Interaction of the disaccharide trehalose with a phospholipid bilayer: a molecular dynamics study

The disaccharide trehalose is well known for its bioprotective properties. Produced in large amounts during stress periods in the life of organisms able to survive potentially damaging conditions, trehalose plays its protective role by stabilizing biostructures such as proteins and lipid membranes. In this study, molecular dynamics simulations are used to investigate the interaction of trehalose with a phospholipid bilayer at atomistic resolution. Simulations of the bilayer in the absence and in the presence of trehalose at two different concentrations (1 or 2 molal) are carried out at 325 K and 475 K. The results show that trehalose is able to minimize the disruptive effect of the elevated temperature and stabilize the bilayer structure. At both temperature, trehalose is found to interact directly with the bilayer through hydrogen bonds. However, the water molecules at the bilayer surface are not completely replaced. At high temperature, the protective effect of trehalose is correlated with a significant increase in the number of trehalose-bilayer hydrogen bonds, predominantly through an increase in the number of trehalose molecules bridging three or more lipid molecules.     

Low concentration of DMSO stabilizes the bilayer gel phase rather than the interdigitated gel phase in dihexadecylphosphatidylcholine membrane

We have investigated effects of dimethylsulfoxide (DMSO) on the phase stability of multilamellar vesicles of the ether-linked 1,2-dihexadecyl-sn-glycero-3-phosphatidylcholine (DHPC-MLV), which is known to be in the interdigitated gel (LbetaI) phase in excess water at 20 degrees C. The results of X-ray diffraction experiments indicate that the DHPC membrane was in the Lbeta, phase at X> or =0.12 (X=mole fraction of DMSO in DMSO/water mixture). The result of differential scanning calorimetry indicate that the gel to liquid-crystalline phase transition temperature increased, but the LbetaI to Pbeta, phase transition temperature decreased with an increase in DMSO concentration. These results show that DMSO stabilizes the bilayer gel phase rather than the LbetaI phase at its low concentration. The solubility of phosphorylcholine, which is the same structure as the headgroup of DHPC, decreased with an increase in DMSO concentration, indicating that the interaction free energy of the hydrophilic segments of the membrane with solvents increases with an increase in DMSO concentration. On the basis of the thermodynamic analysis, the mechanism of the stabilization of the bilayer gel phase of DHPC-MLV by DMSO is discussed. The decrease in the repulsive interaction between the headgroups of the phospholipid induced by the low concentrations of DMSO in water plays an important role in this stabilization.     

Effects of a bacterial trehalose lipid on phosphatidylglycerol membranes

Bacterial trehalose lipids are biosurfactants with potential application in the biomedical/healthcare industry due to their interesting biological properties. Given the amphiphilic nature of trehalose lipids, the understanding of the molecular mechanism of their biological action requires that the interaction between biosurfactant and membranes is known. In this study we examine the interactions between a trehalose lipid from Rhodococcus sp. and dimyristoylphosphatidylglycerol membranes by means of differential scanning calorimetry, X-ray diffraction, infrared spectroscopy and fluorescence polarization. We report that there are extensive interactions between trehalose lipid and dimyristoylphosphatidylglycerol involving the perturbation of the thermotropic gel to liquid-crystalline phase transition of the phospholipid, the increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer, and the modulation of the order of the phospholipid bilayer. The observations are interpreted in terms of structural perturbations affecting the function of the membrane that might underline the biological actions of the trehalose lipid.     

Modes of interaction of cryoprotectants with membrane phospholipids during freezing

The abilities of a variety of compounds to inhibit liposome fusion during freeze/thaw were assessed by resonance energy transfer. Small unilamellar vesicles have been frozen according to three different protocols. Membrane intermixing was seen to be relatively independent of freezing protocol except when glycerol, dimethyl sulfoxide (DMSO), or sarcosine was used as the cryoprotectant. Low concentrations of polyvinylpyrolidone or 4-hydroxyproline enhanced fusion of liposomes, whereas high concentrations of these compounds had no effect. Glycerol, DMSO, proline, betaine, and sarcosine reduced fusion, but only when their concentrations were greater than 1 M. The most effective cryoprotectants were trehalose and sucrose, which both reduced fusion to minimal levels at concentrations of only 0.2 M. We have also used europium to probe the modes of interaction of these compounds with phospholipids. Europium, which is known to bind to the phosphate headgroup, maximized fusion in liposomes subjected to freeze/thaw. This "europium-induced" fusion was progressively reduced by the presence of increasing sucrose, trehalose, or glycerol, suggesting a competition for the headgroup. However, the presence of proline, betaine, or sarcosine did not reduce europium-induced fusion, suggesting that these compounds do not compete for the headgroup. Substitution of polar side chains on the hydrophobic regions of proline or sarcosine eliminate their cryoprotective properties, suggesting that these compounds interact with the acyl chains of the bilayer.     

Di-rhamnolipids improve effect of trehalose on both hypothermic preservation and cryopreservation of rat hepatocytes

Trehalose, a disaccharide of glucose, is a highly hydrophilic small molecule (MW?=?342D) and a bioprotectant normally impermeable to the membrane phospholipid bilayer. Di-rhamnolipids, a major component of rhamnolipids, were applied to increase the effect of trehalose in cryopreservation and hypothermic preservation. We found that di-rhamnolipids (10 mg/L) increased the survival of hepatocytes after cryopreservation or hypothermic preservation as indicated by cell viability using trypan blue exclusion and methyl thiazolyl tetrazolium assay. Correspondingly, after hepatocytes were preserved in the presence of di-rhamnolipids, their hepatospecific functions were comparable to those of freshly cultured cells in terms of intracellular glutathione level, albumin secretion, urea production, and metabolic activities of cytochrome P450 isoforms. Measurement of trehalose intracellular concentration showed that its accumulation increased in the presence of di-rhamnolipids (10 mg/L) but was not altered by two other well-known surfactants, Tween-80, and Pluronic 127. Hence, di-rhamnolipids, which are non-toxic, effective, and commercially available, could be a promising protectant by potentiating the function of trehalose against hypothermic or cryopreservation cell damage.     

Interactions of a bacterial biosurfactant trehalose lipid with phosphatidylserine membranes

Trehalose lipids are biosurfactants produced by rhodococci that, in addition to their well known potential industrial and environmental uses, are gaining interest in their use as therapeutic agents. The study of the interaction of biosurfactants with membranes is important in order to understand the molecular mechanism of their biological actions. In this work we look into the interactions of a bacterial trehalose lipid produced by Rhodococcus sp. with dimyristoylphosphatidylserine membranes by using differential scanning calorimetry, X-ray diffraction and infrared spectroscopy. Differential scanning calorimetry and X-ray diffraction show that trehalose lipid broadens and shifts the phospholipid gel to liquid-crystalline phase transition to lower temperatures, does not modify the macroscopic bilayer organization and presents good miscibility both in the gel and the liquid-crystalline phases. Infrared experiments show that trehalose lipid increases the fluidity of the phosphatidylserine acyl chains, changed the local environment of the polar head group, and decreased the hydration of the interfacial region of the bilayer. Trehalose lipid was also able to affect the thermotropic transition of dimyristoylphosphatidyserine in the presence of calcium. These results support the idea that trehalose lipid incorporates into the phosphatidylserine bilayers and produces structural perturbations which might affect the function of the membrane.     

Influence of plant sterols on the phase properties of phospholipid bilayers

The effects of stigmasterol, sitosterol, campesterol, and cholesterol on the phase properties of dipalmitoylphosphatidylcholine bilayers have been compared by differential scanning calorimetry and x-ray diffraction. The sterols were equally effective at progressively reducing the cooperativity and the enthalpy of the dipalmitoylphosphatidylcholine phase transition as their concentrations in the bilayer were increased. Moreover, both differential scanning calorimetry and x-ray diffraction indicated that the dipalmitoylphosphatidylcholine transition was eliminated by each of the sterols when they were present at a concentration of 33 mole%. This indicates that the interaction between phospholipid and both plant and animal sterols is stoichiometric, each sterol associating with two phospholipid molecules. At concentrations above 33 mole% the sterols were no longer completely solvated by the phospholipid, and sterol-sterol interaction resulted. Cholesterol, even at concentrations as high as 50 mole%, did not disrupt the lamellar structure of the bilayer. When these high concentrations of plant sterols were intercalated into the phospholipid, crystallinity, which presumably derives from sterol-sterol interaction, was detectable in the bilayer by x-ray diffraction. This observation is consistent with previous reports to the effect that the C₁₇ chains of the plant sterols render them less soluble in phospholipid than is cholesterol. It is clear that this solvation difference is of insufficient magnitude to affect the stoichiometry of dipalmitoylphosphatidylcholine-sterol interaction, but it could well account for the less effective modulation of lipid bilayer permeability exhibited by plant sterols in comparison with cholesterol.     

Interfacial properties of hydrophilic surfaces of phospholipid films as determined by the method of contact angles

Hydrophilic films of phospholipids were deposited onto plastic substrates (surface-treated for cell cultures) and shown to adhere sufficiently for measuring their interfacial properties by the method of contact angles. Both by absolute magnitude and by their dependence on temperature, the interfacial properties of these phospholipid films were indistinguishable from those determined for black lipid bilayer membranes with a different method by other authors. According to both their vesicular micromorphology and water permeability, the surface films can be interpreted to consist essentially of multibilayer vesicles with the hydrophilic groups facing outward. Treatment of these films with cell-culture medium containing calf serum results in changes of interfacial properties that are very similar to those effected on virus-transformed 3T3 cells (earlier work). These interfacial effects may be attributed essentially to serum proteins (such as albumin) adsorbing to phospholipid or cellular surfaces. The interfacial properties of nontransformed 3T3 cells are much less affected by serum treatment (earlier work), which correlates closely with their higher serum requirement for proliferation. Comparison of these results with those on the interfacial effects of serum on phospholipid films suggests that at least part of the proliferation-stimulating effect of serum is mediated by changes of interfacial properties of cell membranes upon adsorption of serum proteins such as albumin. Treatment of phospholipid films with concanavalin A, an inhibitor of cell proliferation, does not result in effects on their interfacial properties correlating with those on cellular membranes. This confirms previous suggestions that the latter depends on specific binding of convanavalin A to specific carbohydrates on the cell membrane.     

Interfacial properties of hydrophilic surfaces of phospholipid films as determined by the method of contact angles. Comparison with cell surfaces

Hydrophilic films of phospholipids were deposited onto plastic substrates (surface-treated for cell cultures) and shown to adhere sufficiently for measuring their interfacial properties by the method of contact angles. Both by absolute magnitude and by their dependence on temperature, the interfacial properties of these phospholipid films were indistinguishable from those determined for black lipid bilayer membranes with a different method by other authors. According to both their vesicular micromorphology and water permeability, the surface films can be interpreted to consist essentially of multibilayer vesicles with the hydrophilic groups facing outward. Treatment of these films with cell-culture medium containing calf serum results in changes of interfacial properties that are very similar to those effected on virus-transformed 3T3 cells (earlier work). These interfacial effects may be attributed essentially to serum proteins (such as albumin) adsorbing to phospholipid or cellular surfaces. The interfacial properties of nontransformed 3T3 cells are much less affected by serum treatment (earlier work), which correlates closely with their higher serum requirement for proliferation. Comparison of these results with those on the interfacial effects of serum on phospholipid films suggests that at least part of the proliferation-stimulating effect of serum is mediated by changes of interfacial properties of cell membranes upon adsorption of serum proteins such as albumin. Treatment of phospholipid films with concanavalin A, an inhibitor of cell proliferation, does not result in effects on their interfacial properties correlating with those on cellular membranes. This confirms previous suggestions that the latter depends on specific binding of concanavalin A to specific carbohydrates on the cell membrane.     

Solution effects on the thermotropic phase transition of unilamellar liposomes

We have investigated the effect of two monosaccharides, glucose and fructose, and two disaccharides, sucrose and trehalose, on the thermotropic phase transition of unilamellar extruded vesicles of DPPC. All the sugars investigated raise the main transition temperature (Tm) of some fraction of the lipid, but there are differences between the effect of glucose and the other three sugars. At low concentrations of glucose, Tm is lowered. At high concentrations of glucose there are two transitions, one with a low Tm and one with a high Tm. The data suggest that at low concentrations, all of the glucose present may bind to the bilayer and increase headgroup spacing by physical intercalation or increased hydration. The appearance of a Tm above that of pure hydrated DPPC suggests the possibility of the dehydration of some other population of phospholipid molecules. The other three sugars increase Tm, but at high concentrations of trehalose, sucrose, and fructose a second peak occurs at a low Tm. The other sugars appear to dehydrate the bilayer at low concentrations, but may show some binding or increased hydration of some portion of the lipid at very high concentrations. The sugar effects on unilamellar vesicles are strikingly different from the effects of these sugars on multilamellar vesicles.     

Properties of the protein kinase C-phorbol ester interaction

The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC. The initial calcium-dependent and reversible form of membrane-associated PKC was capable of binding PDBu. Both PKC and PDBu were released from this complex by calcium chelation. Sustained interaction with phospholipid vesicles resulted in a PKC-membrane complex that could not be dissociated by calcium chelation and appeared to result from insertion of PKC into the hydrocarbon portion of the phospholipid bilayer. Membrane insertion was observed at calcium concentrations of 2-500 microM and with membrane compositions of 10-50% acidic phospholipid. However, the extent of insertion was dependent on the binding conditions and was promoted by high phospholipid to PKC ratios, high calcium, the presence of phorbol esters, high membrane charge, and long incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preservation of freeze-dried liposomes by trehalose

One of the practical difficulties with the frequently proposed use of liposomes for delivery of water-soluble substances to cells in whole organisms is that liposomes are relatively unstable during storage. We have studied the ability of trehalose, a carbohydrate commonly found at high concentrations in organisms capable of surviving dehydration, to stabilize dry liposomes. With trehalose both inside and outside the bilayer, almost 100% of trapped solute was retained in rehydrated vesicles previously freeze-dried with 1.8 g trehalose/g dry phospholipid. Trehalose is very effective at inhibiting fusion between liposomes during drying, as assessed by freeze-fracture and resonance energy transfer between fluorescent probes incorporated into the bilayer. However, inhibition of fusion alone does not account for the preservation of the dry liposomes, since the concentration of trehalose required to prevent leakage is more than 10-fold that required to prevent fusion. We provide evidence that stabilization of the dry liposomes requires depression of transition temperature and consequent maintenance of the constituent lipids in the dry liposomes in a liquid crystalline phase.     

Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide.

Dimethylsulfoxide (DMSO) is known to protect isolated enzymes during freezing while destabilizing proteins at high temperatures. This apparent paradox is the subject of a review by Arakawa et al. ((1990) Cryobiology 27, 401-415), who present evidence for a temperature-dependent, hydrophobic interaction between DMSO and non-polar moieties of proteins. The present study investigates the interaction of DMSO with phospholipid bilayers. Phospholipid vesicles containing carboxyfluorescein were exposed to several concentrations of DMSO at various temperatures. Leakage rates increased with DMSO concentration and temperature. This effect was not reduced in the presence of solutes that have been shown to neutralize DMSO toxicity in tissues. The increased leakage rates correlate well with the increased partitioning of DMSO from water to octanol at higher temperatures. Additionally, reductions in the CH2 vibrations of the bilayer are also shown to depend on DMSO concentration and temperature. A similar reduction in CH2 vibrations was observed in solutions of octanol and DMSO, suggesting that this effect is not mediated through an interaction with water. Furthermore, investigation of sulfoxide vibrations indicate that DMSO is not hydrogen bonded to the alcohol moiety of octanol, and therefore the interaction between DMSO and octanol is most likely due to a hydrophobic association. These results are consistent with a destabilization of phospholipid membranes at higher temperatures due to a hydrophobic association between DMSO and the bilayer.     

Conformational state and functional activity of NADPH-dependent cytochrome P-450 reductase isolated and incorporated into phospholipid bilayers

It was shown that the alpha-helix content in both isolated and incorporated into phospholipid bilayer NADPH-dependent cytochrome P-450 reductase is 20%. NADPH or dithionite reduction of the flavoprotein is not followed by conformational changes. The incorporation of the NADPH-dependent cytochrome P-450 reductase molecule into the phospholipid bilayer does not affect its catalytic properties. It was found that the protein does not interact with the phospholipid bilayer of phosphatidyl choline liposomes but incorporates readily into the liposomal membrane from a microsomal phospholipid mixture with a binding constant of 17.4 microM.     

The effect of dimethyl sulphoxide on the structure and phase behaviour of palmitoleoylphosphatidylethanolamine

The thermotropic phase behaviour and structure of a nonbilayer-forming lipid, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, dispersed in water and in aqueous solutions of up to 50 wt% dimethyl sulphoxide (DMSO) have been characterised using synchrotron X-ray diffraction methods. It was found that the presence of DMSO in the solvent induced an increase in the temperature of lamellar-gel to lamellar-liquid-crystal phase transition and a decrease in the temperature of the lamellar-liquid-crystal to inverted-hexagonal phase transition of the phospholipid. The presence of DMSO also caused a decrease in the X-ray repeat spacings of all the phases studied. Electron density profiles of the phospholipid dispersed in water and 50 wt% DMSO in the bilayer gel state were calculated. The presence of 50 wt% DMSO caused the apparent disappearance of the solvent layer separating phospholipid bilayers in the gel state. The results suggest that DMSO contributes to the bilayer electron density profile and that the amphiphilic solvent molecules partition into the interfacial region.     

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of solutes on the freezing properties of and hydration forces in lipid lamellar phases.

Quantitative deuterium nuclear magnetic resonance is used to study the freezing behavior of the water in phosphatidylcholine lamellar phases, and the effect upon it of dimethylsulfoxide (DMSO), sorbitol, sucrose, and trehalose. When sufficient solute is present, an isotropic phase of concentrated aqueous solution may coexist with the lamellar phase at freezing temperatures. We determine the composition of both unfrozen phases as a function of temperature by using the intensity of the calibrated free induction decay signal (FID). The presence of DMSO or sorbitol increases the hydration of the lamellar phase at all freezing temperatures studied, and the size of the increase in hydration is comparable to that expected from their purely osmotic effect. Sucrose and trehalose increase the hydration of the lamellar phase, but, at concentrations of several molal, the increase is less than that which their purely osmotic effect would be expected to produce. A possible explanation is that very high volume fractions of sucrose and trehalose disrupt the water structure and thus reduce the repulsive hydration interaction between membranes. Because of their osmotic effect, all of the solutes studied reduced the intramembrane mechanical stresses produced in lamellar phases by freezing. Sucrose and trehalose at high concentrations produce a greater reduction than do the other solutes.     

On the use of deuterium nuclear magnetic resonance as a probe of chain packing in lipid bilayers

The packing of hydrocarbon chains in the bilayers of lamellar (L alpha) phases of soap/water and phospholipid/water mixtures has been studied by deuterium NMR spectroscopy and X-ray diffraction. A universal correlation is shown to exist between the average C-D bond order parameter SCD of hydrocarbon chains and the average area per chain ach, irrespective of the chemical structure of the surfactant (hydrophilic group, number of chains per molecule, and chain length), composition, and temperature. The practical utility of the correlation is illustrated by its application to the characterization of the distribution of various hydrophobic and amphiphilic solutes in bilayers. The distribution of hydrocarbons within a bilayer is shown to depend upon their molecular structure in a manner which highlights the nature of the molecular interactions involved. For example, benzene is shown to be fairly uniformly distributed across the bilayer with an increasing tendency to distribute into the center at high concentrations. In contrast, the more complex hydrocarbon tetradecane preferentially distributes into the center of the bilayer at low concentrations, while at higher concentrations it intercalates between the surfactant chains. Alcohols such as benzyl alcohol, octanol, and decanol all interact similarly with the bilayer in so far as they are pinned to the polar/apolar interface, presumably by involvement of the hydroxyl group in a hydrogen bond. But the response of the surfactant chains to the void volume created in the center of the bilayer is dependent upon the distance of penetration of the alcohol into the bilayer. For benzyl alcohol, the shortest molecule, this void volume is taken up by the disordering of the chains, while for decanol, the longest molecule, it is absorbed by interdigitation of the chains of apposing monolayers. For octanol, the chain interdigitation mechanism is dominant at low concentrations, but there is a transition to chain disordering at high concentrations. Finally, it is shown that the correlation provides a useful test for statistical mechanical models of chain ordering in lipid bilayers.     

Supported phospholipid bilayers for two-dimensional protein crystallization

Phospholipid bilayers, supported on UV irradiated carbon shadowed nitrocellulose electron microscope grids, have been used to induce two-dimensional crystal growth of IgE and IgG anti-DNP monoclonal antibodies. The UV irradiation renders the grids hydrophilic in a very uniform fashion and allows for the transfer of phospholipid monolayers from an air/water interface in a sequential dipping procedure. The surface coverage achieved was nearly 100% as measured by antibody binding and by the formation of protein arrays on the bilayer covered grids. The supported bilayers appear to be stably held and are appropriate for slow binding conditions and long incubation times with low concentrations of binding protein.     

Trichosanthin induces leakage and membrane fusion of liposome

Trichosanthin (TCS) is a ribosome inactivating protein with multiple pharmacological properties. Here the interaction between TCS and a phospholipid bilayer is investigated to provide evidence for membrane translocation mechanism of TCS. The results show that TCS can destabilize liposomes made by phospholipids with negatively charged head group. The destabilization effect is pH-dependent and happens only under acidic conditions. Membrane fusion is also seen to accompany the destabilizing process. The interaction between a phospholipid bilayer and C7, a mutant of TCS with 7 residues at its C-terminus deleted, has been investigated. Deleting the C-terminus almost completely abolishes the destabilizing effect of TCS on the phospholipid bilayer, which implicates the C-terminus in the interaction between trichosanthin and the membrane.