase transition of the host PG bilayer. With dimyristoyl-PG:Chol mixtures, cooperative chain-melting phase transitions are completely or almost completely abolished at Chol concentrations near 50 mol%, whereas with the dipalmitoyl- and distearoyl-PG:Chol mixtures, cooperative hydrocarbon chain-melting phase transitions are still discernable at Chol concentrations near 50 mol%. We are also unable to detect the presence of significant populations of separate domains of the anhydrous or monohydrate forms of Chol in our binary mixtures, in contrast to previous reports. We ascribe the previously reported large scale formation of Chol crystallites to the fractional crystallization of the Chol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. We further show that the direction and magnitude of the change in the phase transition temperature induced by Chol addition is dependent on the hydrocarbon chain length of the PG studied. This finding agrees with our previous results with phosphatidylcholine bilayers, where we found that Chol increases or decreases the phase transition temperature in a hydrophobic mismatch-dependent manner (Biochemistry 1993, 32:516-522), but is in contrast to our previous results for phosphatidylethanolamine (Biochim. Biophys. Acta 1999, 1416:119-234) and phosphatidylserine (Biophys. J. 2000, 79:2056-2065) bilayers, where no such hydrophobic mismatch-dependent effects were observed. We also show that the addition of Chol facilitates the formation of the lamellar crystalline phase in PG bilayers, as it does in phosphatidylethanolamine and phosphatidylserine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of Chol. Moreover, the formation of the lamellar crystalline phase in PG bilayers at lower temperatures excludes Chol, resulting in an apparent Chol immiscibility in gel-state PG bilayers. We suggest that the magnitude of the effect of Chol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipids dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.     

Solute effects on the colloidal and phase behavior of lipid bilayer membranes: ethanol-dipalmitoylphosphatidylcholine mixtures.

By means of the scanning differential calorimetry, x-ray diffractometry, and the dynamic light scattering, we have systematically studied the phase and packing properties of dipalmitoylphosphatidylcholine vesicles or multibilayers in the presence of ethanol. We have also determined the partial ternary phase diagram of such dipalmitoylphosphatidylcholine/water/ethanol mixtures. The directly measured variability of the structural bilayer parameters implies that ethanol binding to the phospholipid bilayers increases the lateral as well as the transverse repulsion between the lipid molecules. This enlarges the hydrocarbon tilt (by up to 23 degrees) and molecular area (by < or = 40%). Ethanol-phospholid association also broadens the interface and, thus, promotes lipid headgroup solvation. This results in excessive swelling (by 130%) of the phosphatidylcholine bilayers in aqueous ethanol solutions. Lateral bilayer expansion, moreover, provokes a successive interdigitation of the hydrocarbon chains in the systems with bulk ethanol concentrations of 0.4-1.2 M. The hydrocarbon packing density as well as the propensity for the formation of lamellar gel phases simultaneously increase. The pretransition temperature of phosphatidylcholine bilayers is more sensitive to the addition of alcohol (initial shift: delta Tp = 22 degrees C/mol) than the subtransition temperature (delta Ts reversible 5 degrees C/mol), whereas the chain-melting phase transition temperature is even less affected (delta Tm = 1.8 degrees C/mol). After an initial decrease of 3 degrees for the bulk ethanol concentrations below 1.2 M, the Tm value increases by 2.5 degrees above this limiting concentration. The gel-phase phosphatidylcholine membranes below Tm are fully interdigitated above this limiting concentration. The chain tilt on the fringe of full chain interdigitation is zero and increases with higher ethanol concentrations. Above Tm, some of the lipid molecules are solubilized by the bound ethanol molecules. More highly concentrated ethanol solutions (> 7 M) solubilize the phosphatidylcholine bilayers with fluid chains fully and result in the formation of mixed lipid-alcohol micelles.     

New structural model for mixed-chain phosphatidylcholine bilayers

Multilamellar suspensions of a mixed-chain saturated phosphatidylcholine with 18 carbon atoms in the sn-1 chain and 10 carbon atoms in the sn-2 chain have been analyzed by X-ray diffraction techniques. The structural parameters for this lipid in the gel state are quite different than usual phosphatidylcholine bilayer phases. A symmetric and sharp wide-angle reflection at 4.11 A indicates that the hydrocarbon chains in hydrated C(18):C(10)PC bilayers are more tightly packed than in usual gel-state phosphatidylcholine bilayers and that there is no hydrocarbon chain tilt. The lipid thickness is about 12 A smaller than would be expected in a normal bilayer phase, and the area per molecule is 3 times the area per hydrocarbon chain. In addition, the bilayer thickness increases upon melting to the liquid-crystalline state, whereas normal bilayer phases decrease in thickness upon melting. On the basis of these data, we propose a new lipid packing model for gel-state C(18):C(10)PC bilayers in which the long C(18) chain spans the entire width of the hydrocarbon region of the bilayer and the short C(10) chain aligns or abuts with the C(10) chain from the apposing molecule. This model is novel in that there are three hydrocarbon chains per head group at the lipid-water interface. Calculations show that this phase is energetically favorable for mixed-chain lipids provided the long acyl chain is nearly twice the length of the shorter chain. In the liquid-crystalline state C(18):C(10)PC forms a normal fluid bilayer, with two chains per head group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous vesiculation of uncharged phospholipid dispersions consisting of lecithin and lysolecithin

The work presented here demonstrates that the phenomenon of spontaneous vesiculation is not restricted to charged lipids and lipid mixtures, but occurs also in isoelectric phospholipid mixtures consisting of egg phosphatidylcholine (EPC) and egg lysophosphatidylcholine (lyso-EPC). 1H high-resolution NMR and freeze-fracture electron microscopy have been used to characterize the mixed EPC/lyso EPC dispersions in excess H2O. The predominant phase in these mixed phospholipid dispersions is smectic (lamellar) at least up to approximately 70% lysophosphatidylcholine. The type of phospholipid aggregate formed in excess H2O depends on the mole ratio diacyl to monoacyl phosphatidylcholine. The dispersive (lytic) action of lysophosphatidylcholine on phosphatidylcholine bilayers becomes effective at lysophospholipid contents in excess of approximately 10%. Large multilamellar liposomes are disrupted and replaced by smaller particles, mainly unilamellar vesicles. Between 30 and 70% lysophosphatidylcholine a significant proportion of the total phospholipid is present as small unilamellar vesicles (SUV) of a diameter of 23 nm (range: 20-70 nm). At even higher lysophosphatidylcholine contents the fraction of phospholipid present as small mixed micelles with a diameter smaller than about 14 nm grows at the expense of the vesicular structures. There is a second effect of increasing the quantity of lysophosphatidylcholine in phosphatidylcholine bilayers: the presence of lysophosphatidylcholine in excess of 10% renders the phospholipid bilayer more permeable to ions as compared to pure phosphatidylcholine bilayers. The key factor in inducing spontaneous vesiculation is probably not the charge but the wedge-like shape of the lysophospholipid molecule. The molecular shape may give rise to an asymmetric distribution of lysophosphatidylcholine between the two halves of the bilayer, thus stabilizing highly curved bilayers as present in SUV.     

Effects of phenylamide herbicides on the physical properties of phosphatidylcholine membranes

A number of phenylamide herbicides are observed to uncouple electron transport in isolated chloroplasts and mitochondria and alter the H+ permeability of artificial liposomes. Several of these phenylamides were incorporated into phosphatidylcholine multilamellar and small unilamellar vesicles to measure their effects on the physical properties of membranes. X-ray diffraction analysis of the multilamellar vesicles revealed that the herbicides partitioned into the hydrocarbon chain region of the bilayer, but caused only minimal perturbations on hydrocarbon chain packing. 31P-NMR spectroscopy of these multilamellar vesicles showed both a broadening and lowering of the phase transition temperature of the bilayer lipids upon addition of the herbicides. 13C-NMR spectroscopy of small, unilamellar phosphatidylcholine vesicles was performed to measure the effects of the phenylamides on the chemical shifts and the spin-lattice relaxation times of the individual phosphatidylcholine carbon atoms. None of the added compounds had any measurable effect on the 13C-NMR chemical shifts of the phosphatidylcholine. However, the herbicides significantly modified spin-lattice relaxation times of certain of the lipid carbon atoms. These results generally indicate that the herbicides orient in the lipid bilayers such that the hydrocarbon chains of the phenylamides associate with the hydrocarbon chains of the lipid, whereas the phenyl moiety resides in the polar region of the bilayer.     

The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C(95) on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C(95) had no detectable effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C(95). Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C(95). These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C₉₅ on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C₉₅ had no detectible effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C₉₅. Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C₉₅. These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

Phosphatidylcholine bilayers A theoretical model which describes the main and the lower transitions

We present a theoretical model which describes both the main and the lower phase transition in phosphatidylcholine bilayers. The main transition involves a melting of the hydrocarbon chains while the lower transition is seen as a nematic to isotropic transition involving entire lipid molecules (which are rod shaped when projected onto the bilayer plane). This latter transition is consistent with experimental data which suggest the presence of long-axis rotation for temperatures below the main melting transtition. The model is extended to mixtures of phosphatidylcholines and compared with experimental data.     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems

The transient membrane lipid diacylglycerol (DG) is known to modify and destabilize phospholipid bilayers and can lead to the formation of nonbilayer structures. Since cholesterol forms a major fraction of many plasma membranes, we have investigated how it modifies the structural effects of DG on bilayers of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE). We view these systems as modelling the behaviour of local, DG-containing sites in membranes. Using X-ray diffraction, we have characterized the lamellar (L alpha) and inverse hexagonal (HII) structures that these ternary lipid mixtures form in excess aqueous solution. As the DG level increases, the lipid progresses from a single L alpha structure to a mixture of L alpha and HII, and then to a pure HII structure. This allows determination of the DG levels at which the HII transition begins, which we interpret as those levels that destabilize bilayers. In both PC and PE bilayers, the presence of 30 mol% cholesterol reduces the amounts of DG required to destabilize the bilayer structure. The destabilization can be translated into the number of neighbouring lipid molecules that a DG molecule perturbs, and of bilayer areas that it affects. The data show that the presence of cholesterol greatly enhances the perturbing effects of DG. We examine the possible role of DG in enzyme activation and membrane fusion.     

Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)     

A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.     

The effect of cholesterol on the structure of phosphatidylcholine bilayers

The effect of cholesterol on the structure of phosphatidylcholine bilayers was investigated by X-ray diffraction methods. Electron density profiles at 5 Å resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayer was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12–16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.     

Phase behavior and structure of aqueous dispersions of sphingomyelin

The phase behavior of bovine brain sphingomyelin in water has been determined by polarizing light microscopy, differential scanning calorimetry, and X-ray diffraction. Lamellar phases, in which water is intercalated between sheets of lipid molecules arranged in the classical bilayer fashion, are present over much of the phase diagram. An order-disorder transition separates the high temperature, liquid crystalline, lamellar phase from a more ordered lamellar phase at low temperatures. The hydration characteristics of sphingomyelin are similar to the structurally related lecithin in that only limited amounts of water are incorporated above and below the transition. Above the transition at 47 degrees C, a maximum of 35% by weight of water can be incorporated between the lipid bilayers, the total thickness at maximum hydration being 60.2 A, the lipid thickness 38 A, and the surface area per lipid molecule at the interface 60 A(2). Water in excess of 35% by weight is present as a separate phase. Below the phase transition, at 25 degrees C a maximum of 42% by weight of water may be incorporated between the lipid bilayers. On increasing the hydration, the lamellar repeat distance increases from 63.5 A to a limiting value of 76 A. Within this hydration range the calculated lipid thickness decreases from 63.5 to 42.5 A, and the surface area per lipid molecule increases from 36.1 to 53.6 A(2). Although these changes may be accounted for by a structure in which the hexagonally packed ordered hydrocarbon chains tilt progressively with respect to the normal to the bilayer plane on increasing hydration, it is possible that changes in other more complex lamellar structures may be responsible for these variations in lipid thickness and surface area.     

Hydrocarbon chains dominate coupling and phase coexistence in bilayers of natural phosphatidylcholines and sphingomyelins

The structure and thermotropic phase behaviour of aqueous dispersions of egg phosphatidylcholine, egg sphingomyelin, bovine brain sphingomyelin and binary mixtures of phosphatidylcholine and sphingomyelins have been examined by synchrotron X-ray diffraction methods. Small-angle lamellar Bragg peaks and wide-angle X-ray scattering bands have been subjected to peak fitting procedures to identify coexisting gel and fluid as well as fluid-fluid bilayer structures. Molecular species of egg phosphatidylcholine exhibit fluid-fluid immiscibility throughout heating scans from 20 ° to 50 °C. Egg and brain sphingomyelins exhibit gel-fluid bilayer coexistence at temperatures below the main phase transition temperature and fluid-fluid phase coexistence at higher temperatures. Binary mixtures of equimolar proportions of egg phosphatidylcholine and either of the sphingomyelins show gel-fluid phase coexistence at temperatures below the gel phase transition temperature of the respective sphingomyelin. Binary mixtures containing egg sphingomyelin show fluid-fluid immiscibility at all temperatures of the heating scans whereas the fluid phase of mixtures comprising brain sphingomyelin are apparently miscible at all temperatures. An analysis of binary mixtures containing egg sphingomyelin and egg phosphatidylcholine in molar ratios 50:50, 67:33 and 83:17 at 50 °C to identify the composition of the lamellar phases indicated that the two phospholipids are immiscible in bilayers in the fluid phase. The results are discussed in terms of the role of intermolecular hydrogen bonds and hydrocarbon chain composition of sphingomyelins in maintaining coupling across fluid bilayers.     

Study of the interaction of an alpha-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic alpha-helical transmembrane peptide, acetyl-K(2)-L(24)-K(2)-amide (L(24)), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L(24) decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L(24) in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L(24):PC=1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L(24). As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and (2)H NMR spectroscopy study of the interaction of the L(24) and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between (2)H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L(24) suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

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.