Incorporation of Blood-Clotting Proteins into Phospholipid Langmuir Monolayers: A Fluorescence Microscopy Study

Phospholipid monolayers adsorbed at an air-water interface are model cell membranes and have been used in this work to study interactions with blood-clotting proteins. Factor I (non-membrane binding) was used as a control protein, and its association with L-alpha-dipalmitoylphosphatidylcholine Langmuir monolayers was compared to factor VII, a membrane-binding protein. Fluorescence micrographs indicated that factor I penetration of the lipid monolayers in the phase transition region occurred extensively, causing condensation of the lipid film. The association of factor I with phospholipid monolayers was deemed nonspecific. Factor VII was shown to associate with the periphery of lipid domains in the absence of calcium ions, causing flattening of domain edges. In the presence of calcium, factor VII induced expansion of the lipid monolayer. This effect is a specific interaction attributed to exposure of hydrophobic residues upon calcium binding, followed by protein association with lipid hydrocarbon chains. Copyright 2001 Academic Press.     

Monolayer studies of single-chain polyprenyl phosphates

The monolayer properties of some single-chain polyprenyl phosphates (phytanyl, phytyl, and geranylgeranyl phosphates), which we regard as hypothetical primitive membrane lipids, were investigated at the air-water interface by surface pressure-area (pi-A) isotherm measurements. The molecular area/ pressure at various pH conditions dependence revealed the acid dissociation constants (pKa values) of the phosphate. The pKa values thus obtained at the air-water interface (pKa1 = 7.1 and pKa2 = 9.4 for phytanyl phosphate) were significantly shifted to higher pH than those observed in the bilayer state in water (pKa1 = 2.9 and pKa2 = 7.8). The difference in pKa values leads to a stability of the phosphate as both monolayer and bilayer states in a pH range of 2-6. In addition, the presence of ions such as sodium, magnesium, calcium, and lanthanum in the subphase significantly altered the stability of the polyprenyl phosphate monolayers, as shown by the determination of monolayer collapse and compression/expansion hysteresis. Although sodium ions in the subphase showed only a weak effect on the stabilization of the monolayer, addition of magnesium ions or of a small amount of calcium ions significantly suppressed the dissolution of the monolayer into the subphase and increased its mechanical stability against collapse. In contrast, the presence of larger amounts of calcium or of lanthanum ions induced collapse of the monolayers. Based on these experimental facts, a plausible scenario for the formation of primitive cell membrane by transformation of a monolayer to vesicle structures is proposed.     

Interaction of rabbit C-reactive protein with phospholipid monolayers at air/water interface

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. The structure of CRP has been previously established by crystallography, and the significance of its interaction with lipid membranes is accepted in the literature. However, the nature of the interaction between CRP and phospholipids is not yet well understood. In this paper we use monolayer technique to study the characteristics of the interaction of rabbit C-reactive protein (rCRP) with the phospholipid membranes. The results show that rCRP is surface active and can spontaneously insert into the lipid monolayers. The critical pressure for rCRP inserting into the phospholipid monolayers is about 34.5 mN/m, which is not sensitive to the types of the lipid headgroups and the presence of calcium ions in the subphase. The findings of this paper may provide a clue to the further understanding of the mechanism of the interactions between rCRP and the biological membranes.     

Study of adsorption and penetration of E2(279-298) peptide into Langmuir phospholipid monolayers

The peptide corresponding to the sequence (279-298) of the Hepatitis G virus (HGV/GBV-C) E2 protein was synthesized, and surface activity measurements, pi-A compression isotherms, and penetration of E2(279-298) into phospholipid monolayers spread at the air-water interface were carried out on water and phosphate buffer subphases. The results obtained indicated that the pure E2(279-298) Langmuir monolayer exhibited a looser packing on saline-buffered than on pure water subphase and suggest that the increase in subphase ionic strength stabilizes the peptide monolayer. To better understand the topography of the monolayer, Brewster angle microscopy (BAM) images of pure peptide monolayers were obtained. Penetration of the peptide into the pure lipid monolayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) and into mixtures of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) at various initial surface pressures was investigated to determine the ability of these lipid monolayers to host the peptide. The higher penetration of peptide into phospholipids is attained when the monolayers are in the liquid expanded state, and the greater interaction is observed with DMPC. Furthermore, the penetration of the peptide dissolved in the subphase into these various lipid monolayers was investigated to understand the interactions between the peptide and the lipid at the air-water interface. The results obtained showed that the lipid acyl chain length is an important parameter to be taken into consideration in the study of peptide-lipid interactions.     

Surface complexation of DNA with insoluble monolayers. Influence of divalent counterions

DNA interacts with insoluble monolayers made of cationic amphiphiles as well as with monolayers of zwitterionic lipids in the presence of divalent ions. Binding to dioctadecyldimethylammonium bromide (DODAB) or distearoyl-sn-glycero-3-phosphocholine (DSPC) monolayers in the presence of calcium is accompanied by monolayer expansion. For the positively charged DODAB monolayer, this causes a decrease of surface potential, while an increase is observed for the DSPC monolayers. Binding to dipalmitoyl-sn-glycero-3-phosphocholine preserves most of the liquid expanded-liquid condensed coexistence region. The liquid condensed domains adopt an elongated morphology in the presence of DNA, especially in the presence of calcium. The interaction of DNA with phospholipid monolayers is ion specific: the presence of calcium leads to a stronger interaction than magnesium and barium. These results were confirmed by bulk complexation studies.     

Nonequilibrium 2-hydroxyoctadecanoic acid monolayers: effect of electrolytes

2-Hydroxyacids display complex monolayer phase behavior due to the additional hydrogen bonding afforded by the presence of the second hydroxy group. The placement of this group at the position α to the carboxylic acid functionality also introduces the possibility of chelation, a utility important in crystallization including biomineralization. Biomineralization, like many biological processes, is inherently a nonequilibrium process. The nonequilibrium monolayer phase behavior of 2-hydroxyoctadecanoic acid was investigated on each of pure water, calcium chloride, sodium bicarbonate and calcium carbonate crystallizing subphases as a precursor study to a model calcium carbonate biomineralizing system, each at a pH of ～6. The role of the bicarbonate co-ion in manipulating the monolayer structure was determined by comparison with monolayer phase behavior on a sodium chloride subphase. Monolayer phase behavior was probed using surface pressure/area isotherms, surface potential, Brewster angle microscopy, and synchrotron-based grazing incidence X-ray diffraction and X-ray reflectivity. Complex phase behavior was observed for all but the sodium chloride subphase with hydrogen bonding, electrostatic and steric effects defining the symmetry of the monolayer. On a pure water subphase hydrogen bonding dominates with three phases coexisting at low pressures. Introduction of calcium ions into the aqueous subphase ensures strong cation binding to the surfactant head groups through chelation. The monolayer becomes very unstable in the presence of bicarbonate ions within the subphase due to short-range hydrogen bonding interactions between the monolayer and bicarbonate ions facilitated by the sodium cation enhancing surfactant solubility. The combined effects of electrostatics and hydrogen bonding are observed on the calcium carbonate crystallizing subphase.     

Incorporation conditions guiding the aggregation of a glycosylphosphatidyl inositol (GPI)-anchored protein in Langmuir monolayers

This work investigates the process of incorporation of a glycosylphosphatidyl inositol (GPI)-anchored alkaline phosphatase into Langmuir monolayers of dimyristoyl phosphatidic acid (DMPA). Three different methods of protein incorporation were assayed. When the protein solution was injected below the air–water interface after formation of the lipid monolayer a micro-heterogeneous distribution of alkaline phosphatase throughout the interface was observed. Adsorption kinetics studied by fluorescence microscopy, associated with surface pressure measurements, led to the proposition of a model in which the protein penetration is modulated by the surface packing of the monolayer and intermolecular interactions occurring between the phospholipid and the protein. At initial surface pressures higher than 20 mN m⁻¹, the protein is quickly adsorbed on the interface and the lateral diffusion drives the alkyl chains to turn towards the air phase while the polypeptide moiety faces the aqueous subphase.     

Effects of Calcium Ions on Thermodynamic Properties of Mixed Bilirubin/Cholesterol Monolayers

The mixed monolayer behavior of bilirubin/cholesterol was studied through surface pressure-area (?-A) isotherms on aqueous solutions containing various concentrations of calcium ions. Based on the data of ?-A isotherms, the mean area per molecule, collapse pressure, surface compressibility modulus, excess molecular areas, free energy of mixing, and excess free energy of mixing of the monolayers on different subphases were calculated. The results show an expansion in the structure of the mixed monolayer with Ca2+ in subphase, and non-ideal mixing of the components at the air/water interface is observed with positive deviation from the additivity rule in the excess molecular areas. The miscibility between the components is weakened with the increase of concentration of Ca2+ in subphase. The facts indicate the presence of coordination between Ca2+ and the two components. The mixed monolayer, in which the molar ratio of bilirubin to cholesterol is 3:2, is more stable from a thermodynamic point of view on pure water. But the stable 3:2 stoichiometry complex is destroyed with the increase of the concentration of Ca2+ in subphase. Otherwise, the mixed monolayers have more thermodynamic stability at lower surface pressure on Ca2+ subphase.     

Interaction between puroindolines and the major polar lipids of wheat seed endosperm at the air-water interface

To understand the role of the puroindolines (PIN-a and PIN-b) in the defense mechanism and stabilization of lipid films in the gas cell of bread dough, we have isolated the proteins and lipids from wheat seed endosperm and studied their interaction at the air/water interface using a Langmuir trough. The nature and shape of the pressure–area compression isotherms of the lipid monolayer in the presence of puroindolines in the subphase depended on the concentration of protein. A distinct phase separation occurred, when the concentration of protein in the subphase increased. The interfacial elasticity of the lipid monolayer in the presence of puroindolines in the subphase was higher than the pure lipid. Injection of protein beneath the preexisting lipid monolayer resulted in the increase of surface pressure due to the penetration of proteins. The extent of penetration depended on the nature of lipid head groups as well as on the initial surface pressure. The penetration of puroindolines to lipid monolayer was observed to be zero after crossing a critical initial surface pressure. The magnitude of the critical initial surface pressure for anionic lipids was significantly higher than the zwitterionic and nonionic lipids. The experimental results showed that both PIN-a and PIN-b had more affinity for anionic polar lipids than the neutral polar lipids and stabilized the lipid monolayer.     

Calcium-induced phospholipid ordering depends on surface pressure

The effect of sodium and calcium ions on zwitterionic and anionic phospholipids monolayers is investigated using vibrational sum-frequency generation in conjunction with surface pressure measurements and fluorescence microscopy. Sodium ions only subtly affect the monolayer structure, while the effect of calcium is large and depends strongly on the surface pressure. At low surface pressures (approximately 5 mN/m), the presence on Ca2+ results in the unexpected appearance of ordered domains. For pressures between approximately 5 and approximately 25 mN/m, Ca2+ ions induce disorder in the monolayer. For pressures exceeding 25 mN/m, calcium cations expand the monolayer, while simultaneously ordering the lipid chains. Interestingly, effects are similar for both zwitterionic lipids and negatively charged lipids. In both vibrational sum-frequency generation and surface tension measurements, the molecular signature of the association of Ca2+ with the lipids is evident from Ca2+-induced changes in the signals corresponding to area changes of 4 A2/lipid-precisely the surface area of a Ca2+ ion, with evidence for a change in lipid Ca2+ complexation at high pressures.     

An electrochemical study into the interaction between complement-derived peptides and DOPC mono- and bilayers

Electrochemical methods employing the hanging mercury drop electrode were used to study the interaction between variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQH ARASHLGLAR) and dioleoyl phosphatidylcholine (DOPC) monolayers. Capacitance potential and impedance measurements showed that the CNY21 analogues investigated interact with DOPC monolayers coating the mercury drop. Increasing the peptide hydrophobicity by substituting the two histidine residues with leucine resulted in a deeper peptide penetration into the hydrophobic region of the DOPC monolayer, indicated by an increase in the dielectric constant of the lipid monolayer (Deltaepsilon = 2.0 after 15 min interaction). Increasing the peptide net charge from +3 to +5 by replacing the histidines by lysines, on the other hand, arrests the peptide in the lipid head group region. Reduction of electroactive ions (Tl+, Pb2+, Cd2+, and Eu3+) at the monolayer-coated electrode was employed to further characterize the types of defects induced by the peptides. All peptides studied permeabilize the monolayer to Tl+ to an appreciable extent, but this effect is more pronounced for the more hydrophobic peptide (CNY21L), which also allows penetration of larger ions and ions of higher valency. The results for the various ions indicate that charge repulsion rather than ion size is the determining factor for cation penetration through peptide-induced defects in the DOPC monolayer. The effects obtained for monolayers were compared to results obtained with bilayers from liposome leakage and circular dichroism studies for unilamellar DOPC vesicles, and in situ ellipsometry for supported DOPC bilayers. Trends in peptide-induced liposome leakage were similar to peptide effects on electrochemical impedance and permeability of electroactive ions for the monolayer system, demonstrating that formation of transmembrane pores alone does not constitute the mechanism of action for the peptides investigated. Instead, our results point to the importance of local packing defects in the lipid membrane in close proximity to the adsorbed peptide molecules.     

Preparation, characterization, and application of polypropylene-clinoptilolite composites for the selective adsorption of lead from aqueous media

The miscibility, mechanical and morphological properties of mixed Langmuir and Langmuir-Blodgett monolayers prepared from the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and the perfluorinated fatty acid perfluorooctadecanoic acid have been studied as a function of film composition and subphase salinity. It was demonstrated here, for the first time, that the extent of surfactant miscibility in mixed phospholipid-perfluoroacid monolayers, and hence the resulting mechanical properties of the monolayer film, can be controlled by altering the concentration of sodium ions in the underlying subphase. Elevated Na(+) concentrations resulted in lower net attractive interactions between film components, likely through specific ion adsorption to the negatively-charged perfluoroacid, along with decreased film elasticities. These results differ significantly from conventional fatty-acid-carboxylate monolayer systems in which film cohesion is typically enhanced through adsorption of cations to surfactant headgroups. Atomic force microscope images of films deposited onto solid mica substrates revealed that the films deposited from pure water formed multimolecular aggregates of surfactant, which could be attributed to the highly cohesive nature of the films, but the use of salt in the subphase diminished aggregate formation and resulted in the production of homogeneous monolayer films.     

Mixed DPPC/DPTAP monolayers at the air/water interface: influence of indolilo-3-acetic acid and selenate ions on the monolayer morphology

The interactions of mixed monolayers of two lipids, zwitterionic 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and positively charged 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), with phytohormone indolilo-3-acetic acid (IAA) and selenate anions in the aqueous subphase were studied. For this purpose, isotherms of the surface pressure versus the mean molecular area were recorded. Domain formation was investigated by using Brewster angle microscopy (BAM). The method of grazing incidence X-ray diffraction (GIXD) was also applied for the characterization of the organization of lipid molecules in condensed monolayers. It was found that selenate ions contribute to monolayer condensation by neutralizing the positive net charge of mixed monolayers whereas IAA molecules penetrated the lipid monolayer, causing its expansion/fluidization. When both solutes were introduced into the subphase, a competition between them for interaction with the positively charged lipids in the monolayer was observed.     

Bilayer and trilayer crystalline formation by collapsing behenic acid monolayers at gas/aqueous interfaces

X-ray reflectivities and grazing incidence X-ray diffractions of behenic acid (BA) monolayers compressed to the collapse region reveal that the resulting structures are reproducible and exhibit a high degree of order. The structures of the collapsed monolayers depend on the subphase solution. On pure water, the collapsed monolayer forms a stable crystalline trilayer structure. For monolayers spread on Ca2+ solutions, we find that an inverted bilayer structure is formed; that is, stretched BA-Ca-BA (calcium dibehenate, with calcium ions bridging the polar headgroups) forms a monolayer with the hydrophobic tails in contact with the water surface.     

Interaction of the neurotransmitter, neuropeptide Y, with phospholipid membranes: infrared spectroscopic characterization at the air/water interface

The association of neuropeptide Y (NPY) at the air/water interface and with phospholipid monolayers on water as subphase has been investigated using external infrared reflection absorption spectroscopy (IRRAS). Studies of the conformation and orientation of NPY suggest that it adopts an alpha-helical structure and is oriented parallel to the air/water interface in neat peptide monolayers. Both secondary structure and orientation are preserved in mixed lipid/NPY monolayers. Comparison of NPY associated with zwitterionic DPPC and with anionic DMPS suggests that electrostatic attraction plays a major role for peptide binding to the membrane surface.     

Annexin A1 interaction with a zwitterionic phospholipid monolayer: a fluorescence microscopy study

We present the results of a fluorescence microscopy study of the interaction of annexin A1 with dipalmitoylphosphatidylcholine (DPPC) monolayers as a function of the lipid monolayer phase and the pH of the aqueous subphase. We show that annexin A1-DPPC interaction depends strongly on the domain structure of the DPPC monolayer and only weakly on the subphase pH. Annexin A1 is found to be line active, with preferential adsorption at phase boundaries. Also, annexin A1 is found to form networks in the presence of a domain structure in the monolayer. Our results point toward an important contribution of the unique N-terminal domain to the organization of the protein at the interface.     

Thermodynamic characteristics and Langmuir-Blodgett deposition behavior of mixed DPPA/DPPC monolayers at air/liquid interfaces

The role of dipalmitoylphosphatic acid (DPPA) as a transfer promoter to enhance the Langmuir-Blodgett (LB) deposition of a dipalmitoylphosphatidylcholine (DPPC) monolayer at air/liquid interfaces was investigated, and the effects of Ca2+ ions in the subphase were discussed. The miscibility of the two components at air/liquid interfaces was evaluated by surface pressure-area per molecule isotherms, thermodynamic analysis, and by the direct observation of Brewster angle microscopy (BAM). Multilayer LB deposition behavior of the mixed DPPA/DPPC monolayers was then studied by transferring the monolayers onto hydrophilic glass plates at a surface pressure of 30 mN/m. The results showed that the two components, DPPA and DPPC, were miscible in a monolayer on both subphases of pure water and 0.2 mM CaCl2 solution. However, an exception occurs between X(DPPA)=0.2 and 0.5 at air/CaCl2-solution interface, where a partially miscible monolayer with phase separation may occur. Negative deviations in the excess area analysis were found for the mixed monolayer system, indicating the existence of attractive interactions between DPPA and DPPC molecules in the monolayers. The monolayers were stable at the surface pressure of 30 mN/m for the following LB deposition as evaluated from the area relaxation behavior. It was found that the presence of Ca2+ ions had a stabilization effect for DPPA-rich monolayers, probably due to the association of negatively charged DPPA molecules with Ca2+ ions. Moreover, the Ca2+ ions may enhance the adhesion of DPPA polar groups to a glass surface and the interactions between DPPA polar groups in the multilayer LB film structure. As a result, Y-type multilayer LB films containing DPPC could be fabricated from the mixed DPPA/DPPC monolayers with the presence of Ca2+ ions.     

Influence of lipidation of GBV-C/HGV NS3 (513-522) and (505-514) peptide sequences on its interaction with mono and bilayers

Two decapeptide fragments of the non-structural hepatitis G NS3 protein (GBV-C/HGV), 513-522 (RGRTGRGRSG) and 505-514 (SAELSMQRRG), as well as their palmitoylated derivatives were synthesized. The physico-chemical properties of the peptides were analyzed in both the absence and presence of the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), the negative 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) and the positive 1,2-dioeloyl-3-trimethylammonium-propane (DOTAP) lipid monolayers. Based on their high hydrophilic properties, neither parent peptide presented surface activity and their incorporation into lipid monolayers was low. In contrast, their palmitoylated derivatives showed concentration-dependent surface activity and could be inserted into lipid monolayers to varying degrees depending on their sequence. Compression isotherms showed that the presence of palmitoylated peptides in the subphase resulted in a molecular arrangement less condensed than that corresponding to the pure phospholipid. In concordance with the monolayer results, differential scanning calorimetry (DSC) demonstrated that the parent peptides did not have any effect on the thermograms, while the palmitoylated derivatives affected the thermotropic properties of DPPC bilayers.     

Interactions of anthracyclines with zwitterionic phospholipid monolayers at the air-water interface

The present note describes the use of surface pressure measurements (Langmuir monolayer technique) for the analysis of interactions of two different anthracyclines (adriamycin and daunorubicin) with a non-ionic, zwitterionic phospholipid monolayer, at the air-water interface. Because the surface membrane of the cell is the first barrier encountered by the anthracyclines in the treatment of cancer, drug-membrane interactions studied in model (monolayers or bilayers) and natural systems play an important role in the understanding of the bioactivity properties of these molecules. We report here the rate constants of the adsorption process of adriamycin and daunorubicin in the presence of a zwitterionic phospholipid monolayer at the air-water interface. Because interactions with the lipid monolayer strongly depend on the molecular packing of the lipid, we investigated this process at a relatively low surface pressure (7 mN/m), the interactions being favoured by the gaseous and liquid expanded structure of the lipid monolayer. The apparent molecular area of these molecules during the insertion into the lipid film and their interactions with the phospholipid polar head groups was evaluated and the estimated percentage of anthracyclines at the interface after adsorption into the lipid monolayer is briefly discussed. The rate constants for the adsorption and desorption process at the water-monolayer interface have been calculated on the basis of a single-exponential model. The observed difference of these parameters for daunorubicin and adriamycin suggests a different interaction of these anthracyclines during the adsorption to and/or penetration across the phospholipid monolayer.     

Molecular organization in protein-lipid film on the water surface studied by x-ray standing wave measurements under total external reflection

The x-ray standing wave method has been applied to study self-assembling processes in a protein-lipid film formed by injecting the protein-lipid mixture of alkaline phosphatase and phosphatidylinositol under the phospholipid monolayer preliminarily deposited on the water subphase by Langmuir method. X-ray standing wave measurements allowed to determine the composition of the protein-lipid film and to locate ions position in the direction normal to the film surface. The presence of trace Ni contamination incorporated in the protein-lipid film from the water subphase has been established. Numerical analysis of the X-ray standing wave fluorescence data revealed that after injection under the phospholipid monolayer, the protein-lipid mixture separated in a self-assembled manner to layered structure, molecules of alkaline phosphatase arranged themselves into a pure protein layer containing no phospholipid molecules.