Anionic Phospholipid Monolayers Research Articles

Structural Reorganization of Cell Membrane Models Caused by the Anticancer Antibiotic Doxorubicin

The molecular mechanisms of the interaction of anticancer antibiotic doxorubicin with lipid cell membrane models have been investigated using grazing incidence X-ray diffraction (XRD) and X-ray reflectivity (XRR). The model systems were monolayers of four types of phospholipids, related to the main components of animal cell membranes. New information on the processes of damage of phospholipid monolayer lattice caused by doxorubicin is obtained. It is established that the action of doxorubicin on anionic phospholipid monolayers is determined by the electrostatic interaction: positively charged doxorubicin molecules are incorporated between negatively charged phospholipid functional groups. In the case of neutral phospholipids the key role belongs to the hydrophobic interaction: doxorubicin molecules are coordinated with phospholipid hydrocarbon tails in disordered regions.

Interaction of the Antimicrobial Peptides Rhesus θ-Defensin and Porcine Protegrin-1 with Anionic Phospholipid Monolayers.

A combination of Langmuir isotherm, Brewster angle microscopy (BAM), and neutron reflectivity studies have been performed to gain insight into the effects on model bacterial cell membranes of the antimicrobial peptides, Rhesus θ-defensin 1 (RTD-1), and porcine protegrin 1 (PG-1). The peptides were interacted with monolayers spread at the air-water interface and prepared from a 3:1 molar mixture of phosphatidylethanolamine and phosphatidylglycerol used to approximate the cell membranes of Gram positive bacteria. The Langmuir film balance measurements show that both peptides perturb the lipid monolayers causing an increase in surface pressure, and the BAM studies show that each results in the formation of small domains within the lipid films, around 5 μm diameter. The overall change in monolayer surface pressure caused by PG-1, however, is a little more pronounced than that due to RTD-1 (+8.5 mN·m(-1) vs +5.5 mN·m(-1)), and the rate of its initial interaction with the monolayer is a little more rapid than that for RTD-1. The neutron reflectivity studies also show differences for PG-1 and RTD-1, with the model fits to these data showing that the more amphiphilic PG-1 becomes fully embedded within the lipid film-causing an extension of the lipid acyl chains but leaving the thickness of the lipid headgroup layer unaffected-while RTD-1 is seen to insert less deeply-causing the same extension of the lipid acyl chains as PG-1 but also causing a significant increase in thickness of the lipid headgroup layer. The various differing effects of the two peptides on anionic lipid monolayers are discussed in the context of their differing hemolytic activities, and their proposed differing propensities to form transmembrane pores.

Effect of lipid headgroup charge and pH on the stability and membrane insertion potential of calcium condensed gene complexes.

Noncovalently condensed complexes of genetic material, cell penetrating peptides (CPPs), and calcium chloride present a nonviral route to improve transfection efficiency of nucleic acids (e.g., pDNA and siRNA). However, the exact mechanisms of membrane insertion and delivery of macromolecule complexes to intracellular locations as well as their stability in the intracellular environment are not understood. We show that calcium condensed gene complexes containing different hydrophilic (i.e., dTAT, K9, R9, and RH9) and amphiphilic (i.e., RA9, RL9, and RW9) CPPs formed stable cationic complexes of hydrodynamic radii 100 nm at neutral pH. However, increasing the acidity caused the complexes to become neutral or anionic and increase in size. Using zwitterionic and anionic phospholipid monolayers as models that mimic the membrane composition of the outer leaflet of cell membranes and intracellular vesicles and pHs that mimic the intracellular environment, we study the membrane insertion potential of these seven gene complexes (CPP/pDNA/Ca(2+) complexes) into model membranes. At neutral pH, all gene complexes demonstrated the highest insertion potential into anionic phospholipid membranes, with complexes containing amphiphilic peptides showing the maximum insertion. However, at acidic pH, the gene complexes demonstrated maximum monolayer insertion into zwitterionic lipids, irrespective of the chemical composition of the CPP in the complexes. Our results suggest that in the neutral environment the complexes are unable to penetrate the zwitterionic lipid membranes but can penetrate through the anionic lipid membranes. However, the acidic pH mimicking the local environment in the late endosomes leads to a significant increase in adsorption of the complexes to zwitterionic lipid headgroups and decreases for anionic headgroups. These membrane-gene complex interactions may be responsible for the ability of the complexes to efficiently enter the intracellular environment through endocytosis and escape from the endosomes to effectively deliver their genetic payload.

Membrane Insertion Potential of Synthetic Cell Penetrating Peptides

Cell penetrating peptides (CPPs) have been established as excellent candidates for mediating drug delivery into cells. When designing synthetic CPPs for drug delivery applications, it is important to understand their ability to penetrate the cell membrane. In this paper, anionic or zwitterionic phospholipid monolayers at the air-water interface are used as model cell membranes to monitor the membrane insertion potential of synthetic CPPs. The insertion potential of CPPs having different cationic and hydrophobic amino acids were recorded using a Langmuir monolayer approach that records peptide adsorption to model membranes. Fluorescence microscopy was used to visualize alterations in phospholipid packing due to peptide insertion. All CPPs had the highest penetration potential in the presence of anionic phospholipids. In addition, two of three amphiphilic CPPs inserted into zwitterionic phospholipids, but none of the hydrophilic CPPs did. All the CPPs studied induced disruptions in phospholipid packing and domain morphology, which were most pronounced for amphiphilic CPPs. Overall, small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Designers of synthetic CPPs for efficient intracellular drug delivery should consider small nuances in CPP electrostatic and hydrophobic properties.

Dynamic measurements of membrane insertion potential of synthetic cell penetrating peptides.

Cell penetrating peptides (CPPs) have been established as excellent candidates for mediating drug delivery into cells. When designing synthetic CPPs for drug delivery applications, it is important to understand their ability to penetrate the cell membrane. In this paper, anionic or zwitterionic phospholipid monolayers at the air-water interface are used as model cell membranes to monitor the membrane insertion potential of synthetic CPPs. The insertion potential of CPPs having different cationic and hydrophobic amino acids were recorded using a Langmuir monolayer approach that records peptide adsorption to model membranes. Fluorescence microscopy was used to visualize alterations in phospholipid packing due to peptide insertion. All CPPs had the highest penetration potential in the presence of anionic phospholipids. In addition, two of three amphiphilic CPPs inserted into zwitterionic phospholipids, but none of the hydrophilic CPPs did. All the CPPs studied induced disruptions in phospholipid packing and domain morphology, which were most pronounced for amphiphilic CPPs. Overall, small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Designers of synthetic CPPs for efficient intracellular drug delivery should consider small nuances in CPP electrostatic and hydrophobic properties.

The role of protein hydrophobicity in thionin–phospholipid interactions: a comparison of α1 and α2-purothionin adsorbed anionic phospholipid monolayers

The plant defence proteins α1- and α2-purothionin (Pth) are type 1 thionins from common wheat (Triticum aestivum). These highly homologous proteins possess characteristics common amongst antimicrobial peptides and proteins, that is, cationic charge, amphiphilicity and hydrophobicity. Both α1- and α2-Pth possess the same net charge, but differ in relative hydrophobicity as determined by C18 reversed phase HPLC. Brewster angle microscopy, X-ray and neutron reflectometry, external reflection FTIR and associated surface pressure measurements demonstrated that α1 and α2-Pth interact strongly with condensed phase 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) monolayers at the air/liquid interface. Both thionins disrupted the in-plane structure of the anionic phospholipid monolayers, removing lipid during this process and both penetrated the lipid monolayer in addition to adsorbing as a single protein layer to the lipid head-group. However, analysis of the interfacial structures revealed that the α2-Pth showed faster disruption of the lipid film and removed more phospholipid (12%) from the interface than α1-Pth. Correlating the protein properties and lipid binding activity suggests that hydrophobicity plays a key role in the membrane lipid removal activity of thionins.

Interaction Het-S Prion / Membrane to get a Better Insight of Amyloid Toxicity

Many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, spongiform encephalopathies or amyloidopathies, correlate with amyloid formation. Several hypotheses have been proposed to explain the toxicity of amyloids: the existence of intermediates, from oligomers to protofilaments and/or the ability of amyloids to form pores which would destabilize the membranes but also change the cellular homeostasis.Our work aims to characterize the interaction amyloid/membrane in relation with the amyloid toxicity. We have chosen the non toxic Prion Forming Domain 218-289 of Het-s from the filamentous fungi Podospora anserina, as model. By random PCR mutagenesis we generated a toxic mutant in yeast (M8) that interacts in vivo with membrane vesicles. We have demonstrated that in vitro, M8 forms very unusual short amyloid fibers contrary to WT, which polymerizes as long fibers. Furthermore, ATR spectra have shown that M8 toxic mutant is essentially assembled into mixed parallel and anti-parallel β-sheets whereas WT displays a predominant parallel organization. Monolayers of phospholipids at the air-water interface were used as membrane model to investigate the interaction with non toxic amyloid (WT) and toxic amyloid (M8). Characterization of the molecular interactions between amyloid (WT and M8) and different lipid monolayers (DOPG, DOPC, DOPS DOPE and DOPI), at the air-water interface were performed by polarization-modulated infrared reflection absorption spectroscopy, Brewster angle microscopy and ellipsometry. BAM images reveal a high selectivity of the M8 toxic mutant for the anionic phospholipid monolayers (DOPG, DOPI and DOPS). In comparison, WT presents less evidences of interaction with the tested phospholipid monolayers. The secondary structure of amyloids (M8 or WT) interacting with a phospholipid monolayer is mainly made of anti-parallel β-sheets (observed by PMIRRAS) but the orientation of the β-sheet differs between toxic and non toxic mutant.

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers

Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction.

Adsorption of Amyloid β (1-40) Peptide at Liquid Interfaces

The folding of amyloid β (1-40) peptide into β-sheet containing fibrils, which are the main components of deposits and plaques in some neurodegenerative diseases, is thought to play a causative role in Alzheimer's disease. The peptide is amphiphilic and therefore surface active. Interactions with surfaces can play an important role in the secondary structure changes. Langmuir monolayers of zwitterionic (DMPC, DPPC, DMPE, DPPE) as well ionic (DMPG, DPPG, DPTAP, DSTAP) phospholipids have been used to study the influence of the peptide on the lipid packing and, vice versa, the influence of phospholipid monolayers on the peptide secondary structure by infrared reflection absorption spectroscopy and grazing incidence X-ray diffraction. The peptide adsorbs at the air/water (buffer) interface and penetrates into uncompressed phospholipid monolayers. After a special pre-treatment, the peptide forms predominantly a random-coil secondary structure in solution but adopts a β-sheet conformation, which is almost parallel oriented to the surface in the adsorbed state. The peptide does not influence the condensed phospholipid monolayer structure. In contrast to Aβ insertion into zwitterionic or anionic phospholipid monolayers, a non β-sheet structure was detected during the first stage of Aβ insertion into positively charged monolayers. At high lateral pressure, the peptide is squeezed out of the monolayer. It desorbs completely from zwitterionic monolayers and charged monolayers on buffer, but remains adsorbed in β-sheet conformation at charged monolayers on water. In contrast, Aβ remains in a fluid (disordered) and charged monolayer on buffer even above 30 mN m-1. This observation shows that a combination of hydrophobic and electrostatic interactions is necessary to keep the peptide in the adsorbed state at high pressure on buffer.

Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films

Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is inserted and is therefore a candidate motif to participate in interactions with other bilayers or monolayers. In the present work, we have detected intrinsic ability of a peptide based on the sequence of the N-terminal segment of SP-C to interact and insert spontaneously into preformed zwitterionic or anionic phospholipid monolayers. The peptide expands the π–A compression isotherms of interfacial phospholipid/peptide films, and perturbs the lipid packing of phospholipid films during compression-driven liquid-expanded to liquid-condensed lateral transitions, as observed by epifluorescence microscopy. These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been related with the ability of SP-C to facilitate reinsertion of surface active lipid molecules into the lung interface during respiratory compression–expansion cycling.

Aerobic sn-glycerol-3-phosphate dehydrogenase from Escherichia coli binds to the cytoplasmic membrane through an amphipathic alpha-helix.

sn-Glycerol-3-phosphate dehydrogenase (GlpD) from Escherichia coli is a peripheral membrane enzyme involved in respiratory electron transfer. For it to display its enzymic activity, binding to the inner membrane is required. The way the enzyme interacts with the membrane and how this controls activity has not been elucidated. In the present study we provide evidence for direct protein-lipid interaction. Using the monolayer technique, we observed insertion of GlpD into lipid monolayers with a clear preference for anionic phospholipids. GlpD variants with point mutations in their predicted amphipathic helices showed a decreased ability to penetrate anionic phospholipid monolayers. From these data we propose that membrane binding of GlpD occurs by insertion of an amphipathic helix into the acyl-chain region of lipids mediated by negatively charged phospholipids.

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC). In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site. In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.

Simulations of Zwitterionic and Anionic Phospholipid Monolayers

Results of atomistic molecular dynamics simulations of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol monolayers at the air/water interface are presented. Dipalmitoylphosphatidylcholine is zwitterionic and dipalmitoylphosphatidylglycerol is anionic at physiological pH. NaCl and CaCl 2 water subphases are simulated. The simulations are carried out at different surface densities, and a simulation cell geometry is chosen that greatly facilitates the investigation of phospholipid monolayer properties. Ensemble average monolayer properties calculated from simulation are in agreement with experimental measurements. The dependence of the properties of the monolayers on the surface density, the type of the headgroup, and the ionic environment are explained in terms of atomistically detailed pair distribution functions and electron density profiles, demonstrating the strength of simulations in investigating complex, multicomponent systems of biological importance.

Phase separation of phospholipid monolayers induced by membrane penetration of human apolipoprotein H

In this paper we studied the interaction of apolipoprotein H (ApoH), an autoantigen or cofactor in antiphospholipid syndrome, with phospholipid monolayers by means of fluorescence microscopy and surface film balance. Observations with fluorescence microscopy indicate that: (1) ApoH can penetrate into both anionic and neutral phospholipid monolayers; (2) the membrane penetration of ApoH may induce phase separation of the phospholipid monolayers; and (3) ApoH prefers to penetrate into the region of the phospholipid monolayer that is in fluid state.

Penetration of human apolipoprotein H into air/water interface with and without phospholipid monolayers

Apolipoprotein H (β 2-glycoprotein I) is a 54 kDa plasma glycoprotein. The interaction of ApoH with phospholipids has been demonstrated to be involved in the regulation of the function of the activated platelets and the clearance of phosphotidylserine (PS)-expressing cells. In the present work, the monolayer technique is employed to characterize the feature of the penetration of ApoH into air/water interface with and without phospholipid monolayers. The results indicate that ApoH can penetrate into the air/water interface and there is a protein-concentration dependent lag time in the adsorption isotherm. ApoH can insert into the phospholipid monolayers spread at the air/water interface, but no lag time is observed in this case. The fact that ApoH prefers to interact with anionic phospholipid monolayers suggests that the negative charges of the phospholipid monolayers may play an important role in the process of the membrane insertion of ApoH.

Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers

Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescence microscopy. Mass spectometry of F-SP-C indicated that the protein is partially deacylated and labeled with 1 mol fluorescein/1 mol protein. The protein partitioned into the fluid, or liquid expanded, phase. Increasing amounts of F-SP-C in DPPC or DPPC:DPPG monolayers decreased the size and total amounts of the condensed phase at all surface pressures. Calcium (1.6 mM) increased the amount of the condensed phase in monolayers of DPPC:DPPG but not of DPPC alone, and such monolayers were also perturbed by F-SP-C. The study indicates that SP-C perturbs the packing of neutral and anionic phospholipid monolayers even when the latter systems are condensed by calcium, indicating that interactions between SP-C and the lipids are predominantly hydrophobic in nature.

Interactions of annexin V with phospholipid monolayers

To understand the mechanism of annexin V-membrane interactions, we measured the interaction of human recombinant annexin V with phospholipid monolayers with differing head group and acyl group structures. Annexin V interacted with anionic phospholipid monolayers via non-specific electrostatic interactions, which was highly dependent on the surface pressure of monolayer with a sharp maximum. The unique surface pressure dependence of the annexin V-monolayer binding is strikingly similar to that observed for the binding of Ca 2+ to anionic phospholipid monolayers, which indicates that the annexin V-bound Ca 2+ binds two phospholipids at the membrane surface and that factors governing the Ca 2+-phospholipid complex formation regulate the overall annexin V-Ca 2+-membrane interactions.

Membrane leakage induced by synergetic action of Lys-49 and Asp-49 Agkistrodon piscivorus piscivorus phospholipases A 2: Implications in their pharmacological activities

To understand the mechanism by which Lys-49 phospholipase A 2 from the venom of Agkistrodon piscivorus piscivorus exerts its myotoxic activities, we studied the interactions of this non-catalytic phospholipase A 2 with various phospholipid monolayers and liposomes. We measured the interactions of A. p. piscivorus Lys-49 phospholipase A 2 with phospholipid monolayers in terms of the change in surface pressure of monolayer and with liposomes in terms of the leakage of 6-carboxyfluorescein trapped in liposomes. A. p. piscivorus Lys-49 phospholipase A 2 showed the ability to rapidly and extensively induce the leakage of 6-carboxyfluorescein from anionic liposomes. The extent of leakage was sensitive to the ionic strength of medium and the surface charge density of liposomes, indicating the electrostatic nature of the interactions. Also, this protein was able to penetrate into anionic phospholipid monolayers with surface pressure of up to 38 dyn/cm, indicating the involvement of hydrophobic interactions in its binding to anionic liposomes. Another phospholipase A 2 from the same venom, Asp-49 phospholipase A 2, was shown to assist the lytic activity of Lys-49 phospholipase A 2 toward neutral liposomes by hydrolytically generating anionic patches of reaction products on the liposome surface. These results indicate that Lys-49 phospholipase A 2 binds to and disrupts anionic membranes via both electrostatic and hydrophobic interactions and that two phospholipases A 2 from the same venom work synergetically to enhance their pharmacological activities.

The pH dependence of calcium adsorption onto anionic phospholipid monolayers

The adsorption of 45Ca to monolayers of phosphatidylinositol and dicetylphosphoric acid has been measured as a function of subphase pH with simultaneous recordings of surface pressure and interfacial potential. Below pH 3 little calcium was adsorbed and the films are assumed to be unionized. With acid subphases between pH 3 and 6.5 adsorption of calcium occurred initially, but it was then gradually lost due to an ageing process in the films. This time dependent change in the properties of the film was independent of the presence of Ca 2+, but was dependent on the H + concentration in the subphase; it was however not due to an acid hydrolysis of the monolayer. Ca 2+ was permanently adsorbed at pH values above 6.5 with an increasing affinity up to pH 11.