The Effect of Benzyl Alcohol on the Voltage-Current Characteristics of Tethered Lipid Bilayers.

In this study a lipid bilayer membrane model was used in which the bilayer is tethered to a solid substrate with molecular tethers. Voltage-current (V-I) measurements of the tethered bilayer membranes (tBLM) and tBLM with benzyl alcohol (BZA) incorporated in their structures, were measured using triangular voltage ramps of 0-500 mV. The temperature dependence of the conductance deduced from the V-I measurements are described. An evaluation of the activation energies for electrical conductance showed that BZA decreased the activation/ Born energies for ionic conduction of tethered lipid membranes. It is concluded that BZA increased the average pore radius of the tBLM.

Subtle changes in pH affect the packing and robustness of fatty acid bilayers.

Connecting molecular interactions to emergent properties is a goal of physical chemistry, self-assembly, and soft matter science. We show that for fatty acid bilayers, vesicle rupture tension, and permeability to water and ions are coupled to pH via alterations to lipid packing. A change in pH of one, for example, can halve the rupture tension of oleic acid membranes, an effect that is comparable to increasing lipid unsaturation in phospholipid systems. We use both experiments and molecular dynamics simulations to reveal that a subtle increase in pH can lead to increased water penetration, ion permeability, pore formation rates, and membrane disorder. For changes in membrane water content, oleic acid membranes appear to be more than a million times more sensitive to protons than to sodium ions. The work has implications for systems in which fatty acids are likely to be found, for example in the primitive cells on early Earth, biological membranes especially during digestion, and other biomaterials.

The Effect of Cholesterol on the Voltage-Current Characteristics of Tethered Lipid Membranes.

In this study, a lipid bilayer membrane model was used, in which the bilayer is tethered to a solid substrate with molecular tethers. The V-I characteristics of the lipid bilayers were found to be non-linear which suggests the presence of pores that are voltage-dependent. At high applied voltages, the conductance reached a limiting value, presumably indicating a limit on the maximum pore size. A decrease in the spacing between tethers (increasing tether density) caused a decrease in the membrane's conductance at high applied voltage, which is consistent with the maximum pore size being determined by the spacing between the tethers. The inclusion of 10 M% cholesterol within the membrane lipid caused a decrease in the membrane conductance. However, the inclusion of higher levels of cholesterol increased the membrane conductance.

The Effect of Cholesterol on the Dielectric Structure of Lipid Bilayers.

Cholesterol plays an important role in regulating the properties of phospholipid bilayers and many mechanisms have been proposed to explain why cholesterol is so ubiquitous within biological membranes of animals. Here we present the results of studies on the effect of cholesterol on the electrical/dielectric properties of lipid membranes tethered to a solid substrate. These tethered bilayer lipid membranes tBLM were formed on a commercially available chemically modified gold substrate. These lipid bilayers are very robust. Very high-resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure of the lipid bilayers and associated interfaces. The EIS data allowed the dielectric substructure of the lipid bilayers to be determined. The results showed that when cholesterol was present in the tethered membranes at a concentration of 10% (mol/mol); the thickness of the tBLMs increased and the membrane conductance decreased. However, when cholesterol was present in the tethered membrane at more than 30% (mol/mol) the effect of cholesterol was dramatically different; the membranes then became thinner and possessed a much larger electrical conductance. The EIS allowed a distinction to be made between a hydrophobic region in the center of the bilayer and another hydrophobic region further out towards the polar head region, in addition to the polar head region itself. Cholesterol was found to have the largest effect on the inner, hydrophobic region, although the outer hydrophobic region was also affected.

Lipid Membrane Interactions of the Cationic Antimicrobial Peptide Chimeras Melimine and Cys-Melimine.

Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilized melimine and its derivative, with a cysteine on N-terminus, (cys-melimine) on tethered bilayer lipid membranes (tBLMs) was examined using ac electrical impedance spectroscopy. The addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. The results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol, or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the noncysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability, which is then reduced as the melimines aggregate and phase-separate within the membrane. The actions of these antimicrobials are modeled in terms of altering the critical packing parameter (CPP) of the membranes. The variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. The results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for the Gram-positive and Gram-negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy- sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studied here and their modulation of membrane conductivity.

Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes.

Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.

The effect of hydronium ions on the structure of phospholipid membranes.

This work seeks to identify the mechanisms by which hydronium ions (H3O+) modulate the structure of phospholipid bilayers by studying the interactions of H3O+ with phospholipids at the molecular level. For this, we carried out multiple microsecond-long unrestrained molecular dynamics (MD) simulations of a POPC bilayer at different H3O+ concentrations. The results show that H3O+ accumulates at the membrane surface where it displaces water and forms strong and long-lived hydrogen bonds with the phosphate and carbonyl oxygens in phospholipids. This results in a concentration-dependent reduction of the area per lipid and an increase in bilayer thickness. This study provides an important molecular-level insight into the mechanism of how H3O+ modulates the structure of biological membranes and is a critical step towards a better understanding of the effect of low pH on mammalian and bacterial membranes.

The Effect of Benzyl Alcohol on the Dielectric Structure of Lipid Bilayers.

Molecularly tethered lipid bilayer membranes were constructed on a commercially available chemically modified gold substrate. This is a new and promising product that has allowed the construction of very robust lipid bilayers. Very high resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure of the lipid bilayers and associated interfaces. The EIS data were modelled in terms of the dielectric substructure using purpose developed software. The hydrophobic region, where the lipid tails are located, was revealed by the EIS in the frequency range of (1-100) Hz and its thickness was calculated from the capacitance of this region and found to be approximately 3-4 nm. The hydrophilic region, where the polar heads are located, was revealed at higher frequencies and its thickness was estimated to be approximately 1-2 nm. The effect of the local anaesthetic benzyl alcohol (BZA) on the tethered lipid bilayers was investigated. The effect of BZA on the membrane capacitance and conductance allowed the changes in the thickness of the polar head and hydrophobic tails regions to be determined. It was found that the addition of BZA caused a significant increase in the capacitance (corresponding to a decrease in the thickness) of the hydrophobic region and an increase in the membrane electrical conductance. The EIS allowed a distinction between a hydrophobic region in the centre of the bilayer and an outer hydrophobic region. Benzyl alcohol was found to have the largest effect on the outer, hydrophobic region, although the inner hydrophobic region was also consistently affected.

Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology.

This study explains the importance of the phosphate moiety and H3O+ in controlling the ionic flux through phospholipid membranes. We show that despite an increase in the H3O+ concentration when the pH is decreased, the level of ionic conduction through phospholipid bilayers is reduced. By modifying the lipid structure, we show the dominant determinant of membrane conduction is the hydrogen bonding between the phosphate oxygens on adjacent phospholipids. The modulation of conduction with pH is proposed to arise from the varying H3O+ concentrations altering the molecular area per lipid and modifying the geometry of conductive defects already present in the membrane. Given the geometrical constraints that control the lipid phase structure of membranes, these area changes predict that organisms evolving in environments with different pHs will select for different phospholipid chain lengths, as is found for organisms near highly acidic volcanic vents (short chains) or in highly alkaline salt lakes (long chains). The stabilizing effect of the hydration shells around phosphate groups also accounts for the prevalence of phospholipids across biology. Measurement of ion permeation through lipid bilayers was made tractable using sparsely tethered bilayer lipid membranes with swept frequency electrical impedance spectroscopy and ramped dc amperometry. Additional evidence of the effect of a change in pH on lipid packing density is obtained from neutron reflectometry data of tethered membranes containing perdeuterated lipids.

Nanostructural determination of a lipid bilayer tethered to a gold substrate.

Tethered lipid bilayer membranes (tBLM) are planar membranes composed of free lipids and molecules tethered to a solid planar substrate providing a useful model of biological membranes for a wide range of biophysical studies and biotechnological applications. The properties of the tBLM depend on the free lipids and on the chemistry of the tethering molecules. We present a nanoscale characterization of a tBLM composed of deuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (d-DMPC) free lipids, benzyl disulfide undecaethylene glycol phytanol (DLP) tethering molecules, and benzyl disulfiide tetraethylene glycol polar spacer molecules (PSM) used to control the areal density of tethering molecules through coadsorption. The use of selected isotopic substitution provides a way to distinguish the conformation and location of the tethered lipids from the free lipids and to elucidate how the two components influence the structure of the tBLM. These findings provide useful information to optimise the insertion of transmembrane proteins into the tethered bilayer system.

Nanoscale ion sequestration to determine the polarity selectivity of ion conductance in carriers and channels.

The nanoscale spacing between a tethered lipid bilayer membrane (tBLM) and its supporting gold electrode can be utilized to determine the polarity selectivity of the conduction of ion channels and ion carriers embedded in a membrane. The technique relies upon a bias voltage sequestering or eliminating ions, of a particular polarity, into or out of the aqueous electrolyte region between the gold electrode and the tethered membrane. A demonstration is given, using ac swept frequency impedance spectrometry, of the bias polarity dependence of the ionophore conductance of gramicidin A, a cationic selective channel, and valinomycin, a potassium ion selective carrier. We further use pulsed amperometry to show that the intrinsic voltage dependence of the ion conduction is actually selective of the polarity of the transported ion and not simply of the direction of the ionic current flow.

An engineered membrane to measure electroporation: effect of tethers and bioelectronic interface.

This article reports on the construction and predictive models for a platform comprised of an engineered tethered membrane. The platform provides a controllable and physiologically relevant environment for the study of the electroporation process. The mixed self-assembled membrane is formed via a rapid solvent exchange technique. The membrane is tethered to the gold electrode and includes an ionic reservoir separating the membrane and gold surface. Above the membrane, there is an electrolyte solution, and a gold counterelectrode. A voltage is applied between the gold electrodes and the current measured. The current is dependent on the energy required to form aqueous pores and the conductance of each pore. A two-level predictive model, consisting of a macroscopic and a continuum model, is developed to relate the pore dynamics to the measured current. The macroscopic model consists of an equivalent circuit model of the tethered membrane, and asymptotic approximations to the Smoluchowski-Einstein equation of electroporation that is dependent on the pore conductance and the energy required to form aqueous pores. The continuum model is a generalized Poisson-Nernst-Planck (GPNP) system where an activity coefficient to account for steric effects of ions is added to the standard PNP system. The GPNP is used to evaluate the conductance of aqueous pores, and the electrical energy required to form the pores. As an outcome of the setup of the device and the two-level model, biologically important variables can be estimated from experimental measurements. To validate the accuracy of the two-level model, the predicted current is compared with experimentally measured current for different tethering densities.

Transient potential gradients and impedance measures of tethered bilayer lipid membranes: pore-forming peptide insertion and the effect of electroporation.

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >~150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.

Triglyceride-Tethered Membrane Lipase Sensor.

Sensors that can quickly measure the lipase activity from biological samples are useful in enzyme production and medical diagnostics. However, current lipase sensors have limitations such as requiring fluorescent labels, pH control of buffer vehicles, or lengthy assay preparation. We introduce a sparsely tethered triglyceride substrate anchored off of a gold electrode for the impedance sensing of real-time lipase activity. The tethered substrate is self-assembled using a rapid solvent exchange technique and can form an anchored bilayer 1 nm off the gold electrode. This allows for an aqueous reservoir region, providing access to ions transported through membrane defects caused by triglyceride enzymatic hydrolysis. Electrical impedance spectroscopy techniques can readily detect the decrease in resistance caused by enzymatically induced defects. This rapid and reliable lipase detection method can have potential applications in disease studies, monitoring of lipase production, and as point-of-care diagnostic devices.

Measuring Voltage-Current Characteristics of Tethered Bilayer Lipid Membranes to Determine the Electro-Insertion Properties of Analytes.

Tethered bilayer lipid membranes (tBLMs) anchored to a solid substrate can be prepared and individual triangular voltage ramps from zero to 500 mV with a period of 2-10 ms applied to give membrane voltage dependencies with and without the addition of drugs and analytes in order to measure their electro-insertion properties.

Measuring Activation Energies for Ion Transport Using Tethered Bilayer Lipid Membranes (tBLMs).

Model lipid bilayers tethered to a gold substrate with molecular tethers are constructed. The conductance versus temperature dependence curve is then obtained. Here, a method to measure the activation energy for translocation of an ion through existing transmembrane pores in a sparsely tethered bilayer lipid membranes is presented.

Preparing Ion Channel Switch Membrane-Based Biosensors.

Monitoring the changes in membrane conductance using electrical impedance spectroscopy is the platform of membrane-based biosensors in order to detect a specific target molecule. These biosensors represent the amalgamation of an electrical conductor such as gold and a chemically tethered bilayer lipid membrane with specific incorporated ion channels such as gramicidin-A that is further functionalized with detector molecules of interest.

Langmuir-Schaefer Deposition to Create an Asymmetrical Lipopolysaccharide Sparsely Tethered Lipid Bilayer.

Because they are firmly anchored to a noble metal substrate, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cranfield et al. Biophys J 106:182-189, 2014). The challenge to rapidly create asymmetrical tBLMs that include a lipopolysaccharide outer leaflet for bacterial model membrane research can be overcome by the use of a Langmuir-Schaefer deposition protocol. Here, we describe the procedures required to assemble and test asymmetric lipopolysaccharide (LPS) tethered lipid bilayers.

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

Real-time monitoring of heat transfer between gold nanoparticles and tethered bilayer lipid membranes.

Plasmon resonance frequency irradiated gold nanoparticles (GNPs) have gained interest as a laser-targeted treatment for infections, tumors and for the controlled release of drugs in situ. Questions still remain, however, as to the efficiency of heat delivery within biological tissues and how this can be reliably determined. Here, we demonstrate how a nanomaterial-electrode interface that mimics cell membranes can detect the localized heat transfer characteristics arising from plasmon resonance frequency-matched laser excitation of GNPs. We demonstrate that the lipid bilayer membrane can be affected by conjugated GNP induced hyperthermia when irradiated with a laser power output as low as 135 nW/µm2. This is four orders of magnitude lower power than previously reported. By restricting the lateral movement of the lipids in the bilayer membrane, it was shown that the change in membrane conductance as a result of the heat transfer was due to the creation of transient lipidic toroidal pores within the membrane. We further demonstrate that the heat transfer from the GNPs alters diffusion rates of monomers of the gramicidin-A peptide within the lipid leaflets. This work highlights how targeted low laser power GNP hyperthermia treatments, in vivo, could play a dual role of interfering with both cell membrane morphology and dynamics, along with membrane protein function.