Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

Investigation of interactions of a resorcin[4]arene receptor with bilayer lipid membranes (BLMs) for the electrochemical biosensing of mixtures of dopamine and ephedrine

The present article investigates the interactions of a resorcin[4]arene receptor with planar bilayer lipid membranes (BLMs) that can be used for the electrochemical detection of dopamine and ephedrine. BLMs were composed of egg phosphatidylcholine and 35% (w/w) dipalmitoyl phosphatidic acid in which the receptor was incorporated. These BLMs modified with the resorcin[4]arene receptor can be used as one-shot sensors for the direct electrochemical sensing of these energizing-stimulating substances. The interactions of these compounds with the lipid membranes were found to be electrochemically transduced in the form of a transient current signal with a duration of seconds, which reproducibly appeared within 8 and 20 s after exposure of the membranes to dopamine and ephedrine, respectively. The response time for BLMs without the receptor for dopamine was about 3 min, whereas no signals were obtained for ephedrine in the absence of the receptor. The mechanism of signal generation was investigated by differential scanning calorimetric studies. These studies revealed that the adsorption of the receptor is through the hydrophobic tails of the receptor, whereas hydrophilic groups of the receptor were directed towards the electrolyte solution enhancing the ion transport through the lipid membranes. The magnitude of the transient current signal was related to the concentration of the stimulating agent in bulk solution in the micromolar range. No interferences from ascorbic acid were noticed because of the use of the negatively charged lipids in membranes. The present technique can be used as one-shot sensor for the detection of these pharmaceutical substances and future research is targeted to the determination of these chemicals in human biofluids such as urine of athletes.     

Investigation of interactions of a resorcin[4]arene receptor with bilayer lipid membranes (BLMs) for the electrochemical biosensing of mixtures of dopamine and ephedrine.

The present article investigates the interactions of a resorcin[4]arene receptor with planar bilayer lipid membranes (BLMs) that can be used for the electrochemical detection of dopamine and ephedrine. BLMs were composed of egg phosphatidylcholine and 35% (w/w) dipalmitoyl phosphatidic acid in which the receptor was incorporated. These BLMs modified with the resorcin[4]arene receptor can be used as one-shot sensors for the direct electrochemical sensing of these energizing-stimulating substances. The interactions of these compounds with the lipid membranes were found to be electrochemically transduced in the form of a transient current signal with a duration of seconds, which reproducibly appeared within 8 and 20 s after exposure of the membranes to dopamine and ephedrine, respectively. The response time for BLMs without the receptor for dopamine was about 3 min, whereas no signals were obtained for ephedrine in the absence of the receptor. The mechanism of signal generation was investigated by differential scanning calorimetric studies. These studies revealed that the adsorption of the receptor is through the hydrophobic tails of the receptor, whereas hydrophilic groups of the receptor were directed towards the electrolyte solution enhancing the ion transport through the lipid membranes. The magnitude of the transient current signal was related to the concentration of the stimulating agent in bulk solution in the micromolar range. No interferences from ascorbic acid were noticed because of the use of the negatively charged lipids in membranes. The present technique can be used as one-shot sensor for the detection of these pharmaceutical substances and future research is targeted to the determination of these chemicals in human biofluids such as urine of athletes.     

Conducting polymer polypyrrole supported bilayer lipid membranes

Electrochemically synthesized conducting polymer polypyrrole (PPy) film on gold electrode surface was used as a novel support for bilayer lipid membranes (BLMs). Investigations by surface plasmon resonance (SPR) suggest that dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-alpha-phosphatidyl-L-serine (DMPS) can form BLMs on PPy film surface but dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) and didodecyldimethylammonium bromide (DDAB) can not do so, indicating the formation of PPy supported bilayer lipid membranes (s-BLMs) is dependent on the chemical structure of the lipids used. The self-assembly of DMPC induces a smoother topography than the PPy layer with rms roughness decreasing from 4.484 to 2.914 nm convinced by atomic force microscopy (AFM). Impedance spectroscopy measurements confirm that the deposition of BLM substantially increases the resistance of the system indicating a very densely packed BLM structures. The little change of PPy film in capacitance shows that solvent and electrolyte ions still retain within the porous PPy film after BLM deposition. Therefore, the PPy supported BLM is to some extent comparable to conventional BLM with aqueous medium retaining at its two sides. As an example and preliminary application, horseradish peroxidase (HRP) reconstituted into the s-BLM shows the expected protein activity and can transfer electron from or to the underlying PPy support for its response to electrocatalytic reduction of hydrogen peroxide in solution. Thus the system maybe possesses potential applications to biomimetic membrane studies.     

Development of an electrochemical biosensor for the rapid detection of naphthalene acetic acid in fruits by using air stable lipid films with incorporated auxin-binding protein 1 receptor

This work describes the investigations of electrochemical interactions of naphthalene acetic acid (NAA) with stabilized lipid films supported on a methacrylate polymer on a glass fiber filter with incorporated auxin-binding protein 1 receptor for the development of a biosensor for the rapid detection of this compound in fruits. NAA was injected into the flowing streams of a carrier electrolyte solution, the flow of the electrolyte solution stops and an ion current transient was obtained; the magnitude of the signal was correlated to NAA concentration, which could be determined at the micromolar level. NAA preconcentrates at the lipid membrane surface which causes dynamic alterations of the electrostatic fields and phase structure of membranes. The response times were ca. 5 min and naphthalene acetic acid was determined at concentration levels of microM. The effect of potent interferences included a wide range of compounds. The results showed no interferences from these compounds in concentration levels usually found in real samples. The method was applied for the determination of NAA in fruits and the reproducibility of the method was checked in about 100 samples. A quantitative method for the detection of NAA in fruits that can be complimentary to HPLC methods is provided in the present paper. These lipid films can be used as portable sensors for the rapid detection of NAA in fruits by non-skilled personnel.     

Probing the affinity of SecA for signal peptide in different environments

SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including signal peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third alpha-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a Kd of 2.4 microM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signal peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel electrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution and lipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffected by temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP or AMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in an elongated form while inserting the signal peptide into membranes.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Actin-induced perturbation of PS lipid–cholesterol interaction: A possible mechanism of cytoskeleton-based regulation of membrane organization

To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid–lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)–phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid–lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid–lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid–CHOL versus PG lipid–CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid–lipid phase separations in plasma membranes in a similar way by interacting with PS lipids.     

Field-induced reorganization of the neural membrane lipid bilayer: a proposed role in the regulation of ion-channel dynamics

We present a computational model demonstrating that an electric field propagating in the plane of the neural membrane during transmembrane ion movement creates lateral concentration gradients of the lipids. Due to this field-induced reorganization, ethenes of the lipid chains become aligned and polarized. This finding is interpreted within the context of molecular studies of protein folding in biological membranes. We propose that electrostatic interactions between membrane dipoles and charged amino acid residues of the unfolded ion-channel protein regulate protein-folding kinetics (channel closing). These electrostatic interactions thus regulate electrical signaling in neurons.     

Invertase-lipid biocomposite films: preparation, characterization, and enzymatic activity

The formation of biocomposite films of the industrially important enzyme invertase and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein invertase diffuses into the cationic lipid octadecylamine during immersion of the lipid film in the enzyme solution. Entrapment of invertase in the octadecylamine film is highly pH-dependent, underlining the role of attractive electrostatic interactions between the enzyme and the lipid in the biocomposite film formation. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimetry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by fluorescence spectroscopy and biocatalytic activity measurements. The biocatalytic activity of the invertase-lipid biocomposite films was comparable to that of the free enzyme in solution and showed marginally higher temperature stability. Particularly exciting was the excellent reuse characteristics of the biocomposite films, indicating potential industrial application of these films.     

Impedance spectroscopy of bilayer lipid membranes self-assembled on agar support - interaction with HDL

The electrical properties of the supported lipid bilayer membrane (s-BLM) of egg phosphatidylcholine (PC) self-assembled on agar surface were examined. To characterize the insulating properties of s-BLMs, electrochemical impedance spectroscopy (EIS) was used. The analysis of impedance spectra in terms of an equivalent circuit of agar/electrolyte and agar/s-BLM/electrolyte in the frequency range of 0.1 Hz-10 kHz was performed. The high-density lipoproteins (HDL)/s-BLM interaction in the concentration range from 20 microg/ml to 80 microg/ml of HDL was investigated. It is evident that treatment of s-BLM with HDL resulted in an increase of the lipid film resistance and a decrease of membrane capacitance.     

A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.     

Self-assembling bilayer lipid membranes on solid support

Solid-supported bilayer lipid membranes (s-BLMs) that possess some properties similar to those of conventional BLMs can be self-assembled on a freshly cleaved metal wire by a two-step procedure: (i) The tip of a Teflon-coated platinum wire, while immersed in a lipid solution, is cut off with a scalpel; (ii) the new tip of the wire, having become coated with lipid solution, is transferred into 0.1 M KCl. After a few minutes, a stable lipid bilayer forms spontaneously on the tip of the wire, as verified by electrical measurements. An application of such a supported BLM (s-BLM) is reported for the detection of Pb2+ ions. The s-BLM is liquid-crystalline in structure, which makes it amenable to modification for basic studies, as well as for technological applications such as biosensors and molecular electronic devices.     

Antimicrobial selectivity based on zwitterionic lipids and underlying balance of interactions

An important feature of antimicrobial peptides is their ability to distinguish pro- from eukaryotic membranes. In vitro experiments on the antimicrobial peptide NK-2 indicate that the discrimination between zwitterionic phosphatidylethanolamine lipids exposed by prokaryotes and phosphatidylcholine lipids exposed by eukaryotes plays an important role. The underlying mechanism is not understood. Here we present molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration showing that NK-2 binds more strongly to palmitoyloleoylphosphatidylethanolamine (POPE) than to palmitoyloleoylphosphatidylcholine (POPC) bilayers. Finite size effects on the relative free energy have been corrected for with a method that may also be useful in future studies of the affinities of macromolecules for lipid membranes. Our results support the previous hypothesis that the stronger binding to PE compared to PC arises from a better accessibility of the phosphates of the lipids to the cationic peptide in a sense that a similar number of peptide-lipid salt bridges requires to break more favorable electrostatic headgroup-headgroup interactions for PC relative to PE. The transfer of NK-2 from POPC to POPE is found to lead to a decrease in electrostatic peptide-lipid but an increase in lipid-lipid and ion-lipid interactions, correlating with a dehydration of the lipids and the ions but an increased hydration of the peptide. The increase in affinity of NK-2 for POPE compared to POPC hence arises from a complex interplay of competing interactions. This work opens the perspective to study how the affinity of antimicrobial peptides changes with amino acid sequence and lipid composition.     

Electrodynamics of Lipid Membrane Interactions in the Presence of Zwitterionic Buffers

Due to thermal motion and molecular polarizability, electrical interactions in biological systems have a dynamic character. Zwitterions are dipolar molecules that typically are highly polarizable and exhibit both a positive and a negative charge depending on the pH of the solution. We use multilamellar structures of common lipids to identify and quantify the effects of zwitterionic buffers that go beyond the control of pH. We use the fact that the repeat spacing of multilamellar lipid bilayers is a sensitive and accurate indicator of the force balance between membranes. We show that common buffers can in fact charge up neutral membranes. However, this electrostatic effect is not immediately recognized because of the concomitant modification of dispersion (van der Waals) forces. We show that although surface charging can be weak, electrostatic forces are significant even at large distances because of reduced ionic screening and reduced van der Waals attraction. The zwitterionic interactions that we identify are expected to be relevant for interfacial biological processes involving lipid bilayers, and for a wide range of biomaterials, including amino acids, detergents, and pharmaceutical drugs. An appreciation of zwitterionic electrodynamic character can lead to a better understanding of molecular interactions in biological systems and in soft materials in general.     

Electrical interactions of membrane active peptides at lipid/water interfaces

Vital functions of biological membranes are frequently controlled by amphipathic peptides that are associated with the lipid bilayer. The extent of association is largely determined by influences encountered at the interface between the aqueous and lipid moieties, especially involving electrostatic interactions. A basic thermodynamic analysis is presented in terms of a partitioning equilibrium where the membrane is treated as a non-ideal solution of peptide molecules in a two-dimensional lipid solvent. This may then be employed to interpret experimental association isotherms (i.e. the ratio of associated peptide per lipid plotted versus the free aqueous peptide concentration) in the light of a molecular mechanism. Special emphasis is directed towards the evaluation of original titration data under most general circumstances when the association can be monitored using a suitable linear signal (preferentially an optical one). The experimental approaches as well as the merits regarding possible information about the underlying structural and functional features are discussed with pertinent practical examples.     

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.     

Interaction of phosphatidylserine synthase from E. coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies

The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy. The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films. Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (75:25, mol/mol). Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy. PS synthase exhibits a biphasic interaction with the bilayers. The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules. The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface. Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs. The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E. coli membranes.     

Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure

Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16–21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein–lipid interactions: the DYS R16–21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma.