Indolicidin Binding Induces Thinning of a Lipid Bilayer

We use all-atom molecular dynamics simulations on a massive scale to compute the standard binding free energy of the 13-residue antimicrobial peptide indolicidin to a lipid bilayer. The analysis of statistical convergence reveals systematic sampling errors that correlate with reorganization of the bilayer on the microsecond timescale and persist throughout a total of 1.4 ms of sampling. Consistent with experimental observations, indolicidin induces membrane thinning, although the simulations significantly overestimate the lipophilicity of the peptide.     

Bilayer Edges Catalyze Supported Lipid Bilayer Formation

Supported lipid bilayers (SLB) are important for the study of membrane-based phenomena and as coatings for biosensors. Nevertheless, there is a fundamental lack of understanding of the process by which they form from vesicles in solution. We report insights into the mechanism of SLB formation by vesicle adsorption using temperature-controlled time-resolved fluorescence microscopy at low vesicle concentrations. First, lipid accumulates on the surface at a constant rate up to ∼0.8 of SLB coverage. Then, as patches of SLB nucleate and spread, the rate of accumulation increases. At a coverage of ∼1.5 × SLB, excess vesicles desorb as SLB patches rapidly coalesce into a continuous SLB. Variable surface fluorescence immediately before SLB patch formation argues against the existence of a critical vesicle density necessary for rupture. The accelerating rate of accumulation and the widespread, abrupt loss of vesicles coincide with the emergence and disappearance of patch edges. We conclude that SLB edges enhance vesicle adhesion to the surface and induce vesicle rupture, thus playing a key role in the formation of continuous SLB.     

Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM₁ and GM₁-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM₁ positive cooperativity. Surprisingly, we demonstrated fucosyl-GM₁ has approximately 7 times higher CTB binding capacity than GM₁. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM₁ caused the increased binding capacity. This was unintuitive, as GM₁ exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM₁ and GM₂, we observed the mild binding avidity fucosyl-GM₁ activated GM₂ receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM₂ receptors have negligible binding avidity in pure GM₂ bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.     

Binding of Pediocin PA-1 with Anionic Lipid Induces Model Membrane Destabilization

To obtain molecular insights into the action mode of antimicrobial activity of pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D₂O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of pediocin, as registered by the amide I′ band, and phospholipid conformation, as revealed by the methylene ν_s(CH₂) and carbonyl ν(C=O) stretching vibrations. The results show that no interaction between pediocin and DMPC occurs. By contrast, pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.     

Electrical Response to Vibration of a Lipid Bilayer Membrane

The discovery and characterization of a vibration response in a black lipid bilayer membrane is the topic of this paper. An electrical vibration response is obtained when the membrane is under voltage clamp and a weaker, but significant, response is obtained under current clamp. The effect arises from an induced variation in the membrane capacitance. It is further shown that the capacitance variation arises from a change in the membrane area as the membrane undergoes drumhead vibration. Possible physiological significance in mechanoreception is discussed.     

Lipid Bilayer Domain Fluctuations as a Probe of Membrane Viscosity

We argue that membrane viscosity, η_m, plays a prominent role in the thermal fluctuation dynamics of micron-scale lipid domains. A theoretical expression is presented for the timescales of domain shape relaxation, which reduces to the well-known η_m = 0 result of Stone and McConnell in the limit of large domain sizes. Experimental measurements of domain dynamics on the surface of ternary phospholipid and cholesterol vesicles confirm the theoretical results and suggest domain flicker spectroscopy as a convenient means to simultaneously measure both the line tension, σ, and the membrane viscosity, η_m, governing the behavior of individual lipid domains.     

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies^1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation³. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.     

System for Measuring Planar Lipid Bilayer Properties

We present a system for measuring planar lipid bilayer properties. The system is composed of a control unit, an output stage, an LCR meter, pumps for filling reservoirs, a bath with temperature regulation and a measurement chamber with four electrodes. The planar lipid bilayer is automatically formed using a folding method on apertures of different sizes. The automatization is assured by two syringes, which are clamped in actuators. Actuators are driven and controlled by a control unit via RS-232 communication. The temperature of the planar lipid bilayer can be regulated between 15 and 55 °C. The regulation is assured by insertion of the measurement chamber into the temperature-regulated bath. Different shapes of voltage- or current-clamp signals can be applied to the planar lipid bilayer. By measuring the response of the planar lipid bilayer to the applied signal, the capacitance and breakdown voltage of the planar lipid bilayer can be determined. The cutoff frequencies of the system output stage for voltage- and current-clamp methods are 11 and 17 kHz, respectively.     

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)₃-NH₂ (LS2) or Ac-(LSSLLSL)₃-NH₂ (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.     

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)₃-NH₂ (LS2) or Ac-(LSSLLSL)₃-NH₂ (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.     

Asymmetrical Lipid Bilayer Structure for Biological Membranes

IT is generally accepted that the matrix of cellular membranes is a bimolecular leaflet of phospholipid molecules in which the phospholipids are oriented so that their polar heads reside on the outer surfaces of the bilayer, in contact with the aqueous environment, the interior of the sandwich being composed of hydrophobic lipid chains^1–5. To this basic structure proteins cholesterol, glycolipids and other molecules are usually inserted in such a way as to confer on the bilayer the functional properties appropriate for the particular membrane.     

Chloride Transport in Porous Lipid Bilayer Membranes

This paper describes dissipative Cl_- transport in "porous" lipid bilayer membranes, i.e., cholesterol-containing membranes exposed to 1–3 x 10^-7 M amphotericin B. P_DCl (cm·s^-1), the diffusional permeability coefficient for Cl^-, estimated from unidirectional ³⁶Cl^- fluxes at zero volume flow, varied linearly with the membrane conductance (Gm, Ω^-1·cm^-2) when the contributions of unstirred layers to the resistance to tracer diffusion were relatively small with respect to the membranes; in 0.05 M NaCl, P_DCl was 1.36 x 10^-4 cm·s^-1 when Gm was 0.02 Ω^-1·cm^-2. Net chloride fluxes were measured either in the presence of imposed concentration gradients or electrical potential differences. Under both sets of conditions: the values of P_DCl computed from zero volume flow experiments described net chloride fluxes; the net chloride fluxes accounted for ~90–95% of the membrane current density; and, the chloride flux ratio conformed to the Ussing independence relationship. Thus, it is likely that Cl^- traversed aqueous pores in these anion-permselective membranes via a simple diffusion process. The zero current membrane potentials measured when the aqueous phases contained asymmetrical NaCl solutions could be expressed in terms of the Goldman-Hodgkin-Katz constant field equation, assuming that the P_DNa/P_DCl ratio was 0.05. In symmetrical salt solutions, the current-voltage properties of these membranes were linear; in asymmetrical NaCl solutions, the membranes exhibited electrical rectification consistent with constant-field theory. It seems likely that the space charge density in these porous membranes is sufficiently low that the potential gradient within the membranes is approximately linear; and, that the pores are not electrically neutral, presumably because the Debye length within the membrane phase approximates the membrane thickness.     

Permeability of Lipid Bilayer Membranes to Organic Solutes

A sensitive fluorescence technique was used to measure transport of organic solutes through lipid bilayer membranes and to relate permeability to the functional groups of the solute, lipid composition of the membrane, and pH of the medium. Indole derivatives having ethanol, acetate, or ethylamine in the 3-position, representing neutral, acidic, and basic solutes, respectively, were the primary models. The results show: (a) Neutral solute permeability is not greatly affected by changes in lipid composition but presence or absence of cholesterol in the membranes could greatly alter permeability of the dissociable substrates. (b) Indole acetate permeability was reduced by introduction of phosphatidylserine into membranes to produce a net negative charge on the membranes. (c) Permeability response of dissociable solutes to variation in pH was in the direction predicted but not always of the magnitude expected from changes in the calculated concentrations of the undissociated solute in the bulk aqueous phase. Concentration gradients of amines across the membranes caused substantial diffusion potentials, suggesting that some transport of the cationic form of the amine may occur. It is suggested that factors such as interfacial charge and hydration structure, interfacial polar forces, and lipid organization and viscosity, in addition to the expected solubility-diffusion relations, may influence solute flux.     

Association of Model Neurotransmitters with Lipid Bilayer Membranes

Aimed at reproducing the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neurotransmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neurotransmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflection measurements on artificial membranes, so-called sparsely tethered bilayer lipid membranes, reveal the structural aspects of neurotransmitters’ association with zwitterionic and anionic bilayers. We thus establish that serotonin interacts nonspecifically with the membrane at physiologically relevant concentrations, whereas γ-aminobutyric acid does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity, and neutron reflectometry shows that it penetrates the membrane deeply, whereas γ-aminobutyric is excluded from the bilayer.     

Model of a Raft in Both Leaves of an Asymmetric Lipid Bilayer

We present a theory of inhomogeneities in the plasma membrane, or rafts, that can exist in both leaves of the plasma membrane. We note that although neither of the major phospholipid components of the outer leaf, sphingomyelin (SM) nor phosphatidylcholine (PC), evinces a tendency to form phases characterized by nonzero curvature, one of the major components of the inner leaf, phosphatidylethanolamine (PE), displays a strong tendency to do so whereas the other, phosphatidylserine (PS), does not. Therefore, we posit that the concentration difference of PS and PE couples to height fluctuations of the plasma membrane bilayer. This brings about a microemulsion in the inner leaf. Coupling of the concentration difference between PS and PE in the inner leaf and SM and PC in the outer leaf propagates the microemulsion to that leaf as well. The characteristic size of the inhomogeneities is equal to the square-root of the ratio of the bending modulus of the bilayer to its surface tension, a size which is ∼100 nm for the plasma membrane. If the coupling between leaves were to be provided by the interchange of cholesterol, then our model raft would consist of SM and cholesterol in the outer leaf and PS and cholesterol in the inner leaf floating in a sea of PC and PE in both leaves.     

Model of a Raft in Both Leaves of an Asymmetric Lipid Bilayer

We present a theory of inhomogeneities in the plasma membrane, or rafts, that can exist in both leaves of the plasma membrane. We note that although neither of the major phospholipid components of the outer leaf, sphingomyelin (SM) nor phosphatidylcholine (PC), evinces a tendency to form phases characterized by nonzero curvature, one of the major components of the inner leaf, phosphatidylethanolamine (PE), displays a strong tendency to do so whereas the other, phosphatidylserine (PS), does not. Therefore, we posit that the concentration difference of PS and PE couples to height fluctuations of the plasma membrane bilayer. This brings about a microemulsion in the inner leaf. Coupling of the concentration difference between PS and PE in the inner leaf and SM and PC in the outer leaf propagates the microemulsion to that leaf as well. The characteristic size of the inhomogeneities is equal to the square-root of the ratio of the bending modulus of the bilayer to its surface tension, a size which is ∼100 nm for the plasma membrane. If the coupling between leaves were to be provided by the interchange of cholesterol, then our model raft would consist of SM and cholesterol in the outer leaf and PS and cholesterol in the inner leaf floating in a sea of PC and PE in both leaves.     

Transmembrane Lipid Migration in Planar Asymmetric Bilayer Membranes

Planar asymmetric bilayer membranes, formed by apposing a monolayer of the neutral lipid glyceroldioleate (GDO) with one of the negatively charged lipid oleyl acid phosphate (OAP), were used to measure the rate of transmembrane OAP migration. The assay for this lipid flip-flop was the interaction of Ca²⁺ ions with negatively charged lipids which causes membranes to break: when Ca²⁺ is added to the compartment limited initially by the neutral lipid, flip-flop of the charged lipid eventually results in membrane breakdown. At 22 ± 2°C, in the absence of an externally applied electric field, an upper limit to the half time of OAP flip-flop was measured as 18.7 h, with a tentative lower limit of 14.4 h.     

Chronopotentiometric Technique as a Method for Electrical Characterization of Bilayer Lipid Membranes

The basic electrical parameters of bilayer lipid membranes are capacitance and resistance. This article describes the application of chronopotentiometry to the research of lipid bilayers. Membranes were made from egg yolk phosphatidylcholine. The chronopotentiometric characteristic of the membranes depends on the current value. For low current values, no electroporation takes place and the voltage rises exponentially to a constant value. Based on these kinds of chronopotentiometric curves, a method of the membrane capacitance and the membrane resistance calculations are presented.