Interaction of electrically charged lipid monolayers with malate dehydrogenase.

Malate dehydrogenase was adsorbed onto monomolecular lipid films, using a multicompartment trough. The quantity of adsorbed protein and its enzymatic activity were studied with monolayers of various electrical charge densities and subphases of various electrolyte compositions. A closely packed layer of enzyme molecules was adsorbed onto negatively charged films, whereas considerably less protein was adsorbed onto neutral and positively charged monolayers. Electrolytes reduce the quantity of adsorbed protein. The adsorption was found to be irreversible even at high ionic strength. When adsorbed to uncharged lipid films the enzyme is nearly inactive, whereas negatively charged lipid headgroups enhance the specific activity of the enzyme.     

Actin binding to lipid-inserted alpha-actinin.

The interaction of alpha-actinin with lipid films and actin filaments was investigated. First alpha-actinin was incorporated in lipid films at the air/water interface. Injection of alpha-actinin into the subphase of a lipid monolayer led to a significant increase of the surface pressure only for lipid films consisting of a mixture of a negatively charged lipid with a high proportion of diacylglycerol. These alpha-actinin-containing films were transferred onto silanized quartz slides. Photobleaching experiments in the evanescent field allowed quantification of the lateral number density of the lipid-bound alpha-actinin. In combination with the area increase from the monolayer experiments, the photobleaching measurements suggest that alpha-actinin is incorporated into the lipid film in such a way that actin binding sites are accessible from the bulk phase. Binding experiments confirmed that the alpha-actinin selectively binds actin filaments in this configuration. We also showed that, in contrast to actin filaments which are adsorbed directly onto planar surfaces, the alpha-actinin-bound actin filaments are recognized and cleaved by the actin-severing protein gelsolin. Thus we have constructed an in vitro system which opens new ways for investigations of membrane-associated actin-binding proteins and of the physical behavior of actin filaments in the close neighborhood to membranes.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.     

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid‐rich extract and membrane models

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA‐ KAAGQAALGAL‐NH₂, DS 01) with phospholipid (PL) monolayers comprising (i) a lipid‐rich extract of Leishmania amazonensis (LRE‐La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid‐air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE‐La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE‐La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Second harmonic generation of glucose oxidase at the air/water interface

We present a study of the adsorption of the glucose oxidase enzyme (GOx) at the air/water interface, using the nonlinear optical technique of surface second harmonic generation (SSHG). Resonant SSHG experiments were achieved by probing the pi-pi* transition of the flavin adenine dinucleotide (FAD) chromophores embedded in the GOx protein. Because of the subsequent resonance enhancement of the signal, the second harmonic (SH) wave arising from the GOx entities adsorbed at the interface was detectable for protein bulk aqueous concentrations as low as 70 nM. The protein adsorption was followed, and, at high GOx coverage, a change in the orientation of the FAD chromophore was observed, indicating either a rearrangement or a reorientation of the protein at the interface. Inasmuch as GOx is negatively charged at the biological pH of 7, its interactions with charged surfactants were also investigated. As expected, spreading positively charged surfactants onto a partial protein monolayer was found to increase the GOx surface concentration, whereas in the case of negatively charged surfactants, the GOx surface concentration decreased until the SH signal went back to the pure buffer solution response level. With the increasing GOx surface concentration, the rearrangement or reorientation of the protein was also observed.     

Hybrid polar compounds produce a positive shift in the surface dipole potential of self-assembled phospholipid monolayers

Hybrid polar compounds (HPCs) are powerful inducers of terminal differentiation of various types of tumors, including Friend murine erythroleukemia cells (MELCs). They are known to act synergistically with an increase in the extracellular concentration of cations, which causes a positive shift in the negative value of the ionic surface potential. Two HPCs, hexamethylenebisacetamide (HMBA) and suberoylanilide hydroxamic acid (SAHA), were adsorbed on self-assembled phospholipid monolayers supported on a mercury drop and the shift in the surface dipole potential chi of the lipid film due to their adsorption was estimated from charge measurements. At their optimal concentrations for inducing MELC terminal differentiation (5 mM for HMBA and 2.6 microM for SAHA), these HPCs cause a chi shift of about 15-20 mV, positive toward the hydrocarbon tails, both on neutral phosphatidylcholine films and on negatively or positively charged phosphatidylserine films. This strongly suggests that the nonspecific effect of HPCs of different structure in inducing cancer cells to rescue their differentiation program is related to a positive chi shift on the extracellular side of the cell membrane.     

Polyelectrolyte-mediated adsorption of amelogenin monomers and nanospheres forming mono- or multilayers

We have applied optical waveguide lightmode spectroscopy combined with streaming potential measurements and Fourier-transformed infrared spectroscopy to investigate adsorption of amelogenin nanospheres onto polyelectrolytes. The long-term objective was to better understand the chemical nature of these assemblies and to gain further insight into the molecular mechanisms involved during self-assembly. It was found that monolayers of monomers and negatively charged nanospheres of a recombinant amelogenin (rM179) irreversibly adsorbed onto a positively charged polyelectrolyte multilayer films. On the basis of measurements performed at different temperatures, it was demonstrated that intermolecular interactions for the formation of nanospheres were not affected by their adsorption onto polyelectrolytes. Consecutive adsorption of nanospheres resulting in the formation of multilayer structures was possible by using cationic poly(l-lysine) as mediators. N-Acetyl-d-glucosamine (GlcNac) did not disturb the nanosphere-assembled protein's structure, and it only affected the adsorption of monomeric amelogenin. Infrared spectroscopy of adsorbed amelogenin revealed conformational differences between the monomeric and assembled forms of rM179. While there was a considerable amount of alpha-helices in the monomers, beta-turn and beta-sheet structures dominated the assembled proteins. Our work constitutes the first report on a structurally controlled in vitro buildup of an rM179 nanosphere monolayer-based matrix. Our data support the notion that amelogenin self-assembly is mostly driven by hydrophobic interactions and that amelogenin/PEM interactions are dominated by electrostatic forces. We suggest that similar forces can govern amelogenin interactions with non-amelogenins or the mineral phase during enamel biomineralization.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.     

Hydrolysis of phospholipid monolayers by human spermatozoa. Inhibition by male contraceptive gossypol

Monomolecular films of phospholipid were used to study the interaction of intact human spermatozoa with model membranes. Exclusively with negatively charged phosphatidylglycerol monolayers rapid penetration of spermatozoa into the monolayer with subsequent hydrolysis of the lipid was triggered by the addition of 5 mM calcium into the medium. The results suggest the localization of a calcium-dependent phospholipase A2 at the outer acrosomal or plasma membrane of human spermatozoa with its active site exposed to the external environment. Preincubation of the cells with 100 microM gossypol completely abolished the ability of human spermatozoa to hydrolyze or penetrate monolayers of phosphatidylglycerol. The inhibition of the phospholipase activity by gossypol may contribute to the unknown contraceptive mechanisms of this non-steroidal male antifertility agent.     

Phospholipid interactions of synthetic peptides representing the N-terminus of HIV gp41

Peptides representing the N-terminal 23 residues of the surface protein gp41 of LAV1a and LAVmal strains of the human immunodeficiency virus were synthesized and their interactions with phospholipid vesicles studied. The peptides are surface-active and penetrate lipid monolayers composed of negatively charged but not neutral lipids. Similarly, the peptides induce lipid mixing and solute (6-carboxyfluorescein) leakage of negatively charged, but not neutral, vesicles. Circular dichroism and infrared spectroscopy show that at low peptide:lipid ratios (approximately 1:200), the peptides bind to negatively charged vesicles as alpha-helices. At higher peptide:lipid ratios (1:30), a beta conformation is observed for the LAV1a peptide, accompanied by a large increase in light scattering. The LAVmal peptide showed less beta-structure and induced less light scattering. With neutral vesicles, only the beta conformation and a peptide:lipid ratio-dependent increase in vesicle suspension light scattering were observed for both peptides. We hypothesize that the inserted alpha-helical form causes vesicle membrane disruption whereas the surface-bound beta form induces aggregation.     

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.     

Interaction of basic polypeptides with phospholipid monolayers

Adsorption of the polylysine and of the copolypeptides: L-lysine/L-serine and L-lysine/L-phenylalanine on phospholipid monolayers has been investigated. The charge density of the monolayers was varied by using the negatively charged phosphatidyl serine and the neutral phosphatidyl choline at different ratios. The surface concentrations of the adsorbed polypeptides was determined by measuring the surface radiation of their radioactive label.The adsorbing capacity of the monolayer surfaces increases with their negative charge, however with respect to polypeptides the surface activity sequence is pL < pLS < pLφ. From the dependence of adsorption on the ionic strength it was concluded that it is controlled by three types of interaction: (1) electrostatic attraction to the negatively charged surface; (2) electrostatic repulsion between adsorbed polybases; (3) hydrophobic interactions involving specific structural arrangements. This is true even of the apparently neutral PC monolayer where the fixed phosphate groups form an electrical double layer with the more mobile choline groups which can be interpenetrated by the charged groups of the basic polypeptides.     

Interactions of thrombin with fibrinogen adsorbed on methyl-, hydroxyl-, amine-, and carboxyl-terminated self-assembled monolayers

We have examined the initial phase of fibrin formation, thrombin-catalyzed fibrinopeptide cleavage, from adsorbed fibrinogen using surface plasmon resonance and liquid chromatography-mass spectrometry. Fibrinogen adsorption impaired thrombin-fibrinogen interactions compared to the interactions of thrombin with fibrinogen in solution. The properties of the underlying substrate significantly affected the extent and kinetics of fibrinopeptide cleavage, and the conversion of adsorbed fibrinogen to fibrin. Fibrinogen adsorbed on negatively charged surfaces (carboxyl-terminated self-assembled monolayers) released a smaller amount of fibrinopeptides, at a reduced rate relative to those of hydrophobic, hydrophilic, and positively charged surfaces (methyl-, hydroxyl-, and amine-terminated self-assembled monolayers, respectively). Additionally, the conversion of adsorbed fibrinogen to fibrin was comparatively inefficient at the negatively charged surface. These data correlated well with trends previously reported for fibrin proliferation as a function of surface properties. We conclude that thrombin interactions with adsorbed fibrinogen determine the extent of subsequent fibrin proliferation on surfaces.     

Staphylococcus aureusα-Toxin: Characterization of Protein/Lipid Interactions, 2D Crystallization on Lipid Monolayers, and 3D Structure

Staphylococcus aureusα-toxin was characterized with respect to surface activity and its interaction with lipid monolayers. The protein alone had a detergent-like behavior at the air/water interface. Its affinity was higher for negatively charged than for neutral phospholipids. The interaction was pH dependent, showing a maximum increase at pH 7.0. Only a small part of the protein oligomer appeared to be inserted into the monolayers. Crystalline sheets of α-toxin were formed using negatively charged phospholipids. Electron microscopy of such areas, at different tilt angles, allowed reconstruction of a three-dimensional model following image processing. The sheets analyzed consisted of two protein layers arranged on a tetragonal lattice. Under the conditions used to grow the crystals the toxin formed 90-Å-wide cylinders with a height of 70 Å. One of the imposed fourfold axes running perpendicular to the plane of the crystalline layer is positioned at a protein-deficient region which forms a 25-Å-wide pore through the oligomer.     

Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

We monitored the action of phospholipase A₂ (PLA₂) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA₂ molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA₂. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm²/s on the L-DPPC and 4.9 μm²/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm²/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.     

A new kinetic approach for studying phospholipase C (Clostridium perfringens alpha toxin) activity on phospholipid monolayers

The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate. It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized. No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates. By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role. The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate.     

Adsorption of cytochrome c to phospholipid monolayers studied by reflection spectroscopy

The uptake of cytochrome c by charged and neutral lipid monolayers was studied by using reflection spectroscopy. The method was shown to be a very sensitive and useful technique in studies of lipid-protein interactions. It was found that cytochrome c is preferentially bound to monolayers of negatively charged monolayers in the solid phase. Polarized light under oblique incidence was used to determine the average orientation of chromophores in cytochrome c bound to lipid monolayer. The transition moments of chromophore are oriented parallel to the monolayer plane.     

Interactions of colicin A domains with phospholipid monolayers and liposomes: relevance to the mechanism of action

The colicin A polypeptide chain (592 amino acid residues) contains three domains which are linearly organized and participate in the sequential steps involved in colicin action. We have compared the penetrating ability in phospholipid monolayers and the ability to promote vesicle fusion at acidic pH of colicin A and of protein derivatives containing various combinations of its domains. The NH2-terminal domain (171 amino acid residues), required for translocation across the outer membrane, has little affinity for dilauroylphosphatidylglycerol (DLPG) monolayers at all pHs tested. The central domain has a pH-dependent affinity, although lower than that of the entire colicin A. The COOH-terminal domain contains a high-affinity lipid binding site, but in addition an electrostatic interaction is required as a first step in the process of penetration into negatively charged DLPG films. In contrast to the constructs containing the ionophoric domain, the NH2-terminal domain alone has no fusogenic activity for liposomes. These results are discussed with regard to the mechanism of entry and action of colicin A in sensitive cells. Our results suggest the existence of a pH-dependent interaction between the receptor binding domain (amino acid residues 172-388) and the pore-forming domain of colicin A (amino acid residues 389-592).