Interactions between Fengycin and Model Bilayers Quantified by Coarse-Grained Molecular Dynamics

Bacteria, particularly of the genus Bacillus, produce a wide variety of antifungal compounds. They act by affecting the lipid bilayers of fungal membranes, causing curvature-induced strain and eventual permeabilization. One class of these, known as fengycins, has been commercialized for treating agricultural infections and shows some promise as a possible antifungal pharmaceutical. Understanding the mechanism by which fengycins damage lipid bilayers could prove useful to the future development of related antifungal treatments. In this work, we present multi-microsecond-long simulations of fengycin interacting with different lipid bilayer systems. We see fengycin aggregation and uncover a clear aggregation pattern that is partially influenced by bilayer composition. We also quantify some local bilayer perturbations caused by fengycin binding, including curvature of the lipid bilayer and local electrostatic-driven reorganization.     

Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.     

Domain redistribution within ergosterol-containing model membranes in the presence of the antimicrobial compound fengycin

The cyclic lipopeptide fengycin, produced by Bacillus subtilis, exhibits its antimicrobial capabilities by altering the integrity of the cell membrane of plant pathogens. Previous work has correlated fengycin activity with membrane characteristics, such as sterol content. This work focused on the influence of fengycin on supported lipid bilayers containing varying levels of ergosterol. Total internal reflection fluorescence (TIRF) microscopy was used to visualize and distinguish ordered (L_β/L_o) and disordered (L_α/L_d) domains in the model membranes following exposure to low (50 μg) and high (500 μg) fengycin doses. Application of an initial low dose of fengycin to 0% and 3% ergosterol-containing bilayers resulted in redistribution of L_α/L_β and L_o/L_d domains, respectively, which the bilayers compensated and corrected for over time. These membranes were unable to tolerate a second 50 μg dose or a single high fengycin dose. The 6% ergosterol bilayers were able to tolerate sequential low doses of fengycin. Exposure of these bilayers to the high fengycin dose caused a decrease in the number of L_o domains, albeit less than that seen in the 0% and 3% ergosterol bilayers. Bilayers containing 12% ergosterol, exhibited the least amount of change after fengycin exposure. These were the only bilayer to exhibit an increase in area taken up by ordered domains. These results suggest fengycin may preferentially act on the L_β or L_o phase, the area in which ergosterol resides. Bilayers containing low levels of ergosterol appear to be more sensitive to the lipopeptide, suggesting ergosterol plays a role in buffering perturbations caused by fengycin.     

Peptide Nanopores and Lipid Bilayers: Interactions by Coarse-Grained Molecular-Dynamics Simulations

A set of 49 protein nanopore-lipid bilayer systems was explored by means of coarse-grained molecular-dynamics simulations to study the interactions between nanopores and the lipid bilayers in which they are embedded. The seven nanopore species investigated represent the two main structural classes of membrane proteins (α-helical and β-barrel), and the seven different bilayer systems range in thickness from ∼28 to ∼43 Å. The study focuses on the local effects of hydrophobic mismatch between the nanopore and the lipid bilayer. The effects of nanopore insertion on lipid bilayer thickness, the dependence between hydrophobic thickness and the observed nanopore tilt angle, and the local distribution of lipid types around a nanopore in mixed-lipid bilayers are all analyzed. Different behavior for nanopores of similar hydrophobic length but different geometry is observed. The local lipid bilayer perturbation caused by the inserted nanopores suggests possible mechanisms for both lipid bilayer-induced protein sorting and protein-induced lipid sorting. A correlation between smaller lipid bilayer thickness (larger hydrophobic mismatch) and larger nanopore tilt angle is observed and, in the case of larger hydrophobic mismatches, the simulated tilt angle distribution seems to broaden. Furthermore, both nanopore size and key residue types (e.g., tryptophan) seem to influence the level of protein tilt, emphasizing the reciprocal nature of nanopore-lipid bilayer interactions.     

Interaction Between Amyloid-β (1-42) Peptide and Phospholipid Bilayers: A Molecular Dynamics Study

The amyloid-β (Aβ) peptide is a key aggregate species in Alzheimer's disease. Although important aspects of Aβ peptide aggregation are understood, the initial stage of aggregation from monomer to oligomer is still not clear. One potential mediator of this early aggregation process is interactions of Aβ with anionic cell membranes. We used unconstrained and umbrella sampling molecular dynamics simulations to investigate interactions between the 42-amino acid Aβ peptide and model bilayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) lipids and anionic dioleoylphosphatidylserine (DOPS) lipids. Using these methods, we determined that Aβ is attracted to the surface of DPPC and DOPS bilayers over the small length scales used in these simulations. We also found supporting evidence that the charge on both the bilayer surface and the peptide affects the free energy of binding of the peptide to the bilayer surface and the distribution of the peptide on the bilayer surface. Our work demonstrates that interactions between the Aβ peptide and lipid bilayer promotes a peptide distribution on the bilayer surface that is prone to peptide-peptide interactions, which can influence the propensity of Aβ to aggregate into higher-order structures.     

The Role of the Lipid Bilayer in Tau Aggregation

Tau is a microtubule associated protein whose aggregation is implicated in a number of neurodegenerative diseases. We investigate the mechanism by which anionic lipid vesicles induce aggregation of tau in vitro using K18, a fragment of tau corresponding to the four repeats of the microtubule binding domain. Our results show that aggregation occurs when the amount of K18 bound to the lipid bilayer exceeds a critical surface density. The ratio of protein/lipid at the critical aggregation concentration is pH-dependent, as is the binding affinity. At low pH, where the protein binds with high affinity, the critical surface density is independent both of total lipid concentration as well as the fraction of anionic lipid present in the bilayer. Furthermore, the aggregates consist of both protein and vesicles and bind the β-sheet specific dye, Thioflavin T, in the manner characteristic of pathological aggregates. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein and by increasing the local protein concentration, resulting in rapid aggregation. Because anionic lipids are abundant in cellular membranes, these findings contribute to understanding tau-lipid bilayer interactions that may be relevant to disease pathology.     

Effects of cholesterol on the mechanism of fengycin,a biofungicide

Fengycins are a class of antifungal lipopeptides synthesized by the bacteria Bacillus subtilis, commercially available as the primary component of the agricultural fungicide Serenade. They are toxic to fungi but far less to mammalian cells. One key difference between mammalian and fungal cell membranes is the presence of cholesterol only in the former; recent experimental work showed that the presence of cholesterol reduces fengycin-induced membrane leakage. Since our previous all-atom and coarse-grained simulations suggested that aggregation of membrane-bound fengycin is central to its ability to disrupt membranes, we hypothesized that cholesterol might reduce fengycin aggregation. Here, we test this hypothesis using coarse-grained molecular dynamics simulations, with sampling enhanced via the weighted ensemble method. The results indicate that cholesterol subtly alters the size distribution for fengycin aggregates, limits the lateral range of their membrane disordering, and reduces the ability of aggregates to bend the membrane. Taken together, these phenomena may account for cholesterol’s effects on fengycin activity.     

Membrane-thinning effect of curcumin

Interaction of curcumin with lipid bilayers is not well understood. A recent experiment showed that curcumin significantly affected the single-channel lifetime of gramicidin in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer without affecting its single-channel conductance. We performed two experiments to understand this result. By isothermal titration calorimetry, we measured the partition coefficient of curcumin binding to DOPC bilayers. By x-ray lamellar diffraction, we measured the thickness change of DOPC bilayers as a function of the curcumin/lipid ratio. A nonlinear membrane-thinning effect by curcumin was discovered. The gramicidin data were qualitatively interpreted by the combination of isothermal titration calorimetry and x-ray results. We show that not only does curcumin thin the lipid bilayer, it might also weaken its elasticity moduli. The result implies that curcumin may affect the function of membrane proteins by modifying the properties of the host membrane.     

Bilayer edge and curvature effects on partitioning of lipids by tail length: atomistic simulations

The partitioning of lipids among different microenvironments in a bilayer is of considerable relevance to characterization of composition variations in biomembranes. Atomistic simulation has been ill-suited to model equilibrated lipid mixtures because the time required for diffusive exchange of lipids among microenvironments exceeds typical submicrosecond molecular dynamics trajectories. A method to facilitate local composition fluctuations, using Monte Carlo mutations to change lipid structures within the semigrand-canonical ensemble (at a fixed difference in component chemical potentials, Δμ), was recently implemented to address this challenge. This technique was applied here to mixtures of dimyristoylphosphatidylcholine and a shorter-tail lipid, either symmetric (didecanoylphosphatidylcholine (DDPC)) or asymmetric (hexanoyl-myristoylphosphatidylcholine), arranged in two types of structure: bilayer ribbons and buckled bilayers. In ribbons, the shorter-tail component showed a clear enrichment at the highly curved rim, more so for hexanoyl-myristoylphosphatidylcholine than for DDPC. Results on buckled bilayers were variable. Overall, the DDPC content of buckled bilayers tended to exceed by several percent the DDPC content of flat ones simulated at the same Δμ, but only for mixtures with low overall DDPC content. Within the buckled bilayer structure, no correlation could be resolved between the sign or magnitude of the local curvature of a leaflet and the mean local lipid composition. Results are discussed in terms of packing constraints, surface area/volume ratios, and curvature elasticity.     

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.     

Role of cholesterol flip-flop in oxidized lipid bilayers

We performed a series of molecular dynamics simulations of cholesterol (Chol) in nonoxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC-oxidized-lipid-bilayers with 0–50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased molecular dynamics simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration led to a change of phase state from the liquid-disordered to the liquid-ordered phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella-sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol’s flip-flop rate depends on the lipid bilayer type, and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups.     

How type II diabetes-related islet amyloid polypeptide damages lipid bilayers

A leading hypothesis for the decimation of insulin-producing β-cells in type 2 diabetes attributes the cause to islet amyloid polypeptide (IAPP) for its deleterious effects on the cell membranes. This idea has produced extensive investigations on human IAPP (hIAPP) and its interactions with lipid bilayers. However, it is still difficult to correlate the peptide-lipid interactions with its effects on islet cells in culture. The hIAPP fibrils have been shown to interact with lipids and damage lipid bilayers, but appear to have no effect on islet cells in culture. Thus, a modified amyloid hypothesis assumes that the toxicity is caused by hIAPP oligomers, which are not preamyloid fibrils or protofibrils. However, so far such oligomers have not been isolated or identified. The hIAPP monomers also bind to lipid bilayers, but the mode of interaction is not clear. Here, we performed two types of experiments that, to our knowledge, have not been done before. We used x-ray diffraction, in conjunction with circular dichroism measurement, to reveal the location of the peptide bound to a lipid bilayer. We also investigated the effects of hIAPP on giant unilamellar vesicles at various peptide concentrations. We obtained the following qualitative results. Monomeric hIAPP binds within the headgroup region and expands the membrane area of a lipid bilayer. At low concentrations, such binding causes no leakage or damage to the lipid bilayer. At high concentrations, the bound peptides transform to β-aggregates. The aggregates exit the headgroup region and bind to the surface of lipid bilayers. The damage by the surface bound β-aggregates depends on the aggregation size. The initial aggregation extracts lipid molecules, which probably causes ion permeation, but no molecular leakage. However, the initial β-aggregates serve as the seed for larger fibrils, in the manner of the Jarrett-Lansbury seeded-polymerization model, that eventually disintegrate lipid bilayers by electrostatic and hydrophobic interactions.     

Fengycin antibiotics isolated from B-FS01 culture inhibit the growth of Fusarium moniliforme Sheldon ATCC 38932

Strain B-FS01, isolated from rape (Brassica napus) stem infected by Slerotinia sclerotiorum and identified as Bacillus subtilis, exhibited predominantly antagonistic activities against Fusarium moniliforme Sheldon ATCC 38932. Antifungal active compounds (AAC) were isolated and purified from the cultures of strain B-FS01 against ATCC 38932. The HPLC/electron spray ionization/collision-induced dissociation mass spectrum of AAC revealed a cluster of fengycin homologues containing fengycins A, fengycins B and a new type of fengycin. Further toxic assay of AAC in vitro against F. moniliforme indicated that AAC could strongly inhibit the growth of both mycelia and spores. In addition, treatment with AAC significantly modified the maize seed infection by ATCC 38932.     

Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.     

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Abeta(1-40) peptide in solid-supported lipid bilayers

We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

A Novel Variant of Narrow-Spectrum Antifungal Bacterial Lipopeptides That Strongly Inhibit <Emphasis Type="Italic">Ganoderma boninense</Emphasis>

Bacterial antifungal cyclic lipopeptides (ACLs) have become a promising alternative to synthetic fungicide to control pathogenic fungi. Bacillus sp. is known to produce three families of ACL, namely iturin, surfactin, and fengycin. In this paper, we characterized the ACLs produced by B. methylotrophicus HC51 (referred as HC51) mainly regarding its composition and effectivity against fungal plant pathogen. HC51 culture was tested against various pathogenic fungi and the ACLs were extracted and analyzed using liquid chromatography–electrospray ionization mass spectrometry. HC51 showed strong antifungal activity against the plant pathogens Ganoderma sp. and Fusarium sp. Cell-free methanol extract of HC51 contains iturin A and various variants of fengycin. C16 fengycin A was present in four fractions which indicates it as a major component of ACL from HC51. Five variants of fengycin were detected, four of which had been previously reported. We found a novel C17 fengycin F that is characterized by a substitution of l-ornithine into lysine. Considering that l-ornithine is an important building block of fengycin, this substitution suggests the possibility of an alternative pathway for fengycin biosynthesis.     

Atomistic-level study of the interactions between hIAPP protofibrils and membranes: Influence of pH and lipid composition

The pathology of type 2 diabetes mellitus is associated with the aggregation of human islet amyloid polypeptide (hIAPP) and aggregation-mediated membrane disruption. The interactions of hIAPP aggregates with lipid membrane, as well as the effects of pH and lipid composition at the atomic level, remain elusive. Herein, using molecular dynamics simulations, we investigate the interactions of hIAPP protofibrillar oligomers with lipids, and the membrane perturbation that they induce, when they are partially inserted in an anionic dipalmitoyl-phosphatidylglycerol (DPPG) membrane or a mixed dipalmitoyl-phosphatidylcholine (DPPC)/DPPG (7:3) lipid bilayer under acidic/neutral pH conditions. We observed that the tilt angles and insertion depths of the hIAPP protofibril are strongly correlated with the pH and lipid composition. At neutral pH, the tilt angle and insertion depth of hIAPP protofibrils at a DPPG bilayer reach ~52° and ~1.62 nm with respect to the membrane surface, while they become ~77° and ~1.75 nm at a mixed DPPC/DPPG membrane. The calculated tilt angle of hIAPP at DPPG membrane is consistent with a recent chiral sum frequency generation spectroscopic study. The acidic pH induces a smaller tilt angle of ~40° and a shallower insertion depth (~1.24 nm) of hIAPP at the DPPG membrane surface, mainly due to protonation of His18 near the turn region. These differences mainly result from a combination of distinct electrostatic, van der Waals, hydrogen bonding and salt-bridge interactions between hIAPP and lipid bilayers. The hIAPP-membrane interaction energy analysis reveals that besides charged residues K1, R11 and H18, aromatic residues Phe15 and Phe23 also exhibit strong interactions with lipid bilayers, revealing the crucial role of aromatic residues in stabilizing the membrane-bound hIAPP protofibrils. hIAPP-membrane interactions disturb the lipid ordering and the local bilayer thickness around the peptides. Our results provide atomic-level information of membrane interaction of hIAPP protofibrils, revealing pH-dependent and membrane-modulated hIAPP aggregation at the early stage.     

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.