The effect of hydrostatic pressure on S-layer-supported lipid membranes

We report on the behavior of unsupported and surface layer (S-layer)-supported lipid membranes at the application of a uniform hydrostatic pressure. At a hydrostatic pressure gradient higher than 6 N/m(2), unsupported lipid membranes, independent from which side pressurized and S-layer-supported lipid membranes pressurized from the lipid-faced side revealed a pronounced increase in capacitance. A maximal hydrostatic pressure gradient of 11.0 N/m(2) resulted in an almost doubling of the capacitance of the (composite) membranes. S-layer-supported lipid membranes showed a hysteresis in the capacitance versus pressure plot, indicating that this composite structure required a certain time to reorient when the pressure gradient acting from the lipid-faced side was balanced. By contrast, the S-layer-supported lipid membrane pressurized from the protein-faced side revealed only a minute increase in capacitance (C/C(0,max)=1.17+/-0.05), reflecting only minor pressure-induced area expansion. In addition, no hysteresis could be observed, indicating that no rearrangement of the composite membrane occurred. The maximal induced tension was with 4.3+/-0.2 mN/m, significantly higher than that of unsupported (2.5+/-0.3 mN/m) and S-layer-supported lipid membranes pressurized from the lipid-faced side (2.6+/-0.1 mN/m).     

Antimicrobial properties of a lipid interactive alpha-helical peptide VP1 against Staphylococcus aureus bacteria

Theoretical analysis indicates that peptide VP1 forms a membrane interactive amphiphilic alpha-helix with antibacterial properties. Fourier transform infra-red based analyses showed VP1 to be alpha-helical (45%) in the presence of vesicle mimics of membranes from Staphylococcus aureus and to induce increases in the fluidity of these vesicles, as indicated by a rise in wavenumber of circa 0.5 to 1.0 cm(-1). The peptide induced surface pressure increases of 5 mN m(-1) in monolayer mimics of S. aureus membranes confirm the formation of a membrane interactive alpha-helix. These interactions appeared to involve significant hydrophobic and electrostatic contributions as VP1 induced comparable surface pressure changes in anionic (5.5 mN m(-1)) and zwitterionic (4 mN m(-1)) lipid monolayers. It is suggested that whilst efficacy requires further sequence specific information, the peptides generic structure provides the basis for its broad antimicrobial activity.     

Investigations into the membrane interactions of m-calpain domain V

m-calpain is a calcium-dependent heterodimeric protease implicated in a number of pathological conditions. The activation of m-calpain appears to be modulated by membrane interaction, which has been predicted to involve oblique-orientated alpha-helix formation by a GTAMRILGGVI segment located in domain V of the protein's small subunit. Here, we have investigated this prediction. Fourier transform infrared conformational analysis showed that VP1, a peptide homolog of this segment, exhibited alpha-helicity of approximately 45% in the presence of dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine (DMPS) vesicles. The level of helicity was unaffected over a 1- to 8-mM concentration range and did not alter when the anionic lipid composition of these vesicles was varied between 1% and 10% DMPS. Similar levels of alpha-helicity were observed in trifluoroethanol and the peptide appeared to adopt alpha-helical structure at an air/water interface with a molecular area of 164 A(2) at the monolayer collapse pressure. VP1 was found to penetrate dimyristoylphosphatidylcholine/DMPS monolayers, and at an initial surface pressure of 30 mN m(-1), the peptide induced surface pressure changes in these monolayers that correlated strongly with their anionic lipid content (maximal at 4 mN m(-1) in the presence of 10% DMPS). Neutron diffraction studies showed VP1 to be localized at the hydrophobic core of model palmitoyloleylphosphatidylcholine/palmitoyloleylphosphatidylserine (10:1 molar ratio) bilayer structures and, in combination, these results are consistent with the oblique membrane penetration predicted for the peptide. It would also appear that although not needed for structural stabilization anionic lipid was required for membrane penetration.     

Rupture of plasma membrane under tension

We present a study on the rupture behavior of single NIH 3T3 mouse fibroblasts under tension using micropipette aspiration. Membrane rupture was characterized by breaking and formation of an enclosed membrane linked to a tether at the cell apex. Three different rupture modes, namely: single break, initial multiple breaks, and continuous multiple breaks, were observed under similar loading condition. The measured mean tensile strengths of plasma membrane were 3.83 ± 1.94 and 3.98 ± 1.54mN/m for control cells and cells labeled with TubulinTracker, respectively. The tensile strength data was described by Weibull distribution. For the control cells, the Weibull modulus and characteristic strength were 1.86 and 4.40 mN/m, respectively; for cells labeled with TubulinTracker, the Weibull modulus and characteristic strength were 2.68 and 4.48 mN/m, respectively. Based on the experimental data, the estimated average transmembrane proteins-lipid cleavage strength was 2.64 ± 0.64 mN/m. From the random sampling of volume ratio of transmembrane proteins in cell membrane, we concluded that the Weibull characteristic of plasma membrane strength was likely to be originated from the variation in transmembrane proteins-lipid interactions.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on phospholipid monolayers. Protein structure studied using 2D IR and beta correlation analysis

We have applied two-dimensional infrared (2D IR) and betanu correlation spectroscopy to in-situ IR spectroscopy of pulmonary surfactant proteins SP-B and SP-C in lipid-protein monolayers at the air-water interface. For both SP-B and SP-C, a statistical windowed autocorrelation method identified two separate surface pressure regions that contained maximum amide I intensity changes: 4-25 mN/m and 25-40 mN/m. For SP-C, 2D IR and betanu correlation analyses of these regions indicated that SP-C adopts a variety of secondary structure conformations, including alpha-helix, beta-sheet, and an intermolecular aggregation of extended beta-sheet structure. The main alpha-helix band split into two peaks at high surface pressures, indicative of two different helix conformations. At low surface pressures, all conformations of the SP-C molecule reacted identically to increasing surface pressure and reoriented in phase with each other. Above 25 mN/m, however, the increasing surface pressure selectively affected the coexisting protein conformations, leading to an independent reorientation of the protein conformations. The asynchronous 2D IR spectrum of SP-B showed the presence of two alpha-helix components, consistent with two separate populations of alpha-helix in SP-B-a hydrophobic fraction associated with the lipid chains and a hydrophilic fraction parallel to the membrane surface. The distribution of correlation intensity between the two alpha-helix cross peaks indicated that the more hydrophobic helix fraction predominates at low surface pressures whereas the more hydrophilic helix fraction predominates at high surface pressures. The different SP-B secondary structures reacted identically to increasing surface pressure, leading to a reorientation of all SP-B subunits in phase with one another.     

Effect of salt on the interaction of Hal18 with lipid membranes

One of the major obstacles in the development of new antimicrobial peptides as novel antibiotics is salt sensitivity. Hal18, an α-helical subunit of Halocidin isolated from Halocynthia aurantium, has been previously shown to maintain its antimicrobial activity in high salt conditions. The α-helicity of Hal18 in the presence and absence of salt was demonstrated by circular dichroism spectroscopy, which showed that the peptide was mainly unordered containing β-strands and β-turns. However, in the presence of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS) vesicles, Hal18 folded to form α-helices (circa 42?%). Furthermore, the structure was not significantly affected by pH or the presence of metal ions. These data were supported by monolayer results showing Hal18 induced stable surface pressure changes in monolayers composed of DMPC (5?mN?m(-1)) and DMPS (8.5?mN?m(-1)), which again were not effected by the presence of metal ions or pH. It is proposed that the hydrophobic groove within its molecular architecture enables the peptide to form stable associations with lipid membranes. The balance of hydrophobicity along the Hal18 long axis would also support oblique orientation of the peptide at the membrane interface. Hence, this model of membrane interaction would enable the peptide to penetrate deep into the membrane. This concept is supported by lysis data. Overall, it would appear that this peptide is a potential candidate for future AMP design for use in high salt environments.     

Archaeal lipids forming a low energy-surface on air-water interface

Archaea or archaebacteria are the microorganism living in extreme environments such as hot springs and salt lakes. The membrane is featured universally by lipids which possess saturated polyisoprenoid chains in the hydrophobic moiety. This paper concerns the surface properties of Langmuir membranes made of archaeal lipid models (AL) bearing a phytanyl group or (3RS, 7R, 11R)-3,7,11,15-tetramethylhexadecyl group. All of the AL provide a Langmuir membrane on an air-water interface with an abnormally low surface tension (32-37 mN/m at 20-70 degrees C), while the conventional lipids having n-alkyl chains give membranes of 54-56 mN/m. The abnormally low energy surface of AL lipids is considered to arise from the bulky and fluid polyisoprenoid chain.     

Fouling effects of yeast culture with antifoam agents on microfilters

The fouling effects of yeast fermentation broths of Candida utilis in the presence of various commercial antifoam agents (PPG2000, B5600, and G832) up to 4.0 mL/L were studied, using Millipore polyvinylidene fluoride 0.22-mum hydrophilic membranes (GVWP), in a stirred-cell system at 50 kPa and 700 rpm. PPG2000, which has a low value of work of adhesion (W(a) of 0.81 mN/m), gave a steady flux of broth of 29 L/(h m(2)) and was found to have no significant fouling effect on the microfiltration of broth. G832, which has a high W(a), (26.0 mN/m) reduced the flux of the broth to 17 L/(h m(2)); i.e., by 42% when only 1.0 mL/L was used. However, B5600, which has a W(a) of 14.3 mN/m, was found to enhance the flux of broth to 54 L/(h m(2)); i.e., by 86%, due to the preferential adsorption of the B5600 components onto the hydrophobic cell contents released. These results were reinforced by the depressurization experiments performed with both hydrophilic (GVWP) and hydrophobic (GVHP) membranes, using both young and aged broths. B5600 was found to be the optimum antifoam agent in this study in terms of membrane performance and defoaming efficiency. (c) 1997 John Wiley & Sons, Inc.     

Material property characteristics for lipid bilayers containing lysolipid.

The apparent area expansion modulus and tensile strength of egg phosphatidylcholine (EPC) membranes are measured in the presence of monooleoylphosphatidylcholine (MOPC). The apparent area expansion modulus decreases from 171 mN m-1 for pure EPC membrane to 82 mN m-1 for a membrane containing 30 mol % MOPC. This significant decrease of the apparent area expansion modulus is attributed to the change of the membrane area due to the tension-dependent exchange of MOPC between the bathing solution and the membrane. Similar to the apparent area expansion modulus, the tensile strength of the membrane decreases with the increase of the molar concentration of MOPC in the membrane. The tensile strength of pure EPC membrane is 9.4 mN m-1 whereas that for a membrane containing 30 mol % MOPC is only 1.8 mN m-1, and for a membrane containing 50 mol % MOPC it is even smaller, on the order of 0.07 mN m-1. The decrease of the tensile strength is coupled with a decrease of the work for membrane breakdown, which changes from 4.3 x 10(-2) kT for pure EPC membrane to 2 x 10(-6) kT for a membrane with 50 mol % MOPC. Overall, these results show that the decrease of the apparent area expansion modulus in the presence of exchangeable molecules is a fundamental property for all membranes and depends on the area occupied by these molecules. The method presented here provides a unique tool for measuring the area occupied by an exchangeable molecule in the bilayer membrane.     

Interactions of surfactin with membrane models.

Surfactin, an acidic cyclic lipopeptide produced by strains of Bacillus subtilis, is a powerful biosurfactant possessing biological activities. Interactions of ionized surfactin (two negative charges) with lecithin vesicles have been monitored by changes in its CD spectra. These changes are more important in the presence of Ca2+ ions. We have studied the penetration of ionized surfactin into lipid monolayers. Using dimyristoyl phospholipids, the surfactin penetration is more important in DMPC than in DMPE monolayers and is greatly reduced in DMPA monolayers because of electrostatic repulsion. The surfactin penetration is lowered when the acyl chain length of the phospholipids increases. The exclusion pressure varies from 40 mN m-1 for DMPC to 30 mN m-1 for DPPC and 18 mN m-1 for egg lecithin. The presence of Ca2+ ions, which neutralize the charges of both surfactin and lipids in the subphase, leads to an important change of the penetration process that is enhanced in the case of acidic, but also of long chain (higher than C14) zwitterionic phospholipids (DPPC and lecithin). From compression isotherms of mixed surfactin/phospholipid monolayers, it appears that surfactin is completely miscible with phospholipids. The present study shows that surfactin penetrates spontaneously into lipid membranes by means of hydrophobic interactions. The insertion in the lipid membrane is accompanied by a conformation change of the peptide cycle.     

Oxidized phosphatidylcholines promote phase separation of cholesterol-sphingomyelin domains

Lipid lateral segregation in the plasma membrane is believed to play an important role in cell physiology. Sphingomyelin (SM) and cholesterol (Chol)-enriched microdomains have been proposed as liquid-ordered phase platforms that serve to localize signaling complexes and modulate the intrinsic activities of the associated proteins. We modeled plasma membrane domain organization using Langmuir monolayers of ternary POPC/SM/Chol as well as DMPC/SM/Chol mixtures, which exhibit a surface-pressure-dependent miscibility transition of the coexisting liquid-ordered and -disordered phases. Using Brewster angle microscopy and Langmuir monolayer compression isotherms, we show that the presence of an oxidatively modified phosphatidylcholine, 1-palmitoyl-2-azelaoyl-sn-glydecero-3-phosphocholine, efficiently opposes the miscibility transition and stabilizes micron-sized domain separation at lipid lateral packing densities corresponding to the equilibrium lateral pressure of ～32 mN/m that is suggested to prevail in bilayer membranes. This effect is ascribed to augmented hydrophobic mismatch induced by the oxidatively truncated phosphatidylcholine. To our knowledge, our results represent the first quantitative estimate of the relevant level of phospholipid oxidation that can potentially induce changes in cell membrane organization and its associated functions.     

Elasticity and phase behavior of DPPC membrane modulated by cholesterol, ergosterol, and ethanol

Giant vesicles formed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and sterols (cholesterol or ergosterol) in water and water/ethanol solutions have been used to examine the effect of sterol composition and ethanol concentration on the area compressibility modulus (K(a)), overall mechanical behavior, vesicle morphology, and induction of lipid alkyl chain interdigitation. Our results from micropipette aspiration suggest that cholesterol and ergosterol impact the order and microstructure of the gel (L(beta)') phase DPPC membrane. At low concentration (10-15 mol%) these sterols disrupt the long-range lateral order and fluidize the membrane (K(a) approximately 300 mN/m). Then at 18 mol%, these sterols participate in the formation of a continuous cohesive liquid-ordered (L(o)) phase with a sterol-dependent membrane density (K(a) approximately 750 for DPPC/ergosterol and K(a) approximately 1100 mN/m for DPPC/cholesterol). Finally at approximately 40 mol% both cholesterol and ergosterol impart similar condensation to the membrane (K(a) approximately 1200 mN/m). Introduction of ethanol (5-25 vol%) results in drops in the magnitude of K(a), which can be substantial, and sometimes individual vesicles with lowered K(a) reveal two slopes of tension versus apparent area strain. We postulate that this behavior represents disruption of lipid-sterol intermolecular interactions and therefore the membrane becomes interdigitation prone. We find that for DPPC vesicles with sterol concentrations of 20-25 mol%, significantly more ethanol is required to induce interdigitation compared to pure DPPC vesicles; approximately 7 vol% more for ergosterol and approximately 10 vol% more for cholesterol. For lower sterol concentrations (10-15 mol%), interdigitation is offset, but by <5 vol%. These data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L(beta)I) phase bilayer is a major factor in the disruption of cellular activity, which typically occurs between approximately 12 and approximately 16 vol% ethanol in yeast fermentations. We summarize our findings by producing, for the first time, "elasticity/phase diagrams" over a wide range of sterol (cholesterol and ergosterol) and ethanol concentrations.     

Investigation of phospholipid area compression induced by calcium-mediated dextran sulfate interaction.

The association of anionic polyelectrolytes such as dextran sulfate (DS) to zwitterionic phospholipid surfaces via Ca(2+) bridges results in a perturbation of lipid packing at physiologically relevant Ca(2+) concentrations. Lipid area compression was investigated in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar bilayer dispersions by (2)H-NMR and in monolayer studies. Binding of DS to DMPC surfaces via Ca(2+) results in denser lipid packing, as indicated by higher lipid chain order. DMPC order parameters are homogeneously increased throughout the lipid bilayer. Higher order translates into more extended hydrocarbon chains and decreased average lipid area per molecule. Area compression is reported as a function of DS concentration and molecular weight. Altering the NaCl and Ca(2+) concentrations modified electrostatic interactions between DS and phospholipid. A maximal area reduction of DeltaA = 2.7 A(2) per DMPC molecule is observed. The lipid main-phase transition temperature increases upon formation of DMPC/Ca(2+)/DS-complexes. Lipid area compression after addition of DS and Ca(2+) to the subphase was also observed in monolayer experiments. A decrease in surface tension of up to 3.5 mN/m at constant molecular area was observed. DS binds to the lipid headgroups by formation of Ca(2+) bridges without penetrating the hydrophobic region. We suggest that area compression is the result of an attractive electrostatic interaction between neighboring lipid molecules induced by high local Ca(2+) concentration due to the presence of DS. X-ray diffraction experiments demonstrate that DS binding to apposing bilayers reduces bilayer separation. We speculate that DS binding alters the phase state of low-density lipoproteins that associate with polyelectrolytes of the arterial connective tissue in the early stages of arteriosclerosis.     

Enzyme-catalyzed oxidation of cholesterol in pure monolayers at the air/water interface.

Mean molecular area vs. lateral surface pressure isotherms were determined for monolayers containing cholesterol, 4-cholesten-3-one (cholestenone), or binary mixtures of the two. At all lateral surface pressures examined, cholestenone had a larger mean molecular area requirement than cholesterol. Results with the binary mixtures of cholesterol and cholestenone suggested that the sterols did not mix ideally (non additive mean molecular area) with each other in the monolayer; the observed mean molecular area for mixtures was less than would be expected based on ideal mixing. The mixed sterol monolayers also displayed a reduction in the lateral collapse pressure which appeared to be a linear function of the mole fraction of cholestenone in the monolayer, suggesting that cholesterol and cholestenone were completely miscible in the mixed monolayer. The pure cholesterol monolayer was next used to examine the cholesterol oxidase-catalyzed (Brevibacterium sp.) oxidation of cholesterol to cholestenone at different lateral surface pressures at 22 degrees C. The difference in mean molecular area requirements of cholesterol and cholestenone was directly used to convert monolayer area changes (at constant lateral surface pressure) into average reaction rates. It was observed that the average catalytic activity of cholesterol oxidase increased linearly with increased lateral surface pressure in the range of 1 to 20 mN/m. In addition, the enzyme was capable to oxidize cholesterol in monolayers with a lateral surface pressure close to the collapse pressure of cholesterol monolayers (collapse pressure 45 mN/m; oxidation was observed at 40 mN/m). The adsorption of cholesterol oxidase to an inert sterol monolayer film at low surface pressures (around 9 mN/m) was marginal, although clearly detectable at very low (0.5-4 mN/m) lateral surface pressures, suggesting that the enzyme did not penetrate deeply into the monolayer in order to reach the 3 beta-hydroxy group of cholesterol. This interpretation is further supported by the finding that a maximally compressed cholesterol monolayer (40 mN/m) was readily susceptible to enzyme-catalyzed oxidation. It is concluded that cholesterol oxidase is capable of oxidizing cholesterol in laterally expanded monolayers as well as in tightly packed monolayers, where the lateral surface pressure is close to the collapse pressure. The kinetic results suggested that the rate-limiting step in the overall process was the substrate availability per surface area (or surface concentration) at the water/lipid interface.     

Structural and dynamic interfacial properties of the lipoprotein initiating domain of apolipoprotein B

To better understand the earliest steps in the assembly of triglyceride (TG)-rich lipoproteins, we compared the biophysical and interfacial properties of two closely related apolipoprotein B (apoB) truncation mutants, one of which contains the complete lipoprotein initiating domain (apoB20.1; residues 1-912), and one of which, by virtue of a 50 amino acid C-terminal truncation, is incapable of forming nascent lipoproteins (apoB19; residues 1-862). Spectroscopic studies detected no major differences in secondary structure, and only minor differences in conformation and thermodynamic stability, between the two truncation mutants. Monolayer studies revealed that both apoB19 and apoB20.1 bound to and penetrated egg phosphatidylcholine (EPC) monolayers; however, the interfacial exclusion pressure of apoB20.1 was higher than apoB19 (25.1 mN/m vs. 22.8 mN/m). Oil drop tensiometry revealed that both proteins bound rapidly to the hydrophobic triolein/water interface, reducing interfacial tension by approximately 20 mN/m. However, when triolein drops were first coated with phospholipids (PL), apoB20.1 bound with faster kinetics than apoB19 and also displayed greater interfacial elasticity (26.9 +/- 0.8 mN/m vs. 22.9 +/- 0.8 mN/m). These data establish that the transition of apoB to assembly competence is accompanied by increases in surface activity and elasticity, but not by significant changes in global structure.     

Surface activity in vitro: role of surfactant proteins

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.     

Effects of pulmonary surfactant proteins SP-B and SP-C and calcium ions on the surface properties of hydrophobic fractions of lung surfactant

This study focused on two hydrophobic fractions (HF-A and HF-B) isolated from porcine lung surfactant (LS) that had similar phospholipid composition, but HF-A consisted of the hydrophobic LS specific proteins (SP-B and SP-C), in contrast to HF-B. Monolayers spread in a Langmuir trough were formed at the air/water interface of both fractions and the rate of adsorption-desorption and the respreading potential of the LS constituents was studied during six consecutive compression/decompression cycles of the monolayers. By drawing a comparison between the behavior of HF-A and HF-B monolayers on the subphase of 150 mm NaCl, either with or without additional Ca²⁺, we estimated the role of hydrophobic LS proteins and Ca²⁺ ions for LS surface activity. The results demonstrated much higher ability of the HF-A sample, compared to HF-B, to maintain lower surface tension (γ) during monolayer compression and its better respreading capacity during decompression. For instance, at a surface concentration corresponding to 80 Ų per phospholipid molecule, the HF-A monolayers showed a much lower γ _max value (surface tension at 100% of the trough area), being ca. 31.0 mN/m, compared to the HF-B monolayers (γ _max? 62.0 mN/m). The surface tension after compression to 20% of the initial area (γ _min) reached ca. 7.0 and 19.0 mN/m in the HF-A and HF-B monolayers, respectively. Better respreading of the HF-A monolayers compared to the HF-B monolayers was due to the faster adsorption and spreading of LS phospholipids during decompression, facilitated by the hydrophobic proteins. As the phospholipid composition of both fractions was similar, we showed that the hydrophobic surfactant proteins were responsible also for the prevention of the irreversible loss of material from the surface during monolayer compression/decompression. The effects observed demonstrated also that the hydrophobic surfactant proteins were the stronger determinant, compared with Ca²⁺ ions, for the surface tension decrease and respreading of the monolayers during film compression/decompression. For instance, when the HF-A monolayers were spread on a subphase with an additional 5 mm Ca²⁺ ion content, no significant changes were detected in the γ _min and γ _max values between the first and sixth cycle, compared to the monolayers spread on a subphase of 150 mm NaCl only. However, in the absence of positively charged SP-B and SP-C (HF-B sample) in highly compressed monolayers, Ca²⁺ ions were able to cause the effects shown by SP-B and SP-C, although to a less extent. The role of the electrostatic and hydrophobic interactions is discussed for the better respreading of LS components in the presence of LS proteins and Ca²⁺ ions.     

Chlorpromazine associates with phosphatidylserines to cause an increase in the lipid's own interfacial molecular area--role of the fatty acyl composition

Partition coefficients of the drug chlorpromazine were determined for five different molecular species of diacylglycerophosphatidylserine in a monolayer kept at constant surface pressure (20 mN/m). Two models of adsorption of chlorpromazine in phosphatidylserine monolayers were compared. The first model correlated the amount of inserted drug molecules with the induced increase in area. The second model introduced the effect of drug adsorption on the lipid's own area by comparing the effect of increasing temperature on the lipid's own interfacial area. From the second model, the extrapolated work of insertion of one drug molecule per lipid molecule in a monolayer kept at 20 mN/m was correlated to the partition of the drug in liposomes. The work of insertion of chlorpromazine was insignificant in the unsaturated dioleoylphosphatidylserine and was maximum in the saturated distearoylphosphatidylserine monolayers. The presence of one double bond in the acyl chains dramatically reduces the work of insertion of chlorpromazine between lipid molecules and also reduces the effect chlorpromazine induces on the lipids own interfacial area in monolayers.     

Interaction of the antimicrobial peptide cyclo(RRWWRF) with membranes by molecular dynamics simulations

Antimicrobial peptides have gained a lot of interest in recent years due to their potential use as a new generation of antibiotics. It is believed that this type of relatively short, amphipathic, cationic peptide targets the bacterial membrane, and destroys the chemical gradients over the membrane via formation of stable or transient pores. Here we use the NMR structure of cyclo(RRWWRF) in a series of molecular dynamics simulations in membranes at various peptide/lipid ratios. We observe that the NMR structure of the peptide is still stable after 100 ns simulation. At a peptide/lipid ratio of 2:128, the membrane is only a little affected compared to a pure dipalmitoylphosphatidylcholine lipid membrane, but at a ratio of 12:128, the water-lipid interface becomes more fuzzy, the water molecules can reach deeper into the hydrophobic core, and the water penetration free-energy barrier changes. Moreover, we observe that the area per lipid decreases and the deuterium order parameters increase in the presence of the peptide. We suggest that the changes in the hydrophobic core, together with the changes in the headgroups, result in an imbalance of the membrane and that it is thus not an efficient hydrophobic barrier in the presence of the peptides, independent of pore formation.     

The use of monolayers for simple and quantitative analysis of lipid-drug interactions exemplified with dibucaine and substance P.

The interaction between lipids and water soluble amphiphiles was investigated by means of a monolayer technique, monitoring the area increase at constant surface pressure. The area increase could be quantitated and binding isotherms at different surface pressures were measured. A comparison of dibucaine binding to monolayers and bilayers showed that a surface pressure of 32 mN/m best represents the packing density in a lipid bilayer (Seelig, 1987). Binding isotherms measured for charged dibucaine and substance P (SP) were analyzed by means of two different models. If electrostatic effects were ignored the binding of dibucaine and SP showed biphasic Scatchard plots. If, however, electrostatic effects were taken into account by means of the Gouy-Chapman theory, the insertion of both amphiphiles was best described in terms of a partitioning into the monolayer lipids. The hydrophobic binding constant was Kp = 660 +/- 80 M-1 for charged dibucaine inserting into coarse liposomes or monolayers at 32 mN/m (Seelig et al., 1986) and 1-1.8 M-1 for SP inserting into monolayers at 32 mN/m (Seelig and Macdonald, 1989).