3205234
Effects of surfactin on membrane models displaying lipid phase separation.
Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.
2541993
Wetting of fibrin plate and apparent promotion of fibrinolysis by surfactin, a new bacterial peptidelipid surfactant
Aus Kulturen vonBacillus subtilis wurde Surfactin, eine neue Substanz, isoliert und aufgeklärt. Es handelt sich um ein Peptidlipid, welches netzende Eigenschaften besitzt und dadurch die Fibrinolyse beschleunigt.
35500197
Structure and dynamics of surfactin studied by NMR in micellar media.
The NMR structure of the cyclic lipopeptide surfactin from Bacillus subtilis was determined in sodium dodecyl sulfate (SDS) micellar solution. The two negatively charged side chains of surfactin form a polar head opposite to most hydrophobic side chains, accounting for its amphiphilic nature and its strong surfactant properties. Disorder was observed around the fatty acid chain, and 15N relaxation studies were performed to investigate whether it originates from a dynamic phenomenon. A very large exchange contribution to transverse relaxation rate R(2) was effectively observed in this region, indicating slow conformational exchange. Temperature variation and Carr-Purcell-Meiboom-Gill (CPMG) delay variation relaxation studies provided an estimation of the apparent activation energy around 35-43 kJ x mol(-1) and an exchange rate of about 200 ms(-1) for this conformational exchange. 15N relaxation parameters were also recorded in dodecylphosphocholine (DPC) micelles and DMSO. Similar chemical exchange around the fatty acid was found in DPC but not in DMSO, which demonstrates that this phenomenon only occurs in micellar media. Consequently, it may either reflect the disorder observed in our structures determined in SDS or originate from an interaction of the lipopeptide with the detergent, which would be qualitatively similar with an anionic (SDS) or a zwitterionic (DPC) detergent. These structural and dynamics results on surfactin are the first NMR characterization of a lipopeptide incorporated in micelles. Moreover, they provide a model of surfactin determined in a more biomimetic environment than an organic solvent, which could be useful for understanding the molecular mechanism of its biological activity.
38904130
Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives.
Inactivation of enveloped viruses (VSV, SFV, and SHV-1) by surfactin lipopeptides was dependent on the hydrophobicity, i.e. the number of carbon atoms of the fatty acid, and on the charge of the peptide moiety as well as on the virus species. Surfactins with fatty acid chains of 13 carbon atoms showed very low antiviral activity in comparison to C14 and C15 isoforms. C15 surfactin monomethyl ester also inactivated SFV which was resistant to the mixture of surfactin isoforms as produced by Bacillus subtilis. In contrast, the dimethyl ester showed no virus-inactivation capacity. Disintegration of viral structures as determined by electron microscopy after inactivation of VSV and SFV was comparable to the titer reduction. The effect of the surfactin isoforms and methyl esters on erythrocyte hemolysis correlated with the virus-inactivation capacity. Surfactins with a fatty acid chain moiety of 15 carbon atoms and one negative charge showed the highest antiviral activity.
45974730
Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis.
The antiviral activity of surfactin, a cyclic lipopeptide antibiotic and biosurfactant produced by Bacillus subtilis, was determined for a broad spectrum of different viruses, Semliki Forest virus (SFV), herpes simplex virus (HSV-1, HSV-2), suid herpes virus (SHV-1), vesicular stomatitis virus (VSV), simian immunodeficiency virus (SIV), feline calicivirus (FCV), murine encephalomyocarditis virus (EMCV). In vitro experiments showed biphasic virus inactivation kinetics for enveloped viruses during treatment. Inactivation of enveloped viruses, especially herpes- and retroviruses, was much more efficient than that of non-enveloped viruses. For those viruses susceptible to its action, surfactin was active at 25 microM in medium containing 5% fetal calf serum (FCS). Concentrations up to 80 microM of surfactin led to a titre reduction of >4.4 log10 CCID50/ml for HSV-1 in 15 min and for SIV and VSV in 60 min. The inactivation rate increased linearly with the incubation temperature by a factor 2.4/10 degrees C and logarithmically with the concentration. Serum components, probably proteins and/or lipids, influence the effective surfactin concentration. A disruption of the viral lipid membrane and partially of the capsid was observed by electron microscopy. These findings suggest that the antiviral action, postulated also in other investigations, seems to be due to a physicochemical interaction of the membrane-active surfactant with the virus lipid membrane. Surfactin may be useful for application in virus safety enhancement of biotechnological and pharmaceutical products.
44889885
Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis
Surfactin, a cyclic lipopeptide antibiotic and biosurfactant produced by Bacillus subtilis, is well-known for its interactions with artificial and biomembrane systems (e.g., bacterial protoplasts or enveloped viruses). To assess the applicability of this antiviral and antibacterial drug, we determined the cytotoxicity of surfactin with a 50% cytotoxic concentration of 30 to 64 microM for a variety of human and animal cell lines in vitro. Concomitantly, we observed an improvement in proliferation rates and changes in the morphology of mycoplasma-contaminated mammalian cells after treatment with this drug. A single treatment over one passage led to complete removal of viable Mycoplasma hyorhinis cells from various adherent cell lines, and Mycoplasma orale was removed from nonadherent human T-lymphoid cell lines by double treatment. This effect was monitored by a DNA fluorescence test, an enzyme-linked immunosorbent assay, and two different PCR methods. Disintegration of the mycoplasma membranes as observed by electron microscopy indicated the mode of action of surfactin. Disintegration is obviously due to a physicochemical interaction of the membrane-active surfactant with the outer part of the lipid membrane bilayer, which causes permeability changes and at higher concentrations leads finally to disintegration of the mycoplasma membrane system by a detergent effect. The low cytotoxicity of surfactin for mammalian cells permits specific inactivation of mycoplasmas without significant deleterious effects on cell metabolism and the proliferation rate in cell culture. These results were used to develop a fast and simple method for complete and permanent inactivation of mycoplasmas in mammalian monolayer and suspension cell cultures.
34730393
Nature and properties of a cytolytic agent produced by Bacillus subtilis.
Surmmary
The substance responsible for lysis of erythrocytes by cultures of Bacillus subtilis, designated subtilysin, was purified. It contained a peptide of leucine, aspartic acid, glutamic acid and valine and probably a lipid. Subtilysin was activated by Mg2+, Mn2+ and Ca2+. The rate of haemolysis was abruptly increased by chilling the reaction mixture. Haemolysis was inhibited by normal sera; the most inhibitory serum fractions contained α- and β-globulins. Haemolysis was inhibited by low concentrations of phosphatidyl-choline, phosphatidylinositol, phosphatidic acid and sphingomyelin. Subtilysin possessed antibiotic properties and lysed protoplasts and spheroplasts derived from several bacterial species; subtilysin was identical with surfactin, a peptidelipid from B. subtilis cultures that inhibits fibrin clot formation. Kakinuma and co-workers found surfactin to be a heptapeptide having an N-terminal glutamic acid in amide linkage with the carboxyl group of 3-hydroxy-13-methyl-tetradecanoic acid. Surfactin (subtilysin) possesses some properties in common with two other cytolytic agents of bacterial origin, namely, staphylococcal δ-toxin and streptolysin S.
11391484
Optimization of Sterilization of Escherichia coli in Milk by Surfactin and Fengycin Using a Response Surface Method
In this paper, the sensitivity of Escherichia coli to surfactin and fengycin was observed, and the optimization of the antimicrobial activity of surfactin and fengycin to E. coli in milk by a response surface methodology was studied. Results showed that E. coli had high sensitivity to these antibiotics, whose minimal inhibitory concentrations were 15.625 μg·mL−1 and 31.25 μg·mL−1, respectively. The optimization result indicated that E. coli could be sterilized by 5 orders of magnitude when the temperature was 5.5°C, the action time was 15.8 h, and the concentration (surfactin/fengycin weight ratio 1:1) was 14.63 μg·mL−1.
41166407
A novel protoplast-bursting factor (surfactin) obtained from bacillus subtilis IAM 1213. II. The interaction of surfactin with bacterial membranes and lipids.
Abstract 1. 1. The protoplast-bursting activity of surfactin was neutralized by membrane fractions which had been solubilized in dilute alkali solution. 2. 2. After digestion of the membranes by trypsin, the residual fraction, containing high contents of lipids, still retained neutralizing activity against surfactin. 3. 3. Phospholipids such as phosphatidylethanolamine and phosphatidylcholine neutralized the activity of surfactin.
10732651
Biosurfactants as Agents to Reduce Adhesion of Pathogenic Bacteria to Polystyrene Surfaces: Effect of Temperature and Hydrophobicity
Polystyrene surfaces were conditioned with surfactin and rhamnolipid biosurfactants and then assessed regarding the attachment of Staphylococcus aureus, Listeria monocytogenes, and Micrococcus luteus. The effect of different temperatures (35, 25, and 4°C) on the anti-adhesive activity was also studied. Microbial adhesion to solvents and contact angle measurements were performed to characterize bacteria and material surfaces. The results showed that surfactin was able to inhibit bacterial adhesion in all the conditions analyzed, giving a 63–66% adhesion reduction in the bacterial strains at 4°C. Rhamnolipid promoted a slight decrease in the attachment of S. aureus. The anti-adhesive activity of surfactin increased with the decrease in temperature, showing that this is an important parameter to be considered in surface conditioning tests. Surfactin showed good potential as an anti-adhesive compound that can be explored to protect surfaces from microbial contamination.
32706349
Metabolism of kalopanaxsaponin B and H by human intestinal bacteria and antidiabetic activity of their metabolites.
To investigate the relationship between the intestinal bacterial metabolism of kalopanaxsaponin B and H from Kalopanax pictus (Araliaceae), and their antidiabetic effect, kalopanaxsaponin B and H were metabolized by human intestinal microflora and the antidiabetic activity of their metabolites was measured. Human intestinal microflora metabolized kalopanaxsaponin B to kalopanaxsaponin A, hederagenin 3-O-alpha-L-arabinopyranoside and hederagenin. The main metabolites of kalopanaxsaponin B were kalopanaxsaponin A and hederagenin. Kalopanaxsaponin H was metabolized to kalopanaxsaponin A and I, hederagenin 3-O-alpha-L-arabinopyranoside and hederagenin. The main metabolites of kalopanaxsaponin H were kalopanaxsaponin I and hederagenin. Among kalopanaxsaponin B, H and their metabolites, kalopanaxsaponin A showed the most potent antidiabetic activity, followed by hederagenin. However, the main components, kalopanaxsaponin B and H, in K. pictus were inactive.
11601126
Hemolytic activity of new linear surfactin analogs in relation to their physico-chemical properties.
New linear analogs of surfactin have been synthesized. Their physico-chemical parameters were determined. The results indicate that these linear products show surface activities although they are lowered compared to those of cyclic compounds. The hemolytic activities have also been assayed. In contrast with cyclic surfactins, no significant hemolysis occurs for the linear products in the range of concentrations tested. Moreover, a protective effect against Triton X-100 induced hemolysis has been highlighted for linear surfactins. The concentration at which this protective effect happens is correlated directly to the CMC, and inversely to the acyl chain length of the product. In a hypotonic medium, analogs having a long acyl chain tend to increase the hemolysis, meanwhile the product with the shortest chain tends to decrease it.
8118732
ROS-Ca(2+) is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis.
The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca(2+) on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca(2+) concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca(2+) was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ(m)). Then the cytoplasmic Ca(2+) concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.
84127653
Interaction of Surfactin with Membranes: A Computational Approach
Modeling analysis was used to understand the molecular mechanisms of the biological activities of surfactin, in particular, its hemolytic activity. This study highlights the importance of the fatty acid chain hydrophobicity of the surfactin on its activities, the C15 homologue being the most active. This is related to its self-association capacity. The detergent effect is the predominant mechanism involved in the hemolytic activity. A two-step mechanism is suggested, depending on the surfactin concentration. Other mechanisms (cationic channel, mobile carrier) can also be involved in particular conditions.
23561779
Detergent-like action of the antibiotic peptide surfactin on lipid membranes.
Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.
13801815
Leakage and lysis of lipid membranes induced by the lipopeptide surfactin
Surfactin is a lipopeptide produced by Bacillus subtilis which possesses antimicrobial activity. We have studied the leakage and lysis of POPC vesicles induced by surfactin using calcein fluorescence de-quenching, isothermal titration calorimetry and 31P solid state NMR. Membrane leakage starts at a surfactin-to-lipid ratio in the membrane, Rb ≈ 0.05, and an aqueous surfactin concentration of CSw ≈ 2 μM. The transient, graded nature of leakage and the apparent coupling with surfactin translocation to the inner leaflet of the vesicles, suggests that this low-concentration effect is due to a bilayer-couple mechanism. Different permeabilization behaviour is found at Rb ≈ 0.15 and attributed to surfactin-rich clusters, which can induce leaks and stabilize them by covering their hydrophobic edges. Membrane lysis or solubilization to micellar structures starts at Rbsat = 0.22 and CSw = 9 μM and is completed at Rmsol = 0.43 and CSw = 11 μM. The membrane–water partition coefficient of surfactin is obtained as K = 2 × 104 M−1. These data resolve inconsistencies in the literature and shed light on the variety of effects often referred to as detergent-like effects of antibiotic peptides on membranes. The results are compared with published parameters characterizing the hemolytic and antibacterial activity.
23842304
Structural, volumetric, and thermodynamic characterization of a micellar sphere-to-rod transition.
The thermotropic sphere-to-rod transition of nonionic surfactants was characterized in terms of a large set of parameters: the transition temperature and width, the partial volume, coefficient of thermal volume expansion, enthalpy, isobaric heat capacity, and structural parameters, such as radius of gyration and hydrodynamic radius. Data were recorded as a function of concentration of surfactants in H2O and in D2O. To this end, pressure perturbation calorimetry (PPC), small angle neutron scattering (SANS), dynamic light scattering (DLS), differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC) were applied in a study of aqueous solutions containing myristyl, tridecyl, and lauryl maltoside and heptaethyleneglycoltetradecyl ether (C14EO7). Small changes in the thermodynamic and volumetric parameters (e.g., the partial volume change is approximately +2 per thousand) are discussed in detail as the result of three effects governing the transition. (i) Reduction of the water accessible hydrophobic surface area (ASA(ap)) drives the transition. (ii) Shrinking in headgroup size by thermal dehydration triggers the transition. (iii) Hypothesized gradual ordering of the chains may control the effect of chain length on the transition.
30455974
The bacterial lipopeptide surfactin targets the lipid fraction of the plant plasma membrane to trigger immune‐related defence responses
The lipopeptide surfactin secreted by plant‐beneficial bacilli has crucial biological functions among which the ability to stimulate immune‐related responses in host tissues. This phenomenon is important for biological control of plant diseases but its molecular basis is still poorly understood. In this work, we used various approaches to study the mechanism governing the perception of this biosurfactant at the plant cell surface. Combining data on oxidative burst induction in tobacco cells, structure/activity relationship, competitive inhibition, insertion kinetics within plant membranes and thermodynamic determination of binding parameters on model membranes globally indicates that surfactin perception relies on a lipid‐driven process at the plasma membrane level. Such a sensor role of the lipid bilayer is quite uncommon considering that plant basal immunity is usually triggered upon recognition of microbial molecular patterns by high‐affinity proteic receptors.
13612035
Physicochemical studies of the interaction of the lipoheptapeptide surfactin with lipid bilayers of L-alpha-dimyristoyl phosphatidylcholine.
To understand the biological action of surfactin from Bacillus subtilis we investigated its effects on the phase transition of L-alpha-dimyristoyl phosphatidylcholine (DMPC)-vesicles from the crystalline to the fluid state using differential scanning calorimetry; light scattering; small angle neutron scattering and cryo-electron microscopy. DSC-thermograms revealed two phase transition peaks. Light scattering profiles showed two branches with characteristic hysteresis phenomena. With both techniques the same values of the phase transition temperatures T(m1) and T(m2) of 23.5 and 23 degrees C were obtained indicating two forms of DMPC-surfactin aggregates which could be visualized by cryo-electron microscopy. Until 4 mol% surfactin the vesicular form predominated, but was accompanied by bilayered membrane fragments by increasing the biosurfactant concentrations. At surfactin concentrations higher than 15 mol% smaller DMPC-surfactin micelles of ellipsoidal conformation were formed, as demonstrated by small angle neutron scattering. In addition, by "Poor Man's" temperature-jump-relaxation spectroscopy slow transients in the phase transition of vesicular DMPC-surfactin aggregates with relaxation times of 20-30 s were detected which presumably indicate the slow dissipation of intermediate lipid-and surfactin domains formed after the main phase transition on the way to the fluid state. This process is accelerated by surfactin.
33835283
Toluidine blue: aggregation properties and distribution behavior in surfactin micelle solution.
Aggregation properties and distribution behavior of toluidine blue in surfactin solution have been investigated by UV, infrared spectrum and fluorescence measurements. The UV measurement indicates that the location of toluidine blue can facilitate the formation of toluidine blue aggregates in contrast to that in the absence of surfactin. The fluorescence results show that the micropolarity of pyrene initially increases and then decreases with the addition of toluidine blue molecules. The binding constants of toluidine blue with surfactin micelles and the distribution coefficients between the micelles phase and the aqueous phase have been calculated by Benesi-Hildebrand method and pseudo-phase model, respectively. The values of DeltaH degrees < 0 (-22.11 kJ/mol) and DeltaS degrees > 0 (12.58+/-0.07 J/(mol K)) show that electrostatic attractive interaction plays an important role in the location of toluidine blue in surfactin micelles.
34186098
Destruction and solubilization of supported phospholipid bilayers on silica by the biosurfactant surfactin.
The lipopeptide surfactin from Bacillus subtilis strains exhibits strong surface and biological activity, the latter probably because of its interaction with biological membranes. We have investigated the interaction of aqueous solutions of surfactin with supported bilayers of diphosphatidylcholine (DPPC) on silica using neutron reflectometry. We have also used small-angle neutron scattering (SANS) to study the solubilized aggregates formed as a result of the destruction of the supported membrane by surfactin. Although surfactin on its own does not attach to the silica supporting surface, it is taken up from solution by the membrane, confirming that there is an attractive interaction between DPPC and surfactin. The surfactin concentration in the layer can reach up to about 20 mol % relative to DPPC. The membrane is stable provided that the surfactin concentration is below its critical micelle concentration (cmc, 5 x 10(-5) M). Above the cmc, however, the membrane is solubilized and removed from the surface, though not always completely, over a period of hours. There are signs that there is an induction period while the surfactin concentration builds up in the membrane. This would be consistent with the need for a threshold concentration of surfactin in the bilayer. The presence of a surfactin correlation peak in the SANS showed that in the bulk solution, at the same concentrations as used for the deposition, surfactin forms aggregates that must be localized in the DPPC multilamellar vesicles at a separation of about 160 A. The structure could be fitted with an approximate model where the surfactin has an aggregation number of 50 +/- 10 with a radius of about 27 A. Given the very small water thicknesses in the DPPC lamellar aggregates, the surfactin must exist as aggregates in the phospholipid bilayer, and these structures are responsible for solubilizing the DPPC.
43880909
Molecular organization of surfactin-phospholipid monolayers: effect of phospholipid chain length and polar head.
Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin-phospholipid system except for surfactin-DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC>DPPC>DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC>DPPE>DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.
4625803
Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin.
Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and possesses several other interesting biological activities. This work deals with the molecular mechanism of membrane permeabilization by incorporation of surfactin. The surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC). The effect of the addition of cholesterol, dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylethanolamine (POPE) was also checked. It was observed that surfactin was able to induce content leakage at concentrations far below the onset surfactin/lipid ratio for membrane solubilization to occur, which in our system was around 0.92. Electron microscopy showed that vesicles were present after addition of surfactin at a ratio below this value, whereas no vesicles could be observed at ratios above it. Cholesterol and POPE attenuated the membrane-perturbing effect of surfactin, whereas the effect of DPPC was to promote surfactin-induced leakage, indicating that bilayer sensitivity to surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that surfactin destabilizes the inverted-hexagonal structure. Fourier-transform infrared spectroscopy (FTIR) was used to specifically follow the effect of surfactin on different parts of the phospholipid bilayer. The effect on the C=O stretching mode of vibration of POPC indicated a strong dehydration induced by surfactin. On the other hand, the C-H stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization. The reported effects could be useful to explain surfactin-induced 'pore' formation underlying the antibiotic and other important biological actions of this bacterial lipopeptide.
8044611
Penetration of surfactin into phospholipid monolayers: nanoscale interfacial organization.
Atomic force microscopy (AFM) combined with surface pressure-area isotherms were used to probe the interfacial behavior of phospholipid monolayers following penetration of surfactin, a cyclic lipopeptide produced by Bacillus subtilis strains. Prior to penetration experiments, interfacial behavior of different surfactin molecules (cyclic surfactins with three different aliphatic chain lengths--S13, S14, and S15--and a linear surfactin obtained by chemical cleavage of the cycle of the surfactin S15) has been investigated. A more hydrophobic aliphatic chain induces greater surface-active properties of the lipopeptide. The opening of the peptide ring reduces the surface activity. The effect of phospholipid acyl chain length (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine- (DPPC), and distearoylphosphatidylcholine) and phospholipid polar head (DPPC, dipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylserine) on monolayer penetration properties of the surfactin S15 has been explored. Results showed that while the lipid monolayer thickness and the presence of electrostatic repulsions from the interfacial film do not significantly influence surfactin insertion, these parameters strongly modulate the ability of the surfactin to alter the nanoscale organization of the lipid films. We also probed the effect of surfactin structure (influence of the aliphatic chain length and of the cyclic structure of the peptide ring) on the behavior of DPPC monolayers. AFM images and isotherms showed that surfactin penetration is promoted by longer lipopeptide chain length and a cyclic polar head. This indicates that hydrophobic interactions are of main importance for the penetration power of surfactin molecules.
19825689
A study on the interactions of surfactin with phospholipid vesicles.
Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and posses several other interesting biological activities. By means of differential scanning calorimetry and X-ray diffraction the effect of surfactin on the phase transition properties of bilayers composed of different phospholipids, including lipids forming hexagonal-HII phases, has been studied. The interactions of surfactin with phosphatidylcholine and phosphatidylglycerol seem to be optimal in the case of myristoyl acyl chains, which have a similar length to the surfactin hydrocarbon tail. Data are shown that support formation of complexes of surfactin with phospholipids. The ionized form of surfactin seems to be more deeply inserted into negatively charged bilayers when Ca2+ is present, also supporting the formation of surfactin-Ca2+ complexes. In mixtures with dielaidoylphosphatidylethanolamine, a hexagonal-HII phase forming lipid, surfactin displays a bilayer stabilizing effect. Our results are compatible with the marked amphiphilic nature of surfactin and may contribute to explain some of its interesting biological actions; for instance the formation of ion-conducting pores in membranes.
10639754
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.
20868087
Model systems, lipid rafts, and cell membranes.
Views of how cell membranes are organized are presently changing. The lipid bilayer that constitutes these membranes is no longer understood to be a homogeneous fluid. Instead, lipid assemblies, termed rafts, have been introduced to provide fluid platforms that segregate membrane components and dynamically compartmentalize membranes. These assemblies are thought to be composed mainly of sphingolipids and cholesterol in the outer leaflet, somehow connected to domains of unknown composition in the inner leaflet. Specific classes of proteins are associated with the rafts. This review critically analyzes what is known of phase behavior and liquid-liquid immiscibility in model systems and compares these data with what is known of domain formation in cell membranes.
7179753
An outlook on organization of lipids in membranes: searching for a realistic connection with the organization of biological membranes.
Lipid-bilayer membranes are formed by self-assembly processes. The molecular interactions within the bilayer and with the environment impart a unique trans-bilayer lateral pressure profile and provide a set of physical mechanisms for formation of lipid domains and laterally differentiated regions in the plane of the membrane. Results from a number of experimental and theoretical studies of model lipid bilayers are reviewed, emphasizing the significance of these fundamental physical properties for the structure and dynamics of biological membranes. Particular attention is paid to the relevance of postulating the existence of equilibrium thermodynamic phases in biological membranes. This includes a discussion of the possible significance of equilibrium critical points in biological membrane systems that normally exist under non-equilibrium conditions. The need for a new model to replace the celebrated Nicolson-Singer fluid-mosaic model of biological membranes is also discussed.
38807103
Fluorescent probe partitioning in giant unilamellar vesicles of 'lipid raft' mixtures.
Direct visualization of raft-like l(o) (liquid-ordered) domains in model systems and cells using microscopic techniques requires fluorescence probes with known partitioning preference for one of the phases present. However, fluorescent probes may display dissimilar partitioning preferences in different lipid systems and can also affect the phase behaviour of the host lipid bilayer. Therefore a detailed understanding of the behaviour of fluorescent probes in defined lipid bilayer systems with known phase behaviour is essential before they can be used for identifying domain phase states. Using giant unilamellar vesicles composed of the ternary lipid mixture DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)/cholesterol, for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned either on the basis of quantification of the domain area fractions or by using a well-characterized l(d) (liquid-disordered)-phase marker. Fluorescent probes were examined both individually and using dual or triple labelling approaches. Most of the probes partitioned individually into the l(d) phase, whereas only NAP (naphtho[2,3-a]pyrene) and NBD-DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl] preferred the l(o) phase. We found that Rh-DPPE (Lissamine rhodamine B-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, T(mix). Interestingly, the partitioning of DiIC18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy-PC [2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine]. The specific use of each of the fluorescent probes is determined by its photostability, partitioning preference, ability to detect lipid phase separations and induced change in T(mix). We demonstrate the importance of testing a specific fluorescent probe in a given model membrane system, rather than assuming that it labels a particular lipid phase.
675514
Phase equilibria in DOPC/DPPC: Conversion from gel to subgel in two component mixtures.
Biological membranes contain a mixture of phospholipids with varying degrees of hydrocarbon chain unsaturation. Mixtures of long chain saturated and unsaturated lipids with cholesterol have attracted a lot of attention because of the formation of two coexisting fluid bilayer phases in such systems over a broad range of temperature and composition. Interpretation of the phase behavior of such ternary mixtures must be based on a thorough understanding of the phase behavior of the binary mixtures formed with the same components. This article describes the phase behavior of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with 1,2-di-d(31)-palmitoyl-sn-glycero-3-phosphocholine (DPPC) between -20 and 50 degrees C. Particular attention has been paid to the phase coexistence below about 16 degrees C where the subgel phase appears. The changes in the shape of the spectrum (and its spectral moments) during the slow transformation process leads to the conclusion that below 16 degrees C the gel phase is metastable and the gel component of the two-phase mixture slowly transforms to the subgel phase with a slightly different composition. This results in a line of three-phase coexistence near 16 degrees C. Analysis of the transformation of the metastable gel domains into the subgel phase using the nucleation and growth model shows that the subgel domain growth is a two dimensional process.
24290765
Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism.
The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri = 200) and is complete for Ri = 50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.
29997379
Alterations in membrane permeability induced by aminoglycoside antibiotics: studies on liposomes and cultured cells.
Aminoglycoside antibiotics bind to negatively-charged membranes in vitro as well as in vivo. We have examined if this binding could be associated with a change in the properties of membrane permeability. We have used a series of aminoglycoside derivatives and two independent test systems, namely (i) the release of calcein and of Mn2+ from phosphatidylinositol-containing large unilamellar vesicles, and (ii) the influx of Ca2+ into cultured macrophages. We found that certain aminoglycosides (e.g., streptomycin, isepamicin) markedly increase the membrane permeability whereas others (e.g., gentamicin) barely or do not influence it. This increase, when it occurs, is slower or less extensive than observed with pore-forming agents (mellitin, nystatin) or a Ca(2+)-ionophore (ionomycin). It is not observed with an agent [bis(beta-diethylaminoethylether)hexestrol] known to cause membrane fusion, and is not associated with any detectable change in membrane fluidity. In computer-aided conformational analysis of mixed monolayers between phosphatidylinositol and the aminoglycosides studied, it was found that those derivatives inducing an increase in membrane permeability in our experiments adopted an orientation rather perpendicular to the interface, whereas those with no or only a moderate effect were placed in a parallel orientation to this interface. The perpendicular orientation might cause a local condition of disorder which could explain the effects observed.
42181945
Purification of antifungal lipopeptides by reversed-phase high-performance liquid chromatography.
A rapid procedure for the purification of antifungal lipopeptides from Bacillus subtilis, a potential agent for biocontrol of plant diseases, was tested. It consists of a solid-phase extraction on C18 gel followed by reversed-phase chromatography using a biocompatible PepRPC HR 5/5 column with a pharmacia fast protein liquid chromatographic system. This is a very effective method for isolating and fractionating iturin A and surfactin, two lipopeptides of different nature, co-produced by Bacillus subtilis strain S499. The presence of homologous lipopeptides was easily detected.
45246150
Interactions of macrolide antibiotics (Erythromycin A, roxithromycin, erythromycylamine [Dirithromycin], and azithromycin) with phospholipids: computer-aided conformational analysis and studies on acellular and cell culture models.
The potential of 14/15 membered macrolides to cause phospholipidosis has been prospectively assessed, and structure-effects examined, using combined experimental and conformational approaches. Biochemical studies demonstrated drug binding to phosphatidylinositol-containing liposomes and inhibition of the activity of lysosomal phospholipase A1 toward phosphatidylcholine included in the bilayer, in close correlation with the number of cationic groups carried by the drugs (erythromycin A </= roxithromycin < erythromycylamine </= azithromycin). In cultured cells (fibroblasts), phospholipidosis (affecting all major phospholipids except sphingomyelin) was observed after 3 days with the following ranking: erythromycin A </= roxithromycin < erythromycylamine < azithromycin (roxithromycin could, however, not be studied in detail due to intrinsic toxicity). The difference between erythromycylamine and azithromycin was accounted for by the lower cellular accumulation of erythromycylamine. In parallel, based on a methodology developed and validated to study drug-membrane interactions, the conformational analyses revealed that erythromycin A, roxithromycin, erythromycylamine, and azithromycin penetrate into the hydrophobic domain of a phosphatidylinositol monolayer through their desosamine and cladinose moieties, whereas their macrocycle is found close to the interface. This position allows the aminogroups carried by the macrocycle of the diaminated macrolides (erythromycylamine and azithromycin) to come into close contact with the negatively charged phosphogroup of phosphatidylinositol, whereas the amine located on the C-3 of the desosamine, common to all four drugs, is located at a greater distance from this phosphogroup. Our study suggests that all macrolides have the potential to cause phospholipidosis but that this effect is modulated by toxicodynamic and toxicokinetic parameters related to the drug structure and mainly to their cationic character.
22485078
Atomic force microscopy of supported lipid bilayers
Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy (AFM) has become a well-established technique for imaging SLBs at nanometer resolution. A unique feature of AFM is its ability to monitor dynamic processes, such as the interaction of bilayers with proteins and drugs. Here, we present protocols for preparing dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers supported on mica using small unilamellar vesicles and for imaging their nanoscale interaction with the antibiotic azithromycin using AFM. The entire protocol can be completed in 10 h.
39899862
Formation of supported phospholipid bilayers via co-adsorption with beta-D-dodecyl maltoside.
We have investigated the formation of supported model membranes via the adsorption of phospholipid-surfactant mixtures at the Si-water interface by specular neutron reflection. The adsorption of mixed micelles of the nonionic surfactant beta-D-dodecyl maltoside and DOPC or POPC was determined as a function of bulk concentration, and using d25-beta-D-dodecyl maltoside, the composition of DOPC and POPC bilayers was determined. Bilayer thicknesses of 39+/-3 A for DOPC and 41+/-3 A for POPC agree well with data from bulk lamellar phases for both lipids, and the average area per lipid molecule can be varied from 62 to 115 A2 by varying the bulk concentrations used. The amount of surfactant in the bilayer is very sensitive to the bulk volume-to-surface area ratio, but it can be fully eliminated by ensuring a sufficiently large dilution/rinsing volume of the solution.
33060882
Phospholipase A2 hydrolysis of supported phospholipid bilayers: a neutron reflectivity and ellipsometry study.
We have investigated the phospholipase A(2) catalyzed hydrolysis of supported phospholipid bilayers using neutron reflection and ellipsometry. At the hydrophilic silica-water interface, hydrolysis of phosphatidylcholine bilayers by phospholipase A(2) from Naja mossambica mossambica venom is accompanied by destruction of the bilayer at an initial rate, which is comparable for DOPC and DPPC but is doubled for POPC. The extent of bilayer destruction at 25 degrees C decreases from DOPC to POPC and is dramatically reduced for DPPC. Neutron reflectivity measurements indicate that the enzyme penetrates into the bilayers in increasing order for DOPC, POPC, and DPPC, while the amount of enzyme adsorbed at the interface is smallest for DPPC and exhibits a maximum for POPC. Penetration into the hydrophobic chain region in the bilayer is further supported by the fact that the enzyme adsorbs strongly and irreversibly to a hydrophobic monolayer of octadecyltrichlorosilane. These results are rationalized in terms of the properties of the reaction products and the effect of their accumulation in the membrane on the kinetics of enzyme catalysis.
15861971
The adsorption of prothrombin to phosphatidylserine multilayers quantitated by ellipsometry.
We investigated by means of an automated ellipsometer the adsorption of prothrombin from a buffer solution by multilayers of 14:0/14:0- and 18:1/18:1-phosphatidylserine (PS) stacked on chromium slides. In this instrument thickness and refractive index of the adsorbed phospholipid and proteins are monitored continuously. Two equations are derived to relate the mass of stacked phospholipids and the mass of protein adsorbed to the thickness and refractive index. These equations are based upon the Lorentz-Lorenz relation among the molar refractivities, refractive indices, and the densities of binary mixtures. Experimental validation of these equations is performed by measuring stacked multilayers of known mass of phosphatidylserine and the adsorption of [125I] albumin and [3H]prothrombin on these multilayers. Using these equations we measured the dissociation constants Kd and the number of binding sites nb of prothrombin. Values of Kd = 0.15 x 10(-8) M and nb = 122 molecules of PS/molecule of prothrombin were observed for di C14:0 PS and values of Kd = 0.45 x 10(-8) M and nb = 54 molecules of PS/molecule of prothrombin for di C18:1 PS. These data compare well to data obtained by other methods available in the literature.
31068316
Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence.
The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the physical state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan molecule. Experiments performed using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the molecule is not responsible for the observed effects. Also, pH titration in the range from pH 4 to 10 shows that the spectral variations are independent of the charge of the polar head. A two-state model of dipolar relaxation is used to qualitatively explain the behavior of Laurdan. It is concluded that the presence of water molecules in the phospholipid matrix are responsible for the spectral properties of Laurdan in the gel phase. In the liquid crystalline phase there is a relaxation process that we attribute to water molecules that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addition rule.
254433
Membrane lipid domains and dynamics as detected by Laurdan fluorescence
Abstract2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation. In bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). TheGP value can be used for the quantitation of each phase. The wavelength dependence of theGP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined.GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in natural membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 Å.
35778798
Direct Observation of Lipid Domains in Free-Standing Bilayers Using Two-Photon Excitation Fluorescence Microscopy
The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as the shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this review article, we address the most relevant findings reported from our laboratory regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) under two-photon excitation fluorescence microscopy is particularly addressed, especially, the possibility of obtaining information on the phase state of different lipid domains directly from the fluorescent images.
24751861
Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach.
We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.
11006762
Assessing the (a)symmetry of concentration-effect curves: empirical versus mechanistic models.
Modeling the shape of concentration-effect curves is of prime importance in pharmacology. Geometric descriptors characterizing these curves (the upper and lower asymptotes, the mid-point, the mid-point slope, and the point of inflection) are used for drug comparison or for assessing the change in agonist function after a system modification. The symmetry or asymmetry around the mid-point of a concentration-effect curve is a fundamental property that, regretfully, is often overlooked because, generally, models yielding exclusively symmetric curves are used. In the present review, empirical and mechanistic models are examined in their ability to fit experimental data. The geometric parameters of a survey of empirical models, the Hill equation, a logistic variant that we call the modified Hill equation, the Richards function, and the Gompertz model are determined. To analyze the relationship between asymmetry and mechanism, some examples from the ionic channel field, in an increasing degree of complexity, are used. It is shown that asymmetry arises from ionic channels with multiple binding sites that are partly occupied. The operational model of agonism is discussed both in its empirical general formulation and including the signal transduction mechanisms through G-protein-coupled receptors. It is shown that asymmetry results from systems where receptor distribution is allowed. Developed mathematical models are compared for describing experimental data on alpha-adrenoceptors. The existence or not of a relationship between the shape of the curves and receptor reserve is discussed.
26448216
Conformational analysis of gramicidin-gramicidin interactions at the air/water interface suggests that gramicidin aggregates into tube-like structures similar as found in the gramicidin-induced hexagonal HII phase.
The energetics of interaction and the type of aggregate structure in lateral assemblies of up to five gramicidin molecules in the beta 6.3 helical conformation at the air/water interface was calculated using conformational analysis procedures. It was found that within the aggregate two types of gramicidin interaction occur. One leading to a linear organization with a mean interaction energy between monomers of -6 kcal/mol and one in a perpendicular direction leading to a circularly organization with a lower mean interaction energy of -10 kcal/mol. Extrapolation towards larger gramicidin assemblies predicts that gramicidin itself could form tubular structures similar to those found in the gramicidin-induced HII phase. The tryptophans appear to play an essential role in the tubular organization of the gramicidin aggregate, since they determine the cone shape of the monomer and contribute to the structure of the monomer and oligomer by stacking interactions. These results, which are discussed in the light of experimental observations of gramicidin self-association in model membranes and the importance of the tryptophans for HII phase formation, further support the view (Killian, J.A. and De Kruijff, B. (1986) Chem. Phys. Lipids 40, 259-284) that gramicidin is a first example of a new class of hydrophobic polypeptides which can form cylindrical structures within the hydrophobic core of the membrane.
7439916
Molecular Determinants of the Interaction Between the C‐Terminal Domain of Alzheimer's β‐Amyloid Peptide and Apolipoprotein E α‐Helices
Abstract : In a previous work, we predicted and demonstrated that the 29‐42‐residue fragment of β‐amyloid peptide (Aβ peptide) has in vitro capacities close to those of the titled fragment of viral fusion proteins. We further demonstrated that apolipoprotein E2 and E3 but not apolipoprotein E4 can decrease the fusogenic activity of Aβ(29‐42) via a direct interaction. Therefore, we suggested that this fragment is implicated in the neurotoxicity of Aβ and in the protective effects of apolipoprotein E in Alzheimer's disease. Because structurally related apolipoproteins do not interact with the Aβ C‐terminal domain but inhibit viral fusion, we suggested that interactions existing between fusogenic peptides and apolipoproteins are selective and responsible for the inhibition of fusion. In this study, we simulated interactions of all amphipathic helices of apolipoproteins E and A‐I with Aβ and simian immunodeficiency virus (SIV) fusogenic fragments by molecular modeling. We further calculated cross‐interactions that do not inhibit fusion in vitro. The results suggest that interactions of hydrophobic residues are the major event to inhibit the fusogenic capacities of Aβ(29‐42) and SIV peptides. Selectivity of those interactions is due to the steric complementarity between bulky hydrophobic residues in the fusogenic fragments and hydrophobic residues in the apolipoprotein C‐terminal amphipathic helices.
36668735
Location of diphenylhexatriene (DPH) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth.
The average membrane location of a series of diphenylhexatriene (DPH)-derived membrane probes was analyzed by measuring the quenching of DPH fluorescence with a series of nitroxide-labeled lipids in which the depth of the nitroxide group is varied. All DPH derivatives were located deeply within the bilayer. Some derivatives were anchored at a shallower depth than free DPH by attachment to cationic or anionic groups. However, the absolute change in DPH depth upon attachment to such groups was relatively modest (<4 A). In fact, protonated DPH fatty acid and a DPH fatty acyl group attached to a phosphatidylcholine were found to locate slightly more deeply than free DPH. The location of DPH derivatives can be explained by the length of the DPH group and its tendency to orient predominantly parallel to the fatty acyl chains of the bilayer. These factors allow a charged group attached to one end of a DPH molecule to be accommodated at the polar surface while maintaining a deep DPH location. Basically, it appears that most DPH derivatives probe the same region in the bilayer. We conclude previously reported differences in fluorescence polarization of free and anchored forms of DPH may reflect a direct effect of anchoring on motion rather than an effect on average DPH location. Other experiments showed the localization of DPH probes was found to be similar in the presence and absence of cholesterol. This implies that previously observed cholesterol-induced effects on DPH fluorescence polarization also largely reflect differences in DPH motion, not DPH location. From the quenching results it was also possible to define rules governing the location of a variety of chemical groups in membranes by comparison of the results obtained with DPH derivatives to those of similar derivatives of other fluorescent groups. Finally, an important goal of this study was to compare different methods of analysis of quenching data: parallax analysis, distribution (Gaussian) analysis (using a single Gaussian), and a second-order polynomial analysis. To evaluate the accuracy of these methods, the apparent depths of a series of fluorescence probes previously analyzed by parallax analysis was reanalyzed with all three methods. There was good agreement unless the fluorescent molecule was very shallow or very deep. In such cases, only parallax analysis gave physically reasonable results. This is likely to be due to the lack of a sufficient number of quenchers spanning a wide enough range for other analyses to compensate for deviations from ideal curves. Parallax analysis was also compared to distribution (Gaussian) analysis using a double Gaussian fit to account for quenching from the trans leaflet (Ladokhin, A. (1997) Methods Enzymol. 278, 462-473). Again more physically reasonable results were obtained from parallax analysis, likely due to non-Gaussian behavior of the depth dependence of quenching. Notwithstanding these observations, the significant number of cases where Gaussian curve fitting methods for quenching analysis are most powerful are discussed.
23428149
Use of fluorescent probes to monitor molecular order and motions within liposome bilayers.
This article reviews the use of fluorescent probes to monitor the order and dynamics within the acyl chain region of liposome lipid bilayers. Fluorescence anisotropy is first defined and the theoretical framework that allows interpretation of steady-state or dynamic measurements in terms of molecular details is reviewed. The general advantages and/or limitations of fluorescent versus other methods of monitoring membrane order and dynamics are discussed. The properties of two classes of fluorescence probes are then described. The linear probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and parinaric acid (PA) and their derivatives are seen as particularly useful when quantitative interpretation of observations in terms of details of bilayer dynamics and order are critical. Of these, DPH is the more widely and easily used, although parinaric acid has advantages for certain applications. The non-linear probes considered include the anthroyloxyl fatty acids and the recently introduced fluorenyl fatty acid probes. While the geometry and electronic configurations of these probes do not allow for detailed molecular interpretations, these probes can provide unique qualitative information about the state of the lipid bilayer at various positions along the acyl chains.
38065453
Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker.
When small, unilamellar lipid vesicles containing a high concentration of the fluorescent dye 6-carboxyfluorescein are incubated with either frog retinas or human lymphocytes, fluroescence distributes widely throughout each cell. Since "self-quenching" largely prevents the dye from fluorescing as long as it remains sequestered in vesicles, it is clear that a considerable amount of dye is released from the vesicles and diluted into the much larger volume of the cell.
2961179
Tamoxifen perturbs lipid bilayer order and permeability: comparison of DSC, fluorescence anisotropy, laurdan generalized polarization and carboxyfluorescein leakage studies.
The perturbation of the lipid bilayer structure by tamoxifen may contribute to its multiple mechanisms of anticancer action not related to estrogen receptors. This study evaluates the effect of tamoxifen on structural characteristics of model membranes using differential scanning calorimetry (DSC), fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-[trimethylammonium)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), as well as 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) generalized polarization. The comparative measurements in multilammelar vesicles (MLV) prepared from dipalmitoylphosphatidylcholine (DPPC) revealed that tamoxifen decreases the phase transition temperature (Tm) paralleled by a broadening of the phase transition profile. In large unilamellar vesicles (LUV) prepared from egg yolk phosphatidylcholine (EPC), tamoxifen increased the lipid bilayer order predominantly in the outer bilayer region. From membrane permeability measurements, we conclude that the tamoxifen-induced release of entrapped carboxyfluorescein (CF) results from a permanent bilayer disruption and the formation of transient holes in the lipid bilayer.
21621142
Fluorescence method for measuring the kinetics of fusion between biological membranes.
An assay is presented that allows continuous and sensitive monitoring of membrane fusion in both artificial and biological membrane systems. The method relies upon the relief of fluorescence self-quenching of octadecyl Rhodamine B chloride. When the probe is incorporated into a lipid bilayer at concentrations up to 9 mol% with respect to total lipid, the efficiency of self-quenching is proportional to its surface density. Upon fusion between membranes labeled with the probe and nonlabeled membranes, the decrease in surface density of the fluorophore results in a concomitant, proportional increase in fluorescence intensity, allowing kinetic and quantitative measurements of the fusion process. The kinetics of fusion between phospholipid vesicles monitored with this assay were found to be the same as those determined with a fusion assay based on resonance energy transfer [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099]. Octadecyl Rhodamine B chloride can be readily inserted into native biological membranes by addition of an ethanolic solution of the probe. Evidence is presented showing that the dilution of the fluorophore, occurring when octadecyl Rhodamine containing influenza virus is mixed with phospholipid vesicles at pH 5.0, but not pH 7.4, resulted from virus-vesicle fusion and was not related to processes other than fusion. Furthermore, by use of this method, the kinetics of fusion between Sendai virus and erythrocyte ghosts and virus-induced fusion of ghosts were readily revealed. Dilution of the probe was not observed upon prior treatment of fluorescently labeled Sendai virus with trypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
25508416
Rhodopsin-phospholipid reconstitution by dialysis removal of octyl glucoside.
Recombinant membranes were prepared from phospholipid-free rhodopsin and egg phosphatidylcholine (PC) under a wide variety of conditions employing an octyl beta-D-glucoside (OG) dialysis procedure. Two bands were consistently observed after sucrose density centrifugation of these recombinants. The major band, which was protein rich, had a molar phospholipid:protein ratio that was in the range of 30:1 to 50:1, even when the molar phospholipid:protein ratio of the solubilized solution prior to OG removal was as high as 300:1. Similar results were obtained when dioleoyl-PC, 1-palmitoyl-2-oleoyl-PC, disk lipids, or diphytanoyl-PC was used instead of egg PC. These results can be explained in terms of a lower stability of the OG-phospholipid micelles relative to the OG-phospholipid-rhodopsin micelles. Of the phospholipids that were used in the OG dialysis procedure, only saturated dimyristoyl-PC produced a protein-rich recombinant band with a phospholipid:protein ratio close to that of the initial solubilized solution. In contrast to the results obtained by using OG, when solubilized disks supplemented with egg PC were reconstituted from sodium cholate or dodecyltrimethyl-ammonium bromide, the resulting recombinant membranes had initial and final phospholipid:protein ratios which were similar.
20102655
Solubilization of phosphatidylcholine bilayers by octyl glucoside.
The solubilization of large, unilamellar egg phosphatidylcholine vesicles by the nonionic detergent octyl glucoside (OG) was investigated by nuclear magnetic resonance (NMR), fluorescence anisotropy, turbidity, electron microscopy, and centrifugation followed by compositional analysis. The solubilization process is well described by the three-stage model previously proposed for other detergents. In stage I, the OG partitions between the bilayer and aqueous phases with a molar partition coefficient of 59 +/- 6. The presence of OG in the bilayers produces a small "fluidizing" effect, as indicated by changes in the NMR and fluorescence anisotropy parameters. A rearrangement that forms large mixed bilayers occurs in the latter part of stage I. Stage II, the conversion of detergent-saturated bilayers into mixed micelles, begins at a ratio of total OG concentration minus the critical micelle concentration to total phosphatidylcholine concentration of approximately 1.5 and continues until this ratio reaches about 3.0. The correction for the critical micelle concentration of the OG is necessary for comparison of experimental results obtained at different lipid concentrations. The mixed bilayer-mixed micelle interconversion is quantified by the centrifugation experiments and by 31P NMR. The agreement between the two methods is excellent. Advantages of the NMR method are discussed. In stage III, which was not studied in detail here, all of the phosphatidylcholine is present as mixed micelles. Evidence is presented that the various structures present in the dispersions are in equilibrium with one another during these experiments.
23618802
A quantitative model describing the selective solubilization of membrane domains.
The classical three-stage model of membrane solubilization, including mixed membranes, membrane-micelle coexistence, and mixed micelles, is not applicable to demixed, domain-forming membranes and must, therefore, fail to describe the phenomenon of detergent-resistant membranes (DRMs). In lack of a quantitative model, it has often been assumed that ordered, detergent-depleted domains are inert, whereas fluid domains are solubilized. We establish a quantitative model based on equilibrium thermodynamics that is analogous to the three-stage model but comprises three components (two lipids and one detergent) in four phases (liquid-ordered and liquid-disordered membranes, micelles, and detergent in aqueous solution). For a given set of total concentrations and input parameters (initial abundance of ordered domains, solubilization boundaries of the pure lipids, etc.), it serves to calculate the phase boundaries and partial concentrations of all components in all phases. The results imply that the abundance and composition of ordered domains may vary substantially upon addition of detergent, both before and during solubilization of the fluid phase. It seems that gel-phase or order-preferring lipids are thermodynamically "resistant" regardless of the presence of a second, fluid phase. However, thermodynamic or kinetic resistance is not sufficient for obtaining DRMs because the resistant particles may be too small to be isolated. Cholesterol may be crucial for rendering the fragments large enough and, furthermore, enhance the formation of ordered domains by nonideal interactions with the detergent.
20879057
Membrane resistance to Triton X-100 explored by real-time atomic force microscopy.
Lateral segregation of lipids and proteins in biological membranes leads to the formation of detergent-resistant domains, also called "rafts". Understanding the mechanisms governing the biomembrane's resistance to solubilization by detergents is crucial in biochemical research. Here, we used real-time atomic force microscopy (AFM) imaging to visualize the behavior of a model supported lipid bilayer in the presence of different Triton X-100 (TX-100) concentrations. Mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) supported bilayers were prepared by vesicle fusion. Real-time AFM imaging revealed that, at concentrations below the critical micelle concentration (CMC), TX-100 did not solubilize the bilayer, but the DPPC domains were eroded in a time-dependent manner. This effect was attributed to the DPPC molecular packing disorganization by the detergent starting from the DOPC/DPPC interface. Just above the CMC, the detergent led to a complete solubilization of the DOPC matrix, leaving the DPPC domains unaltered. At higher TX-100 concentrations, the DOPC was also immediately removed just after detergent addition, and the DPPC domains remaining on the mica surface appeared to be more swollen and were gradually solubilized. This progressive solubilization of the DPPC remaining phase did not start at the edge of the domains but from holes appearing and expanding at the center of DPPC patches. The swelling of the DPPC domains was directly correlated with TX-100 concentration above the CMC and with detergent intercalation between DPPC molecules. We are convinced that this approach will provide a key system to elucidate the physical mechanisms of membrane solubilization by nonionic detergents.
27916944
Temperature-dependence of the solubilization of dipalmitoylphosphatidylcholine (DPPC) by the non-ionic surfactant Triton X-100, kinetic and structural aspects.
Most of the studies on the solubilization of model membranes conducted thus far involved model membranes made of liquid-crystalline phospholipids. Relatively little is known on the influence of temperature and of the phase of the lipid bilayers on their solubilization by detergents. The aim of the present study was to gain knowledge about the temperature and phase dependence of the solubilization of phospholipid bilayers by the non-ionic detergent Triton X-100 (TR). Detailed investigation of the kinetics of the solubilization of dipalmitoylphosphatidylcholine (DPPC), as well as of palmitoyloleoylphosphatidylcholine (POPC) by TR at different temperatures reveals that: (i) solubilization of DPPC is a relatively slow process, especially below Tm. This means that in order to prevent misleading conclusions it is important to monitor the solubilization after a steady state is established. (ii) Both the steady state structure and size of DPPC/TR aggregates and the kinetics of solubilization depend on temperature. (iii) The TR concentration required for solubilization of POPC bilayers is an increasing function of temperature, although no phase change of bilayers occurs in the studied temperature range. (iv) Detailed studies of the temperature-induced changes of the aggregates present in DPPC/TR or POPC/TR mixtures suggest that the state of aggregation at any temperature above 23 degrees C represents equilibrium. By contrast, for DPPC/TR mixtures at 4 degrees C all the processes are very slow, which complicates the interpretation of results obtained through the common practice of studying "rafts" by investigating detergent-resistant membranes.
4824978
Interaction between artificial membranes and enflurane, a general volatile anesthetic: DPPC-enflurane interaction.
The structural modifications of the dipalmitoylphosphatidylcholine (DPPC) organization induced by increasing concentration of the volatile anesthetic enflurane have been studied by differential scanning calorimetry, small-angle, and wide-angle x-ray scattering. The interaction of enflurane with DPPC depends on at least two factors: the enflurane-to-lipid concentration ratio and the initial organization of the lipids. At 25 degrees C (gel state), the penetration of enflurane within the lipids induces the apparition of two different mixed lipid phases. At low anesthetic-to-lipid molar ratio, the smectic distance increases whereas the direction of the chain tilt changes from a tilt toward next-neighbors to a tilt between next-neighbors creating a new gel phase called L(beta')(2NNN). At high ratio, the smectic distance is much smaller than for the pure L(beta') DPPC phase, i.e., 50 A compared to 65 A, the aliphatic chains are perpendicular to the membrane and the fusion temperature of the phase is 33 degrees C. The electron profile of this phase that has been called L(beta)(i), indicates that the lipids are fully interdigitated. At 45 degrees C (fluid state), a new melted phase, called L(alpha)(2), was found, in which the smectic distance decreased compared to the initial pure L(alpha)(1) DPPC phase. The thermotropic behavior of the mixed phases has also been characterized by simultaneous x-ray scattering and differential scanning calorimetry measurements using the Microcalix calorimeter of our own. Finally, titration curves of enflurane effect in the mixed lipidic phase has been obtained by using the fluorescent lipid probe Laurdan. Measurements as a function of temperature or at constant temperature, i.e., 25 degrees C and 45 degrees C give, for the maximal effect, an enflurane-to-lipid ratio (M/M), within the membrane, of 1 and 2 for the L(alpha)(2) and the L(beta)(i) lamellar phase respectively. All the results taken together allowed to draw a pseudo-binary phase diagram of enflurane-dipalmitoylphosphatidylcholine in excess water.
11819718
Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes.
Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.
41311112
MODULATION AND DYNAMICS OF PHASE PROPERTIES IN PHOSPHOLIPID MIXTURES DETECTED BY LAURDAN FLUORESCENCE *
Steady‐state and dynamic fluorescence properties of 6‐lauroyl‐2‐dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl‐ and dipalmitoyl‐phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time‐resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties of each individual phase are largely influenced by the presence of the other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.
384015
Heat Stress Causes Spatially-Distinct Membrane Re-Modelling in K562 Leukemia Cells
Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS) resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy.
24051667
Visualizing lipid structure and raft domains in living cells with two-photon microscopy
The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell–cell contacts and cover 10–15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 μm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.
11274785
Giant vesicles, Laurdan, and two-photon fluorescence microscopy: evidence of lipid lateral separation in bilayers.
Publisher Summary This chapter describes giant vesicles, Laurdan, and two-photon fluorescence microscopy. The combination of Laurdan, giant unilamellar vesicles (GUVs), and two-photon fluorescence microscopy has been extremely useful in producing a microscopic picture of lipid-phase coexistence in the GUV bilayer model system. Laurdan is a unique probe, giving simultaneous information about morphology and phase state of lipid domains from fluorescence images. The critical issues in membrane biophysics today are centered on the molecular dynamics of the bilayer structure. The interplay between lipids that results in the formation of domains on a bilayer surface and the interactions among these domains and relevant membrane-associated biomolecules is of particular interest. The advantages of using a microscope as the optical arrangement are clear. The light collection efficiency of a well-designed microscope is greatly enhanced over other optical arrangements. In addition, the flexibility of fluorescence microscopes creates for the spectroscopist a malleable optical compartment that can be designed and readily redesigned as needed.
29587159
Lipid rafts reconstituted in model membranes.
One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.
1766404
Cholesterol interactions with fluid-phase phospholipids: effect on the lateral organization of the bilayer.
The lateral organization of lipids and proteins in cell membranes is recognized as an important factor in several cellular processes. Cholesterol is thought to function as a modulator of the lateral segregation of lipids into cholesterol-poor and cholesterol-rich domains. We investigated how the affinity of cholesterol for different phospholipids, as seen in cholesterol partitioning between methyl-beta-cyclodextrin and large unilamellar vesicles, was reflected in the lateral organization of lipids in complex bilayers. We especially wanted to determine how the low-T(m) lipid affected the lateral structure. Partition experiments showed that cholesterol had a higher affinity for N-oleoyl-sphingomyelin (OSM) than for palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers, but the highest preference was for N-palmitoyl-sphingomyelin (PSM)-containing bilayers. Partial phase diagrams of POPC/PSM/cholesterol and OSM/PSM/cholesterol bilayers at 23 degrees C and 37 degrees C were used to gain insight into the lateral organization of lipids in bilayers. Analysis of phase diagrams revealed that the phospholipid composition of cholesterol-poor and cholesterol-rich domains reflected the affinity that cholesterol exhibited toward bilayers composed of different lipids. Therefore, the determined affinity of cholesterol for different phospholipid bilayers was useful in predicting the cholesterol-induced lateral segregation of lipids in complex bilayers.
29962907
Propofol, a general anesthetic, promotes the formation of fluid phase domains in model membranes.
The molecular site of anesthetic action remains an area of intense research interest. It is not clear whether general anesthetics act through direct binding to proteins or by perturbing the membrane properties of excitable tissues. Several studies indicate that anesthetics affect the properties of either membrane lipids or proteins. However, gaps remain in our understanding of the molecular mechanism of anesthetic action. Recent developments in membrane biology have led to the concept of small-scale domain structures in lipid and lipid--protein coupled systems. The role of such domain structures in anesthetic action has not been studied in detail. In the present study, we investigated the effect of anesthetics on lipid domain structures in model membranes using the fluorescent spectral properties of Laurdan (6-dodecanoyl-2-dimethylamino naphthalene). Propofol, a general anesthetic, promoted the formation of fluid domains in model membranes of dipalmitoyl phosphatidyl choline (DPPC) or mixtures of lipids of varying acyl chains (DPPC:DMPC dimyristoyl phosphatidyl choline 1:1). The estimated size of these domains is 20--50 A. Based on these studies, we speculate that the mechanism of anesthetic action may involve effects on protein--lipid coupled systems through alterations in small-scale lipid domain structures.
22331991
Effect of fenitrothion on dipalmitoyl and 1-palmitoyl-2-oleoylphosphatidylcholine bilayers.
The effects of the organophosphorous insecticide fenitrothion (phosphorothioic acid, O,O-dimethyl O-(3-methyl-4-nitrophenyl) ester; FS) on the physical state of pure dipalmitoyl (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes were investigated. FS lowers the phase transition temperature of DPPC. It has no large effects on the DPPC gel phase, but it increases the order of the liquid-crystalline state of DPPC and POPC. FS also decreases 1,6-diphenyl-1,3,5-hexatriene (DPH) lifetime (tau) in the DPPC and POPC liquid-crystalline states. Since a direct quenching of DPH emission by FS was ruled out, tau shortening is assigned to an increased water penetration in the bilayer. The effect of FS is different from most perturbing agents for which an increased order is accompanied by a higher tau. Furthermore, quenching of DPH by KI was increased by FS in POPC liposomes indicating an increased accessibility of the quencher to the hydrophobic core where DPH distributes. The effect of FS on dipole relaxation at the hydrophilic-hydrophobic interface of POPC bilayers was studied with 2-dimethylamino-6-lauroylnaphthalene (Laurdan). FS produces a decrease in Laurdan tau and a narrowing of its emission band. FS significantly increases the generalized polarization values at both emission band ends. These results indicate that FS may allow the coexistence of microdomains that have different physical properties.
27606980
Phase separation is induced by phenothiazine derivatives in phospholipid/sphingomyelin/cholesterol mixtures containing low levels of cholesterol and sphingomyelin.
Lipid rafts are membrane structures enriched in cholesterol, sphingomyelin and glycolipids. In majority raft-mimicking model systems high contents of cholesterol and sphingomyelin (approximately 30 mol%) are used. Existence of raft-like structures was, however, reported also in model and natural membranes containing low levels of cholesterol and sphingomyelin. In the present work differential scanning calorimetry and fluorescence spectroscopy with the use of Laurdan probe was employed to demonstrate the existence of phase separation in model systems containing DPPC with addition of 5 mol% or 10 mol% of both cholesterol and sphingomyelin. Additionally, the influence of three phenothiazine derivatives on phase separation in mixed DPPC/cholesterol/sphingomyelin bilayers was investigated. Chlorpromazine, thioridazine and trifluoperazine were able to induce phase separation in DPPC and DPPC/cholesterol/sphingomyelin bilayers in temperatures below lipid main phase transition. However, only trifluoperazine induced phase separation in temperatures close to or above main phase transition. Trifluoperazine also induced phase separation in bilayers composed of egg yolk PC or DOPC mixed with cholesterol and sphingomyelin. We concluded that presence of lipid domains can be observed in model membranes containing low levels of cholesterol and sphingomyelin. Among three phenothiazine derivatives studied, only trifluoperazine was able to induce a permanent phase separation in phosphatidylcholine/cholesterol/sphingomyelin systems.
24772798
Temperature dependence of the topology of supported dimirystoyl-distearoyl phosphatidylcholine bilayers.
Topology of fluid and gel domains in the supported bilayer two-component system formed from equimolar mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) was determined by AFM, at various temperatures corresponding to the gel and the gel + fluid region of the phase diagram. The data show that, in the disconnected fluid part of the DMPC/DSPC gel-liquid crystal-phase-separation region, the size of fluid domains markedly exceeds that predicted from spectroscopic experiments or from Monte Carlo simulations. They provide a direct evidence for the transition from the disconnected fluid to the disconnected gel region of the phase diagram, again with gel-phase domains much larger than expected. Finally, images of the gel phase at different temperatures suggest that structural rearrangements of the phospholipids can disrupt the continuity of the supported bilayer.
34234622
X-ray photoelectron spectroscopy analysis of whole cells and isolated cell walls of gram-positive bacteria: comparison with biochemical analysis
The surface chemical composition of whole cells and isolated cell walls of four coryneform bacteria and of a Bacillus brevis strain has been determined by X-ray photoelectron spectroscopy (XPS). The XPS data were converted into concentrations of model compounds: peptides, polysaccharides, and hydrocarbonlike compounds. The composition of the surface of B. brevis differed markedly from that of coryneforms: the peptide concentration was about twice higher in the former case, which is attributed to the presence of an S-layer at the cell surface; in contrast, the surface of coryneforms was rich in hydrocarbonlike compounds (about 40%), which was concomitant with a high water contact angle. The peptide surface concentration of the isolated cell walls of the five strains deduced from XPS data fitted well with the total peptide content determined by biochemical analysis, which supports the validity of XPS to determine the overall macromolecular composition of the bacterial cell surface. Compared to biochemical analysis of isolated cell walls, XPS analysis of whole cells provides information which concerns directly the cell surface (2- to 5-nm-thick layer) and is less subject to alteration via losses of cell wall constituents or contamination by intracellular compounds.
23618866
Solubilization of supported lipid membranes by octyl glucoside observed by time-lapse atomic force microscopy.
Detergents are very useful for the purification of membrane proteins. A good detergent for protein extraction has to prevent denaturation by unfolding, and to avoid aggregation. Therefore, gaining access to the mechanism of biomembranes' solubilization by detergents is crucial in biochemical research. Among the wide range of detergents used to purify membrane proteins, n-octyl beta-D-glucopyranoside (OG) is one of the most important as it can be easily removed from final protein extracts. Here, we used real-time atomic force microscopy (AFM) imaging to visualize the behavior of a model supported lipid bilayer in the presence of OG. Two kinds of supported model membranes were prepared by fusion of unilamellar vesicles: with an equimolar mixing of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) or with DPPC alone. Time-lapse AFM experiments evidenced that below its critical micelle concentration (CMC), OG was not able to solubilize the bilayer but the gel DPPC domains were instantly dissolved into the DOPC matrix. This result was interpreted as a disorganization of the DPPC molecular packing induced by OG. When membranes were incubated with OG at concentrations higher than CMC, the detergent immediately provoked the complete and immediate desorption of the whole bilayer for both compositions: DPPC and DOPC/DPPC. After a while, some patches appeared onto the bare mica surface. This redeposition activity, together with fusion events, progressively led to the recovery of a continuous bilayer. These results provide a new insight on the unique properties of OG employed in membrane reconstitution protocols.
4685092
Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization.
Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.
36470564
Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: implications for interpreting phase behavior.
The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (l(d)) domains on the surface of GUVs composed of 40:60 mol% DOPC/DPPC, over a wide range of temperatures (14-25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine(TM) rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.
33148501
Triton promotes domain formation in lipid raft mixtures.
Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds.
23317916
The interaction of phosphatidylcholine bilayers with Triton X-100.
The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.
24908373
Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100.
Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.
39701516
All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713.
The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1μM free FE and in 15% of the vesicles at 10 μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance.
9596362
Real-time partitioning of octadecyl rhodamine B into bead-supported lipid bilayer membranes revealing quantitative differences in saturable binding sites in DOPC and 1:1:1 DOPC/SM/cholesterol membranes.
Quantitative analysis of the staining of cell membranes with the cationic amphiphile, octadecyl rhodamine B (R18), is confounded by probe aggregation and changes to the probes' absorption cross section and emission quantum yield. In this paper, flow cytometry, quantum-dot-based fluorescence calibration beads, and FRET were used to examine real-time transfer of R18 from water to two limiting models of the cellular plasma membrane, namely, a single-component disordered membrane, dioleoyl-L-alpha-phosphatidylcholine (DOPC), and a ternary mixture of DOPC, cholesterol, and sphingomyelin (DSC) membranes, reconstituted on spherical and monodisperse glass beads (lipobeads). The quenching of R18 was analyzed as the probe concentration was raised from 0 to 10 mol % in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that were reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parametrized the propagation of concentration-dependent quenching as a function of real-time binding of R18 to lipobeads. In this way, phenomenological kinetics of serum-albumin-mediated transfer of R18 from the aqueous phase to DOPC and DSC membranes could be evaluated under optimal conditions where the critical aggregation concentration (CAC) of the probe is defined as 14 nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to that for DSC membranes. The difference is comparable to the disparity in the average molecular areas of DOPC and DSC membranes. Finally, this analysis shows little difference in the spectral overlap integrals of the emission spectrum of a fluorescein derivative donor and the absorption spectrum of either monomeric or simulated spectrum of dimeric R18. This approach represents a first step toward a nanoscale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.
25767829
Aminoglycoside antibiotics induce aggregation but not fusion of negatively-charged liposomes.
The binding of aminoglycoside antibiotics to acidic phospholipids of membranes is an essential step in the development of both their renal and auditory toxicities, which could be associated with critical modifications of the membrane properties. This work examines the capacity of aminoglycosides to induce membrane aggregation and fusion. Three techniques were used in parallel: (i) measurement of the dequenching rate of a lipid-soluble fluorescent probe (octadecylrhodamine B) incorporated at self-quenched concentration in membranes; (ii) measurement of the increase in the energy transfer between two fluorescent derivatives of phospholipids; and (iii) electron microscopy of negatively-stained replicas. The results were compared with those obtained with spermine (an aggregating polycation) and melittin (a fusogenic peptide). The three approaches indicate that aminoglycosides induce liposomes aggregation, but not fusion. Aggregation is related to the capacity of each drug studied to bind phosphatidylinositol, as evaluated by its energy of interaction with this acidic phospholipid, and to its toxic potential. Membrane aggregation occurring in vivo could therefore contribute to, or be a determinant of this toxicity, which could rationally be screened for new derivatives by the methods applied here.
6284848
Vesicle-micelle structural transition of phosphatidylcholine bilayers and Triton X-100.
The structural transition stages induced by the interaction of the non-ionic surfactant Triton X-100 on phosphatidylcholine unilamellar vesicles were studied by means of static and dynamic light-scattering, transmission-electron-microscopy (t.e.m.) and permeability changes. A linear correlation was observed between the effective surfactant/lipid molar ratios (Re) ('three-stage' model proposed for the vesicle solubilization) and the surfactant concentration throughout the process. However, this correlation was not noted for the partition coefficients of the surfactant between the bilayer and the aqueous medium (K). Thus a sharp initial K increase was observed until a maximum value was achieved for permeability alterations of 50% (initial step of bilayer saturation). Further surfactant additions resulted in a fall in the K values until 100% of bilayer permeability. Additional amounts of surfactant led to an increase in K until bilayer solubilization. Hence, a preferential incorporation of surfactant molecules into liposomes governs the initial interaction steps, leading to the initial stage of bilayer saturation with a free surfactant concentration that was lower than its critical micelle concentration (c.m.c.). Additional amounts of surfactant increased the free surfactant until the c.m.c. was reached, after which solubilization started to occur. Thus the initial step of bilayer saturation was achieved for a smaller surfactant concentration than that for the Resat, although this concentration was the minimum needed for solubilization to start. Large unilamellar vesicles began to form as the surfactant exceeded 15 mol% (50% bilayer permeability), the maximum vesicle growth being attained for 22 mol% (400 nm). Thereafter, static light-scattering started to decrease gradually, this fall being more pronounced after 40 mol%. The t.e.m. picture for 40 mol% (Resat.) showed unilamellar vesicles, although with traces of smaller structures. From 50 mol% the size distribution curves began to show a bimodal distribution. The t.e.m. pictures for 50-64 mol% revealed tubular structures, together with open bilayer fragments. Thereafter, increasing amounts of surfactant (65-69 mol%) led to planar multilayered structures which gradually tended to form concentric and helicoidal conformations. The scattered intensity decreased to a low constant value at more than 71-72 mol%. However, the surfactant concentration for the Re(sol) (72.6 mol %) still presented traces of aggregated structures, albeit with mono-modal size-distribution curves (particle size of 50 nm). This vesicle size corresponded to the liposome solubilization via mixed-micelle formation.
31537662
A method to detect leakage of DNA intercalators through liposome membranes.
A method to detect leakage of water-soluble intercalators such as ethidium bromide and propidium iodide from liposomes (egg-lecithin and egg-lecithin/cholesterol) by means of fluorescence is presented. The addition of excess DNA outside the liposomes brings about intercalation of released dye with the DNA. The increase in fluorescence intensity that results from the dye-DNA complex is used to detect the degree of dye release. Titration and surfactant-induced leakage measurements display the suitability of the method. Spontaneous leakage measurements show that propidium has much lower leaking rates than those of ethidium. This is probably due to the less lipophilicity of propidium as estimated from the partition coefficients between octanol and medium. Stabilizing the egg-lecithin membranes with cholesterol strongly reduces the leaking rates for ethidium while no effect is detected for the slow leaking rates of propidium. Lowering the temperature from 25 to 6 degrees C results in lower leaking rates for both dyes.
97315666
Solubilization of Lecithin Vesicles by a Cationic Surfactant: Intermediate Structures in the Vesicle-Micelle Transition Observed by Cryo-Transmission Electron Microscopy
Abstract The morphological change of lecithin vesicles during solubilization with the cationic surfactant cetyltrimethylammonium chloride (CTAC) was studied by means of cryo-transmission electron microscopy (cryo-TEM). The observed changes in aggregate size and structure were in most respects similar to those observed for systems containing lecithin and nonionic surfactants. Addition of low concentrations of CTAC to sonicated small unilamellar vesicles do as previously found for nonionics, give rise to a significant increase in the vesicle size. The growth of vesicles was monitored in time resolved cryo-TEM measurements and was clearly shown to proceed through an intermediate state comprising open bilayered discs. Contrary to other systems the addition of CTAC to the present system gives rise, at a certain concentration range, to the formation of a new intermediate structure. The samples stabilize in vesicles or open fragments of bilayers filled with holes to form a mesh-structure. Such structures, previously not documented by cryo-TEM, may be viewed as intermediates between bilayers and cylindrical micelles. The effects of CTAC on aggregate size and structure described above pertain to systems containing 100 mM NaCl. In the absence of salt the vesicle growth at low surfactant concentrations is strongly supressed, and only a dissolution of vesicles directly into mixed cylindrical micelles is observed.
39379443
Flexible loops of thread-like micelles are formed upon interaction of L-alpha-dimyristoyl-phosphatidylcholine with the biosurfactant surfactin as revealed by cryo-electron tomography.
Vesicles of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) are known to disintegrate upon treatment with surfactin, a lipoheptapeptide biosurfactant from Bacillus subtilis OKB 105, as was observed by static light scattering (SLS) and cryo-transmission electron microscopy (cryo-TEM) recently. The lysis of DMPC bilayers occurs strongly dependent on the surfactin concentration according to a three-stage model. Unilamellar DMPC vesicles are disrupted to form sheet-like lamellar intermediates at a moderate surfactant concentration, but undergo a transition towards smaller particles of unknown structure at a higher surfactant concentration according to earlier neutron scattering experiments. Here we present direct structural evidence from cryo-electron tomography data that thread-like micelles with a uniform diameter of 6.5 nm are organized into loops of different sizes at a surfactin concentration of > 15 mol%.
23205290
The vesicle-to-micelle transformation of phospholipid-cholate mixed aggregates: a state of the art analysis including membrane curvature effects.
We revisited the vesicle-to-micelle transformation in phosphatidylcholine-cholate mixtures paying special attention to the lipid bilayer curvature effects. For this purpose, we prepared unilamellar vesicles with different starting sizes (2r(v)=45-120nm). We then studied mixtures of the unilamellar vesicles (1-8mmol kg(-1)) and sodium cholate (0-11.75mmolkg(-1)) by static and dynamic light scattering. The transformation generally comprises at least two, largely parallel phenomena; one increases and the other decreases the average mixed aggregate size. In our view, cholate first induces bilayer fluctuations that lead to vesicle asphericity, and then to lipid bilayer poration followed by sealing/reformation (or fusion). The cholate-containing mixed bilayers, whether in vesicular or open form, project thread-like protrusions with surfactant enriched ends even before complete bilayer solubilisation. Increasing cholate concentration promotes detachment of such protrusions (i.e. mixed micelles formation), in parallel to further softening/destabilising of mixed amphipat bilayers over a broad range of concentrations. Vesicles ultimately fragment into mixed thread-like micelles. Higher cholate relative concentrations yield shorter thread-like mixed micelles. Most noteworthy, the cholate-induced bilayer fluctuations, the propensity for large aggregate formation, the transformation kinetics, and the cholate concentration ensuring complete lipid solubilisation all depend on the starting mean vesicle size. The smallest tested vesicles (2r(v)=45nm), with the highest bilayer curvature, require ~30% less cholate for complete solubilisation than the largest tested vesicles (2r(v)=120nm).
22934471
Modulation of the spontaneous curvature and bending rigidity of lipid membranes by interfacially adsorbed amphipathic peptides.
Amphipathic alpha-helical peptides are often ascribed an ability to induce curvature stress in lipid membranes. This may lead directly to a bending deformation of the host membrane, or it may promote the formation of defects that involve highly curved lipid layers present in membrane pores, fusion intermediates, and solubilized peptide-micelle complexes. The driving force is the same in all cases: peptides induce a spontaneous curvature in the host lipid layer, the sign of which depends sensitively on the peptide's structural properties. We provide a quantitative account for this observation on the basis of a molecular-level method. To this end, we consider a lipid membrane with peptides interfacially adsorbed onto one leaflet at high peptide-to-lipid ratio. The peptides are modeled generically as rigid cylinders that interact with the host membrane through a perturbation of the conformational properties of the lipid chains. Through the use of a molecular-level chain packing theory, we calculate the elastic properties, that is, the spontaneous curvature and bending stiffness, of the peptide-decorated lipid membrane as a function of the peptide's insertion depth. We find a positive spontaneous curvature (preferred bending of the membrane away from the peptide) for small penetration depths of the peptide. At a penetration depth roughly equal to half-insertion into the hydrocarbon core, the spontaneous curvature changes sign, implying negative spontaneous curvature (preferred bending of the membrane toward the peptide) for large penetration depths. Despite thinning of the membrane upon peptide insertion, we find an increase in the bending stiffness. We discuss these findings in terms of how the peptide induces elastic stress.