Miscibility of an acylated hepatitis A synthetic antigen derivative [palmitoyl VP3(110-121)] with lipids: a monolayer study

Mixed monolayers of an acylated derivative of hepatitis A synthetic peptide VP3(110-121) with neutral, cationic or anionic lipids were spread at the air/water interface. Deviations from ideality as well as thermodynamic values were calculated at different surface pressures using the free-excess energy, the interaction parameter and the enthalpy. The miscibility at the collapse point was also checked. Maximum deviations from ideality were found for mixtures containing the anionic lipid phosphatidylglycerol (PG), and it seems that the monolayer composition is not stable through compression, as the peptide is ejected from the film. Films containing neutral [dipalmitoylphosphatidylcholine (DPPC)] or cationic [stearylamine (SA)] lipids showed more regular behaviour. As the peptide has a net negative charge it is probable that electrostatic interactions are in part responsible of the good miscibility of palmitoyl VP3(110-121) with SA. In order to prepare liposomes containing palmitoyl VP3(110–121), lipids such as SA or DPPC/SA will be a more suitable choice than anionic lipids such as PG. Received: 26 May 2000 Accepted: 22 September 2000     

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.     

Effects of lipid chain length on molecular interactions between paclitaxel and phospholipid within model biomembranes

Molecular interactions between an anticancer drug, paclitaxel, and phosphatidylcholine (PC) of various chain lengths were investigated in the present work by the Langmuir film balance technique and differential scanning calorimetry (DSC). Both the lipid monolayer at the air-water interface and lipid bilayer vesicles (liposomes) were employed as model biological cell membranes. Measurement and analysis of the surface pressure versus molecular area curves of the mixed monolayers of phospholipids and paclitaxel under various molar ratio showed that phospholipids and paclitaxel formed a nonideal miscible system at the interface. Paclitaxel exerted an area-condensing effect on the lipid monolayer at small molecular surface areas and an area-expanding effect at large molecular areas, which could be explained by the intermolecular forces and geometric accommodation between the two components. Paclitaxel and phospholipids could form thermodynamically stable monolayer systems: the stability increased with the chain length in the order DMPC (C14:0)>DPPC (C16:0)>DSPC (C18:0). Investigation of paclitaxel penetration into the pure lipid monolayer showed that DMPC had a higher ability to incorporate paclitaxel and the critical surface pressure for paclitaxel penetration also increased with the chain length in the order DMPC>DPPC>DSPC. A similar trend was testified by DSC studies on vesicles of the mixed paclitaxel/phospholipids bilayer. Paclitaxel showed the greatest interaction with DMPC while little interaction could be measured in the paclitaxel/DSPC liposomes. Paclitaxel caused broadening of the main phase transition without significant change at the peak melting temperature of the phospholipid bilayers, which demonstrated that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer. The interaction between paclitaxel and phospholipid was nonspecific and the dominant factor in this interaction was the van der Waals force or hydrophobic force. As the result of the lower net van der Waals interaction between hydrocarbon chains for the shorter acyl chains, paclitaxel interacted more readily with phospholipids of shorter chain length, which also increased the bilayer intermolecular spacing.     

Interactions between phosphatidylcholines and cholesterol in monolayers at the air/water interface

Mixtures of cholesterol and synthetic phospholipids, differing in saturation of phosphatidylcholine (PC) acyl chains, such as distearoyl phosphatidylcholine (DSPC), stearoyl-oleoyl phosphatidylcholine (SOPC) and dioleoyl phosphatidylcholine (DOPC) have been studied as floating Langmuir monolayers at the air/water interface. In order to examine the influence of a polar group, distearoyl phosphatidylethanolamine (DSPE) was chosen. The films were spread at room temperature on aqueous subphases and characterized by the surface pressure-area (pi-A) isotherms and compression modulus (C(s)(-1)) values. The interactions were examined by analyzing the mean molecular areas and quantified by the excess free energy of mixing values. The obtained results indicate that the affinity of cholesterol to saturated/unsaturated phosphatidylcholines does not differ significantly, and revealed strong influence of the kind of a polar group on the cholesterol-phospholipid interactions. On the other hand, the apolar group structure was found to modify the stoichiometry of sterol-PC complexes.     

Interactions between nonpolar surfaces coated with the nonionic surfactant hexaoxyethylene dodecyl ether C12E6 and the origin of surface charges at the air/water interface

The interactions between nonpolar surfaces coated with the nonionic surfactant hexaoxyethylene dodecyl ether C12E6 were investigated using two techniques and three different types of surfaces. As nonpolar surfaces, the air/water interface, silanated negatively charged glass, and thiolated uncharged gold surfaces were chosen. The interactions between the air/water interfaces were measured with a thin film pressure balance in terms of disjoining pressure as a function of film thickness. The interactions between the solid/liquid interfaces were determined using a bimorph surface force apparatus. The influence of the nature of the surface on the interaction forces was investigated at surfactant concentrations below and above the cmc. The adsorption of the nonionic surfactant on the uncharged thiolated surface does not, as expected, lead to any buildup of a surface charge. On the other hand, adsorption of C12E6 on the charged silanated glass and the charged air/water interface results in a lowering of the surface charge density. The reduction of the surface charge density on the silanated glass surfaces is rationalized by changes in the dielectric permittivity around the charged silanol groups. The reason for the surface charge observed at the air/water interface as well as its decrease with increasing surfactant concentration is discussed and a new mechanism for generation of OH- ions at this particular interface is proposed.     

Protein-lipid interactions at the air/water interface

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.     

Interaction of a laminin derived peptide with phosphatidyl choline/phosphatidyl glycerol

The physicochemical interactions between an active peptide sequence derived from laminin and phospholipids have been studied. The main aim is to determine the suitability of this peptide fragment to be linked to liposome's with the purpose to develop targeting devices. Results indicate that this peptide is able to insert in lipid monolayers and also to form monomolecular layers at an air/water interface. Besides, miscibility studies carried out through compression isotherms of mixed monolayers [dipalmitoyl phosphatidylcholine (DPPC)/peptide], indicate a strong interaction at 60-80% DPPC molar composition. Studies carried out with lipid bilayers indicate that the interaction is restricted to the external face of the vesicles. Moreover, the presence of this peptide in the incubation media promotes a low level of carboxyfluorescein (CF) leakage and no fusion of vesicles. These results indicate that the association of this sequence to vesicles do not produce damage of the bilayer and can be used as potential targeting vector.     

Ordering effects of cholesterol on sphingomyelin monolayers investigated by high-resolution broadband sum-frequency generation vibrational spectroscopy

After adding cholesterol, the sphingosine backbones (red) of the three nature SMs become more ordered, and the N-linked acyl chain (blue) remains unaltered.     

Self-assembly and lipid interactions of diacylglycerol lactone derivatives studied at the air/water interface

Synthetic diacylglycerol lactones (DAG-lactones) have been shown to be effective modulators of critical cellular signaling pathways. The biological activity of these amphiphilic molecules depends in part upon their lipid interactions within the cellular plasma membrane. This study explores the thermodynamic and structural features of DAG-lactone derivatives and their lipid interactions at the air/water interface. Surface-pressure/area isotherms and Brewster angle microscopy revealed the significance of specific side-groups attached to the terminus of a very rigid 4-(2-phenylethynyl)benzoyl chain of the DAG-lactones, which affected both the self-assembly of the molecules and their interactions with phospholipids. The experimental data highlight the formation of different phases within mixed DAG-lactone/phospholipid monolayers and underscore the relationship between the two components in binary mixtures of different mole ratios. Importantly, the results suggest that DAG-lactones are predominantly incorporated within fluid phospholipid phases rather than in the condensed phases that form, for example, by cholesterol. Moreover, the size and charge of the phospholipid headgroups do not seem to affect DAG-lactone interactions with lipids.     

Negatively charged phospholipids trigger the interaction of a bacterial Tat substrate precursor protein with lipid monolayers

Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection-absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron-sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate-membrane interactions.     

Mesophases in monolayers of fatty acids and phospholipids

Langmuir monolayers at the air/water interface are distinguished by their richness of phases. These can be assigned to smectic categories, although the dominant competing interactions are not yet well elaborated. For single chain compounds, there are van der Waals attractions of the chains and repulsive forces of the head groups competing in determining the local structure. For larger molecules like phospholipids there appear also to be important dipolar interactions of the head groups. In order to study these interactions, we report surface X-ray diffraction studies of films of fatty acids and phospholipids at the air/water interface, with varying environmental parameters, as well as head group size and shape. We observe a wealth of mesophases and also the influence of molecular chirality on the structure. The defined control of the monolayer structure can be made use of to control crystal formation by diffusion from the subphase and nucleation at the interface. This is studied spectroscopically, microscopically and via electron diffraction of transferred films of cyanine dyes. In that case one observes epitaxy between the dye crystal and surfactant layer, and the relative lattices can be varied via suitable dye mixtures. A very peculiar interfacial influence results from the orientation of molecular dipoles at the interface. This causes long-range repulsive interactions leading to periodic superlattices in two phase coexistence regions. These forces are observed and quantified via fluorescence microscopy.     

Thermodynamic and infrared analyses of the interaction of chlorpromazine with phospholipid monolayers

An investigation has been made of the interaction between chlorpromazine (CPZ) and monolayers of 1,2-dipalmitoyl-sn-3-glycerophosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-3-glycero[phospho-rac-(1-glycerol)] (DPPG), both at the air/water interface and in transferred Langmuir-Blodgett films. The Gibbs free energy, DeltaG, and the compressibility modulus (C(S)(-1)), obtained from the surface pressure isotherms, indicated changes in the in-plane interactions of CPZ/DPPG mixed monolayers, with positive values of DeltaG. The arrangement of CPZ in the zwitterionic DPPC monolayers causes a weaker interaction in CPZ/DPPC mixed monolayers, with the DeltaG fluctuating around zero. IR measurements in transferred monolayers showed that CPZ did not affect the conformational order of the acyl chains, its effects being limited to the bands corresponding to the headgroups. Furthermore, since no shift was observed for the acyl chain bands, the phase transition induced by CPZ is not a liquid expanded (LE) to liquid condensed (LC) transition, as the latter is associated with chain ordering. Taken together, the IR and compressibility results demonstrate that the effect from CPZ cannot be correlated with temperature changes in the subphase for pure monolayers, in contrast to models proposed by other authors.     

The study of the interaction of a model alpha-helical peptide with lipid bilayers and monolayers

We studied the interaction of the alpha-helical peptide acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)) with tethered bilayer lipid membranes (tBLM) and lipid monolayers formed at an air-water interface. The interaction of L(24) with tBLM resulted in adsorption of the peptide to the surface of the bilayer, characterized by a binding constant K(c)=2.4+/-0.6 microM(-1). The peptide L(24) an induced decrease of the elasticity modulus of the tBLM in a direction perpendicular to the membrane surface, E(radial). The decrease of E(radial) with increasing peptide concentration can be connected with a disordering effect of the peptide to the tBLM structure. The pure peptide formed a stable monolayer at the air/water interface. The pressure-area isotherms were characterized by a transition of the peptide monolayer, which probably corresponds of the partial intercalation of the alpha-helixes at higher surface pressure. Interaction of the peptide molecules with lipid monolayers resulted in an increase of the mean molecular area of phospholipids both in the gel and liquid crystalline states. With increasing peptide concentration, the temperature of the phase transition of the monolayer shifted toward lower temperatures. The analysis showed that the peptide-lipid monolayer is not an ideally miscible system and that the peptide molecules form aggregates in the monolayer.     

Quantum chemical study of carbohydrate-phospholipid interactions

Carbohydrates on host membranes are fundamental to many important biological processes. Here, we seek a basic understanding of the nature of the interactions between carbohydrates and phospholipids to dissect their roles in molecular recognition. A hybrid quantum mechanics/quantum mechanics (QM/QM) scheme with two different levels of treatment was used to explore the conformations and energetics of carbohydrate-phospholipid complexes. We investigate the interactions of two phospholipids (POPC and DOPC) with mannose using density functional theory. Carbohydrate-phospholipid interactions are probed with respect to competing interactions with water. Our hybrid QM/QM approach demonstrates that mannose interactions with phospholipids can result in alterations in charge distributions and conformations of phospholipids. The results clearly reveal the interplay between conventional and nonconventional hydrogen bonding; moreover, nonpolar interactions are shown to be crucial in the recognition and further stabilization of carbohydrate-phospholipid complexes. The influence of the acyl chain on phospholipid headgroup orientation is clearly evident in our investigation. The significance of the conventional OH···O and nonconventional CH···O and CH···C interactions in the stabilization of the intermolecular complexes is deduced from the molecular electron density topology using Bader's atoms-in-molecules theory. Finally, we have compared the QM energies with molecular mechanics energies for the same interactions to aid in the refinement of the all-atom lipid-carbohydrate force fields.     

Synthesis of caprazamycin analogues and their structure--activity relationship for antibacterial activity

Synthesis of palmitoyl caprazol 7, which possesses a simple fatty acyl side chain at the 3' '-position of the diazepanone moiety, was carried out and their antibacterial activity was evaluated. The key elements of our approach include the improved synthesis of the key 5'-beta-O-aminoribosyl-glycyluridine derivative, installation of the palmitoyl side chain to the cyclization precursor, and the construction of the diazepanone by an intramolecular reductive amination. The second generation synthesis of (+)-caprazol was also established. Palmitoyl caprazol 7 exhibited antibacterial activity against Mycobacterium smegmatis ATCC607 (MIC = 6.25 microg/mL) with potency similar to that of the caprazamycins (CPZs). Palmitoyl caprazol 7 and N6'-desmethyl palmitoyl caprazol 28 also exhibited antibacterial activity against drug-resistant bacteria including methyciline-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) strains (MIC = 3.13-12.5 microg/mL).     

Interaction of α-Synuclein with Phospholipids and the Associated Restructuring of Interfacial Lipid Water: An Interface-Selective Vibrational Spectroscopic Study

Interaction of α-Synuclein (αS) with biological lipids is crucial for the onset of its fibrillation at the cell membrane/water interface. Probed herein is the interaction of αS with membrane-mimicking lipid monolayer/water interfaces. The results depict that αS interacts negligibly with zwitterionic lipids, but strongly affects the pristine air/water and charged lipid/water interfaces by perturbing the structure and orientation of the interfacial water. The net negative αS (−9 in bulk water; pH 7.4) reorients the water as hydrogen-up (H-up) at the air/water interface, and electrostatically interacts with positively charged lipids, making the interface nearly net neutral. αS also interacts with negatively charged lipids: the net H-up orientation of the interfacial water decreases at the anionic lipid/water interface, revealing a domain-specific interaction of net negative αS with the negatively charged lipids at the membrane surface.     

Interaction of lipophilic gemcitabine prodrugs with biomembrane models studied by Langmuir-Blodgett technique

The stability and bioavailability of anticancer agents, such as gemcitabine, can be increased by forming prodrugs. Gemcitabine is rapidly deaminated to the inactive metabolite (2('),2(')-difluorodeoxyuridine), thus to improve its stability a series of increasingly lipophilic gemcitabine prodrugs linked through the 4-amino group to valeroyl, lauroyl, and stearoyl acyl chains were synthesized. Studies of monolayer properties are important to improve understanding of biological phenomena involving lipid/gemcitabine or lipid/gemcitabine derivative interactions. The interfacial behavior of monolayers constituted by DMPC plus gemcitabine or lipophilic gemcitabine prodrugs at increasing molar fractions was studied at the air/water interface at temperatures below (10 degrees C) and above (37 degrees C) the lipid phase transition. The effect of the hydrophobic chain length of gemcitabine derivatives on the isotherm of pure DMPC was investigated by surface tension measurement, and the results are reported as molar fractions as a function of mean molecular area per molecule. The results show that the compounds interact with DMPC producing mixed monolayers that are subject to an expansion effect, depending on the prodrug chain length. The results give useful hints of the interaction of these prodrugs with biological membranes and increase knowledge on the incorporation site of such compounds, as a function of their lipophilicity, in a lipid carrier; they may lead to improved liposomal formulation design.     

Local high-density distributions of phospholipids induced by the nucleation and growth of smectic liquid crystals at the interface

Amphiphilic molecules adsorbed at the interface could control the orientation of liquid crystals (LCs) while LCs in turn could influence the distributions of amphiphilic molecules. The studies on the interactions between liquid crystals and amphiphilic molecules at the interface are important for the development of molecular sensors. In this paper, we demonstrate that the development of smectic LC ordering from isotropic at the LC/water interface could induce local high-density distributions of amphiphilic phospholipids. Mixtures of liquid crystals and phospholipids in chloroform are first emulsified in water. By fluorescently labeling the phospholipids adsorbed at the interface, their distributions are visualized under fluorescent confocal microscope. Interestingly, local high-density distributions of phospholipids showing a high fluorescent intensity are observed on the surface of LC droplets. Investigations on the correlation between phospholipid density, surface tension and smectic LC ordering suggest that when domains of smectic LC layers nucleate and grow from isotropic at the LC/water interface as chloroform slowly evaporates at room temperature, phospholipids transition from liquid-expanded to liquid-condensed phases in response to the smectic ordering, which induces a higher surface tension at the interface. The results will provide an important insight into the interactions between liquid crystals and amphiphilic molecules at the interface.     

Binding of chitosan to phospholipid vesicles studied with isothermal titration calorimetry

We thermodynamically characterize the interaction of chitosan with small liposomes and the binding and organization of the polysaccharide on the membrane of the vesicles. By means of isothermal titration calorimetry (ITC), we obtain the enthalpy variations arising from binding of the positively ionized chitosan to neutral and negatively charged liposomes. The strong electrostatic interaction of the polysaccharide with the negative charges at the membrane gives rise to highly exothermic signal until charge compensation is reached. The equilibrium constant, the interaction stoichiometry, and the molar enthalpy of binding chitosan monomers to phospholipids from the external leaflet of the vesicle membrane are obtained from the isotherm curve fitting assuming independent binding sites. The strong exothermic signal indicates that the electrostatically driven binding of chitosan to the membrane is energetically favored, leading to further stabilization of the vesicle suspension. The higher the net negative charge of the vesicles, the more pronounced the adsorption of chitosan is, leading to weaker chain organization of the adsorbed chitosan at the membrane. At the point of charge saturation, vesicle aggregation takes place and we show that this behavior does not always lead to charge reversal at the membrane. Models for the binding behavior and structural organization of chitosan are proposed based on the experimental results from ITC, ζ-potential, and dynamic light scattering.     

Effect of the polar headgroup of phospholipids on their interaction with actin

It is generally admitted that actin filaments are anchored to a membrane by membranar actin-binding-proteins. However, we found that actin may also interact directly with membrane phospholipids. The actin-phospholipid complex has been investigated at the air-water interface using a film balance technique. In order to probe the effect of the phospholipid headgroup on the actin-phospholipid interaction, we focus mainly on phospholipids that have the same acyl chain length but different headgroups. For all the phospholipids, the apparent area per molecule (the total surface divided by the number of lipid molecules) increases after the injection of the protein into the subphase, which suggests an intercalation of actin between the phospholipid molecules. This effect seems to be more important for DMPE and DMPS than for DMPG, suggesting that the headgroup plays an important role in this intercalation. The critical surface pressure associated to the liquid expanded-liquid condensed (LE-LC) phospholipid transition increases with the concentration of G-actin and thus suggests that G-actin acts as an impurity, simply competing as a surfactant at the air-water interface. On the other hand, F-actin affects the LE to LC transition of phospholipids differently. In this case, the LE to LC transition is broader and F-actin slightly decreases the critical surface pressure, which suggests that electrostatic interactions are involved.