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The utilization of biomimetic membranes supported by advanced self-assembled monolayers is gaining attraction as a promising sensing tool. Biomimetic membranes offer exceptional biocompatibility and adsorption capacity upon degradation, transcending their role as mere research instruments to open new avenues in biosensing. This study focused on anchoring a sparsely tethered bilayer lipid membrane onto a self-assembled monolayer composed of a biodegradable polymer, functionalized with poly(ethylene glycol)-cholesterol moieties, for lipid membrane integration. Real-time monitoring via quartz crystal microbalance, coupled with characterization using surface-enhanced infrared absorption spectroscopy and electrochemical impedance spectroscopy, provided comprehensive insights into each manufacturing phase. The resulting lipid layer, along with transmembrane pores formed by gramicidin A, exhibited robust stability. Electrochemical impedance spectroscopy analysis confirmed membrane integrity, successful pore formation, and consistent channel density. Notably, gramicidin A demonstrated sustained functionality as an ion channel upon reconstitution, with its functionality being effectively blocked and inhibited in the presence of calcium ions. These findings mark significant strides in developing intricate biodegradable nanomaterials with promising applications in biomedicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H + permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gramicidin A (gA) is a short hydrophobic β-helical peptide that forms cation-selective channels in lipid membranes in the course of transbilayer dimerization. The length of the gA helix is smaller than the thickness of a typical lipid monolayer. Consequently, elastic deformations of the membrane arise in the configurations of gA monomers, conducting dimer, and the intermediate state of coaxial pair, where gA monomers from opposing membrane monolayers are located one on top of the other. The gA channel is characterized by the average lifetime of the conducting state. The elastic properties of the membrane influence the average lifetime, thus making gA a convenient sensor of membrane elasticity. However, the utilization of gA to investigate the elastic properties of mixed membranes comprising two or more components frequently relies on the assumption of ideality, namely that the elastic parameters of mixed-lipid bilayers depend linearly on the concentrations of the components. Here, we developed a general approach that does not rely on the aforementioned assumption. Instead, we explicitly accounted for the possibility of inhomogeneous lateral distribution of all lipid components, as well as for membrane-mediated lateral interactions of gA monomers, dimer, coaxial pair, and minor lipid components. This approach enabled us to derive unknown elastic parameters of lipid monolayer from experimentally determined lifetimes of gA channel in mixed-lipid bilayers. A general algorithm was formulated that allows the unknown elastic parameters of a lipid monolayer to be obtained using gA as a mechanical sensor.

Ion channels are membrane proteins that allow ionic signals to pass through channel pores for biofunctional modulations. However, biodevices that integrate bidirectional biological signal transmission between a device and biological converter through supported lipid bilayers (SLBs) while simultaneously controlling the process are lacking. Therefore, in this study, we aimed to develop a hybrid biotransducer composed of ATP synthase and proton channel gramicidin A (gA), controlled by a sulfonated polyaniline (SPA) conducting polymer layer deposited on a microelectrode, and to simulate a model circuit for this system. We controlled proton transport across the gA channel using both electrical and chemical input signals by applying voltage to the SPA or introducing calcium ions (inhibitor) and ethylenediaminetetraacetic acid molecules (inhibitor remover). The insertion of gA and ATP synthase into SLBs on microelectrodes resulted in an integrated biotransducer, in which the proton current was controlled by the flux of adenosine diphosphate molecules and calcium ions. Lastly, we created an XOR logic gate as an enzymatic logic system where the output proton current was controlled by Input A (ATP synthase) and Input B (calcium ions), making use of the unidirectional and bidirectional transmission of protons in ATP synthase and gA, respectively. We combined gA, ATP synthase, and SPA as a hybrid bioiontronics system to control bidirectional or unidirectional ion transport across SLBs in biotransducers. Thus, our findings are potentially relevant for a range of advanced biological and medical applications.

A perennial problem encountered when using small molecules (drugs) to manipulate cell or protein function is to assess whether observed changes in function result from specific interactions with a desired target or from less specific off-target mechanisms. This is important in laboratory research as well as in drug development, where the goal is to identify molecules that are unlikely to be successful therapeutics early in the process, thereby avoiding costly mistakes. We pursued this challenge from the perspective that many bioactive molecules (drugs) are amphiphiles that alter lipid bilayer elastic properties, which may cause indiscriminate changes in membrane protein (and cell) function and, in turn, cytotoxicity. Such drug-induced changes in bilayer properties can be quantified as changes in the monomer↔dimer equilibrium for bilayer-spanning gramicidin channels. Using this approach, we tested whether molecules in the Pathogen Box (a library of 400 drugs and drug-like molecules with confirmed activity against tropical diseases released by Medicines for Malaria Venture to encourage the development of therapies for neglected tropical diseases) are bilayer modifiers. 32% of the molecules in the Pathogen Box were bilayer modifiers, defined as molecules that at 10 µM shifted the monomer↔dimer equilibrium toward the conducting dimers by at least 50%. Correlation analysis of the molecules' reported HepG2 cell cytotoxicity to bilayer-modifying potency, quantified as the shift in the gramicidin monomer↔dimer equilibrium, revealed that molecules producing <25% change in the equilibrium had significantly lower probability of being cytotoxic than molecules producing >50% change. Neither cytotoxicity nor bilayer-modifying potency (quantified as the shift in the gramicidin monomer↔dimer equilibrium) was well predicted by conventional physico-chemical descriptors (hydrophobicity, polar surface area, etc.). We conclude that drug-induced changes in lipid bilayer properties are robust predictors of the likelihood of membrane-mediated off-target effects, including cytotoxicity., (© 2023 Peyear and Andersen.)

Herein, we report that chromone-containing allylmorpholines can affect ion channels formed by pore-forming antibiotics in model lipid membranes, which correlates with their ability to influence membrane boundary potential and lipid-packing stress. At 100 µg/mL, allylmorpholines 1 , 6 , 7 , and 8 decrease the boundary potential of the bilayers composed of palmitoyloleoylphosphocholine (POPC) by about 100 mV. At the same time, the compounds do not affect the zeta-potential of POPC liposomes, but reduce the membrane dipole potential by 80-120 mV. The allylmorpholine-induced drop in the dipole potential produce 10-30% enhancement in the conductance of gramicidin A channels. Chromone-containing allylmorpholines also affect the thermotropic behavior of dipalmytoylphosphocholine (DPPC), abolishing the pretransition, lowering melting cooperativity, and turning the main phase transition peak into a multicomponent profile. Compounds 4 , 6 , 7 , and 8 are able to decrease DPPC's melting temperature by about 0.5-1.9 °C. Moreover, derivative 7 is shown to increase the temperature of transition of palmitoyloleoylphosphoethanolamine from lamellar to inverted hexagonal phase. The effects on lipid-phase transitions are attributed to the changes in the spontaneous curvature stress. Alterations in lipid packing induced by allylmorpholines are believed to potentiate the pore-forming ability of amphotericin B and gramicidin A by several times.

Previously we described the discovery of a Bacillus spp. specific peptidase activity related to d-stereospecific peptidases (DSPs). The peptidase showed a strong preference for d-leucine and d-valine amino acids. These amino acids are present in the structure of the non-ribosomal peptide (NRP) antibiotics gramicidin A, B and C and polymyxin E. To examine if the Bacillus spp. DSP-related peptidase can hydrolyze these NRPs, the effect of gramicidin A and C and polymyxin E on peptidase activity in Bacillus anthracis culture supernatant was monitored. It was found that both gramicidins inhibited the DSP-related activity in a competitive manner. MALDI-TOF analysis revealed that upon incubation with B. anthracis culture supernatant gramicidin A hydrolyzation products appeared. This study shows that the Bacillus spp. specific DSP-like peptidase was potentially produced by the bacteria to gain intrinsic resistance against NRP antibiotics. These results are of utmost importance in research towards antimicrobial resistance, whereas transfer of DSP-related activity to other clinically relevant pathogens can be a serious threat to human health., (© 2022 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Ion channels are important proteins for physiological information transfer and functional control. To predict the microscopic origins of their voltage-conductance characteristics, here we applied dissipation-corrected targeted molecular dynamics in combination with Langevin equation simulations to potassium diffusion through the gramicidin A channel as a test system. Performing a nonequilibrium principal component analysis on backbone dihedral angles, we find coupled protein-ion dynamics to occur during ion transfer. The dissipation-corrected free energy profiles correspond well to predictions from other biased simulation methods. The incorporation of an external electric field in Langevin simulations enables the prediction of macroscopic observables in the form of I - V characteristics.

Gramicidin A (gA) is a hydrophobic pentadecapeptide readily incorporating into a planar bilayer lipid membrane (BLM), thereby inducing a large macroscopic current across the BLM. This current results from ion-channel formation due to head-to-head transbilayer dimerization of gA monomers with rapidly established monomer-dimer equilibrium. Any disturbance of the equilibrium, e.g., by sensitized photoinactivation of a portion of gA monomers, causes relaxation toward a new equilibrium state. According to previous studies, the characteristic relaxation time of the gA-mediated electric current decreases as the current increases upon elevating the gA concentration in the membrane. Here, we report data on the current relaxation kinetics for gA analogs with N-terminal valine replaced by glycine or tyrosine. Surprisingly, the relaxation time increased rather than decreased upon elevation of the total membrane conductance induced by these gA analogs, thus contradicting the classical kinetic scheme. We developed a general theoretical model that accounts for lateral interaction of monomers and dimers mediated by membrane elastic deformations. The modified kinetic scheme of the gramicidin dimerization predicts the reverse dependence of the relaxation time on membrane conductance for gA analogs, with a decreased dimerization constant that is in a good agreement with our experimental data. The equilibration process may be also modulated by incorporation of other peptides ("impurities") into the lipid membrane., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization., Competing Interests: All the authors declare that there is no conflict of interests regarding the publication of this paper., (Copyright © 2021 Frances Evans et al.)

The presence of biosurfactants produced by a Bacillus strain in corn steep liquor (CSL), a wastewater stream of the corn milling process, has been recently discovered. However, the species responsible for their production has not been identified at the moment. Therefore, in this work, the Bacillus strain isolated from CSL, with capacity to produce biosurfactants, was subjected to amplification and sequence analysis of the 16S rRNA, being identified as Aneurinibacillus aneurinilyticus. This strain has been proved to be endospore forming and thermophile, what would explain its presence in the commercial CSL. It was observed that the strain under evaluation has the ability to produce both cell-bound and extracellular biosurfactant extracts, which were characterized in this work. The electrospray ionization mass spectrometry (ESI) analysis of the biosurfactant extracts revealed that the extracellular biosurfactant produced by Aneurinibacillus aneurinilyticus is composed by a mixture of lipopeptides, containing C16 and C18 fatty acids and amino acids, including valine, phenylalanine, proline, cysteine, histidine, aspartic acid/asparagine, alanine, glycine, leucine/isoleucine, with biomarkers between 1025-458 m/z. Conversely, the cell-bound biosurfactant extract produced by Aneurinibacillus aneurinilyticus was composed by the cyclic decapeptide gramicidin S, with a characteristic peak at 571 m/z, and lipopeptides with characteristic peaks between 1034-705 m/z, containing alanine, glycine, cysteine, serine, proline, aspartic acid/asparagine, similarly to the amino acid sequence of the extracellular biosurfactant extract., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Partitioning of bioactive molecules, including drugs, into cell membranes may produce indiscriminate changes in membrane protein function. As a guide to safe drug development, it therefore becomes important to be able to predict the bilayer-perturbing potency of hydrophobic/amphiphilic drugs candidates. Toward this end, we exploited gramicidin channels as molecular force probes and developed in silico and in vitro assays to measure drugs' bilayer-modifying potency. We examined eight drug-like molecules that were found to enhance or suppress gramicidin channel function in a thick 1,2-dierucoyl- sn -glycero-3-phosphocholine (DC 22:1 PC) but not in thin 1,2-dioleoyl- sn -glycero-3-phosphocholine (DC 18:1 PC) lipid bilayer. The mechanism underlying this difference was attributable to the changes in gramicidin dimerization free energy by drug-induced perturbations of lipid bilayer physical properties and bilayer-gramicidin interactions. The combined in silico and in vitro approaches, which allow for predicting the perturbing effects of drug candidates on membrane protein function, have implications for preclinical drug safety assessment.

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/μm 2 . 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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof Bruce Cornell is Director – Science and Technology at Surgical Diagnostics P/L., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

We report that the channel activities of gramicidin A in a supported lipid bilayer (SLB) were modulated by membrane fusion with exosomes. The mechanism of the modulation was an increase in the number of exosomes inserting into the SLB membrane, rather than enhancements of the single channel activity of gramicidin A. The modulation of apparent channel activities was applicable to the exosome fusion assay. This assay revealed that the membrane fusion of HEK-293 and MCF-7 exosomes was enhanced at pH 6.0, and the initial rates of membrane fusion for MCF-7 exosomes were higher than those for HEK-293 cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Prerequisite for chemical shift (CS) and CS tensor calculations are highly refined structures defining the molecular surroundings of the nuclei under study. Here, we present geometry optimizations with 13 C and 15 N CS constraints for large bio-molecules like peptides and proteins. The method discussed here provides both, refined structures and chemical shift tensors. Furthermore, since the experimental resonances of aligned systems are related to CS tensors, they strongly depend on the orientation and motion of molecules, their fragments, functional groups and moieties. For efficient CS calculations we apply a semi-empirical approach-the bond polarization theory (BPT). The BPT relies on linear bond polarization parameters and we present a new set of parameters based on ab initio second-order Møller-Plesset perturbation theory calculations. The new parametrization extends the applicability of the BPT approach to a wide range of organic molecules and bio-polymers. Here, the method has been applied to the protein ubiquitin and the membrane-active peptide gramicidin A (dimer) in oriented bilayers. The calculated 13 C and 15 N CS values of best-refined structures published until now gave a large scatter with respect to the experiment. It will be shown that BPT CS optimizations can reduce these errors to values near the experimental uncertainty. In combination with molecular dynamics with orientational constraints it is possible to study motional dynamics and BPT calculations can provide residual chemical shift anisotropies.

Using a recently developed binary bilayer system (BBS) consisting of two patches of laterally contacting bilayers, umbrella sampling molecular dynamics (MD) simulations were performed for quantitative characterization of protein-lipid interactions. The BBS is composed of 1,2-dilauroyl- sn -glycero-3-phosphocholine (DLPC) and 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) with an embedded model membrane protein, a gramicidin A (gA) channel. The calculated free energy difference for the transfer of a gA channel from DLPC (hydrophobic thickness ≈ 21.5 Å) to DMPC (hydrophobic thickness ≈ 25.5 Å) bilayers, Δ G (DLPC → DMPC), is -2.2 ± 0.7 kcal/mol. This value appears at odds with the traditional view that the hydrophobic length of the gA channel is ∼22 Å. To understand this discrepancy, we first note that recent MD simulations by different groups have shown that lipid bilayer thickness profiles in the vicinity of a gA channel differ qualitatively from the deformation profile predicted from continuum elastic bilayer models. Our MD simulations at low and high gA:lipid molar ratios and different membrane compositions indicate that the gA channel's effective hydrophobic length is ∼26 Å. Using this effective hydrophobic length, Δ G (DLPC → DMPC) determined here is in excellent agreement with predictions based on continuum elastic models (-3.0 to -2.2 kcal/mol) where the bilayer deformation energy is approximated as a harmonic function of the mismatch between the channel's effective hydrophobic length and the hydrophobic thickness of the bilayer. The free energy profile for gA in the BBS includes a barrier at the interface between the two bilayers which can be attributed to the line tension at the interface between two bilayers with different hydrophobic thicknesses. This observation implies that translation of a peptide between two different regions of a cell membrane (such as between the liquid ordered and disordered phases) may include effects of a barrier at the interface in addition to the relative free energies of the species far from the interface. The BBS allows for direct transfer free energy calculations between bilayers without a need of a reference medium, such as bulk water, and thus provides an efficient simulation protocol for the quantitative characterization of protein-lipid interactions at all-atom resolution.

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes., (Copyright © 2019 Biophysical Society. All rights reserved.)

The two major classes of antidepressants, tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), inhibit neurotransmitter reuptake at synapses. They also have off-target effects on proteins other than neurotransmitter transporters, which may contribute to both desired changes in brain function and the development of side effects. Many proteins modulated by antidepressants are bilayer spanning and coupled to the bilayer through hydrophobic interactions such that the conformational changes underlying their function will perturb the surrounding lipid bilayer, with an energetic cost (Δ G def ) that varies with changes in bilayer properties. Here, we test whether changes in Δ G def caused by amphiphilic antidepressants partitioning into the bilayer are sufficient to alter membrane protein function. Using gramicidin A (gA) channels to probe whether TCAs and SSRIs alter the bilayer contribution to the free energy difference for the gramicidin monomer⇔dimer equilibrium (representing a well-defined conformational transition), we find that antidepressants alter gA channel activity with varying potency and no stereospecificity but with different effects on bilayer elasticity and intrinsic curvature. Measuring the antidepressant partition coefficients using isothermal titration calorimetry (ITC) or cLogP shows that the bilayer-modifying potency is predicted quite well by the ITC-determined partition coefficients, and channel activity is doubled at an antidepressant/lipid mole ratio of 0.02-0.07. These results suggest a mechanism by which antidepressants could alter the function of diverse membrane proteins by partitioning into cell membranes and thereby altering the bilayer contribution to the energetics of membrane protein conformational changes., (© 2019 Kapoor et al.)

Artificial and natural lipid membranes that elicit transmembrane signaling is are useful as a platform for channel-based biosensing. In this account we summarize our research on the design of transmembrane signaling associated with lipid bilayer membranes containing nanopore-forming compounds. Channel-forming compounds, such as receptor ion-channels, channel-forming peptides and synthetic channels, are embedded in planar and spherical bilayer lipid membranes to develop highly sensitive and selective biosensing methods for a variety of analytes. The membrane-bound receptor approach is useful for introducing receptor sites on both planar and spherical bilayer lipid membranes. Natural receptors in biomembranes are also used for designing of biosensing methods., (© 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Species that are currently listed under the genus Brevibacillus (formerly, Bacillus brevis cluster) have been a rich source of antimicrobial peptides for many decades. The first known peptide antibiotic, gramicidin, is presumed to be produced by a Brevibacillus sp. Members of the genus are widely spread in nature. They can be found in a variety of environments including intestinal tracts of animals, seawater, and soil. Some Brevibacillus strains have been used commercially as probiotics. Bioactive peptides produced by Brevibacillus spp. include antibacterial, antifungal and anti-invertebrate agents. Brevibacillus antimicrobial peptides are synthesized through ribosomal or nonribosomal pathway; these two groups can be further categorized based on specific structural features such as cyclization and presence of lipid chain. Some of the antimicrobial compounds produced by this genus share structural similarities that were overlooked previously. For example, the structural similarity between BT peptide, brevibacillin, and bogorol was revealed only recently. Here we review and classify Brevibacillus antimicrobial peptides and summarize their bioactivities and potential applications.

The activity of cell membrane inclusions (such as ion channels) is influenced by the host lipid membrane, to which they are elastically coupled. This coupling concerns the hydrophobic thickness of the bilayer (imposed by the length of the channel, as per the hydrophobic matching principle) but also its slope at the boundary of the inclusion. However, this parameter has never been measured so far. We combine small-angle x-ray scattering data and a complete elastic model to measure the slope for the model gramicidin channel and show that it is surprisingly steep in two membrane systems with very different elastic properties. This conclusion is confirmed and generalized by the comparison with recent results in the simulation literature and with conductivity measurements.

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 10 3 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.

An important limitation of standard classical molecular dynamics simulations is the inability to make or break chemical bonds. This restricts severely our ability to study processes that involve even the simplest of chemical reactions, the transfer of a proton. Existing approaches for allowing proton transfer in the context of classical mechanics are rather cumbersome and have not achieved widespread use and routine status. Here we reconsider the combination of molecular dynamics with periodic stochastic proton hops. To ensure computational efficiency, we propose a non-Boltzmann acceptance criterion that is heuristically adjusted to maintain the correct or desirable thermodynamic equilibria between different protonation states and proton transfer rates. Parameters are proposed for hydronium, Asp, Glu, and His. The algorithm is implemented in the program CHARMM and tested on proton diffusion in bulk water and carbon nanotubes and on proton conductance in the gramicidin A channel. Using hopping parameters determined from proton diffusion in bulk water, the model reproduces the enhanced proton diffusivity in carbon nanotubes and gives a reasonable estimate of the proton conductance in gramicidin A.

Peptide foldamers have been studied for over two decades and numerous sequence patterns have been shown to form well-defined three-dimensional arrangements in solution. In particular, helices of various geometries have been described. In this article, different concepts concerning the construction of helical foldameric peptides, for which the possibility of governing the sense of the formed helix was evidenced, are presented and discussed., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The canonical mechanism of gramicidin (gA) channel formation is transmembrane dimerization of nonconducting subunits that reside in opposite bilayer leaflets. The channels do not open and close; they appear and disappear due to subunit association and dissociation. Many different types of experiments support this monomer ↔ dimer mechanism. Recently, however, this mechanism was challenged, based on experiments with lipid vesicle-incorporated gA under conditions where vesicle fusion could be controlled. In these experiments, sustained channel activity was observed long after fusion had been terminated, which led to the proposal that gA single-channel current transitions result from closed-open transitions in long-lived bilayer-spanning dimers. This proposal is at odds with 40 years of experiments, but involves the key assumption that gA monomers do not exchange between bilayers. We tested the possibility of peptide exchange between bilayers using three different types of experiments. First, we demonstrated the exchange of gA between 1,2-dierucoyl-sn-glycero-3-phosphocholine (DC 22:1 PC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DC 18:1 PC) lipid vesicles using a fluorescence assay for gA channel activity. Second, we added gA-free DC 22:1 PC vesicles to both sides of planar DC 18:1 PC bilayers preincubated with gA, which reduced channel activity up to 10-fold. Third, we added gA-containing DC 22:1 PC vesicles to one or both sides of DC 18:1 PC planar bilayers, which produced much higher channel activity when the gA-containing vesicles were added to both sides of the bilayer, as compared to one side only. All three types of experiments show that gA subunits can exchange between lipid bilayers. The exchange of subunits between bilayers thus is firmly established, which becomes a crucial consideration with respect to the mechanism of channel formation., (Copyright © 2017 Biophysical Society. All rights reserved.)

General anesthetics revolutionized medicine by allowing surgeons to perform more complex and much longer procedures. This widely used class of drugs is essential to patient care, yet their exact molecular mechanism(s) are incompletely understood. One early hypothesis over a century ago proposed that nonspecific interactions of anesthetics with the lipid bilayer lead to changes in neuronal function via effects on membrane properties. This model was supported by the Meyer-Overton correlation between anesthetic potency and lipid solubility and despite more recent evidence for specific protein targets, in particular ion-channels, lipid bilayer-mediated effects of anesthetics is still under debate. We therefore tested a wide range of chemically diverse general anesthetics on lipid bilayer properties using a sensitive and functional gramicidin-based assay. None of the tested anesthetics altered lipid bilayer properties at clinically relevant concentrations. Some anesthetics did affect the bilayer, though only at high supratherapeutic concentrations, which are unlikely relevant for clinical anesthesia. These results suggest that anesthetics directly interact with membrane proteins without altering lipid bilayer properties at clinically relevant concentrations. Voltage-gated Na + channels are potential anesthetic targets and various isoforms are inhibited by a wide range of volatile anesthetics. They inhibit channel function by reducing peak Na + current and shifting steady-state inactivation toward more hyperpolarized potentials. Recent advances in crystallography of prokaryotic Na + channels, which are sensitive to volatile anesthetics, together with molecular dynamics simulations and electrophysiological studies will help identify potential anesthetic interaction sites within the channel protein itself.

The influence of the membrane on transmembrane proteins is central to a number of biological phenomena, notably the gating of stretch activated ion channels. Conversely, membrane proteins can influence the bilayer, leading to the stabilization of particular membrane shapes, topological changes that occur during vesicle fission and fusion, and shape-dependent protein aggregation. Continuum elastic models of the membrane have been widely used to study protein-membrane interactions. These mathematical approaches produce physically interpretable membrane shapes, energy estimates for the cost of deformation, and a snapshot of the equilibrium configuration. Moreover, elastic models are much less computationally demanding than fully atomistic and coarse-grained simulation methodologies; however, it has been argued that continuum models cannot reproduce the distortions observed in fully atomistic molecular dynamics simulations. We suggest that this failure can be overcome by using chemically and geometrically accurate representations of the protein. Here, we present a fast and reliable hybrid continuum-atomistic model that couples the protein to the membrane. We show that the model is in excellent agreement with fully atomistic simulations of the ion channel gramicidin embedded in a POPC membrane. Our continuum calculations not only reproduce the membrane distortions produced by the channel but also accurately determine the channel's orientation. Finally, we use our method to investigate the role of membrane bending around the charged voltage sensors of the transient receptor potential cation channel TRPV1. We find that membrane deformation significantly stabilizes the energy of insertion of TRPV1 by exposing charged residues on the S4 segment to solution., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

To change conformation, a protein must deform the surrounding bilayer. In this work, a three-dimensional continuum elastic model for gramicidin A in a lipid bilayer is shown to describe the sensitivity to thickness, curvature stress, and the mechanical properties of the lipid bilayer. A method is demonstrated to extract the gramicidin-lipid boundary condition from all-atom simulations that can be used in the three-dimensional continuum model. The boundary condition affects the deformation dramatically, potentially much more than typical variations in the material stiffness do as lipid composition is changed. Moreover, it directly controls the sensitivity to curvature stress. The curvature stress and hydrophobic surfaces of the all-atom and continuum models are found to be in excellent agreement. The continuum model is applied to estimate the enrichment of hydrophobically matched lipids near the channel in a mixture, and the results agree with single-channel experiments and extended molecular dynamics simulations from the companion article by Beaven et al. in this issue of Biophysical Journal., (Published by Elsevier Inc.)

Integral membrane protein function can be modulated by the host bilayer. Because biological membranes are diverse and nonuniform, we explore the consequences of lipid diversity using gramicidin A channels embedded in phosphatidylcholine (PC) bilayers composed of equimolar mixtures of di-oleoyl-PC and di-erucoyl-PC (dC 18:1 +dC 22:1 , respectively), di-palmitoleoyl-PC and di-nervonoyl-PC (dC 16:1 +dC 24:1 , respectively), and di-eicosenoyl-PC (pure dC 20:1 ), all of which have the same average bilayer chain length. Single-channel lifetime experiments, molecular dynamics simulations, and a simple lipid compression model are used in tandem to gain insight into lipid redistribution around the channel, which partially alleviates the bilayer deformation energy associated with channel formation. The average single-channel lifetimes in the two-component bilayers (95 ± 10 ms for dC 18:1 +dC 22:1 and 195 ± 20 ms for dC 16:1 +dC 24:1 ) were increased relative to the single-component dC 20:1 control bilayer (65 ± 10 ms), implying lipid redistribution. Using a theoretical treatment of thickness-dependent changes in channel lifetimes, the effective local enrichment of lipids around the channel was estimated to be 58 ± 4% dC 18:1 and 66 ± 2% dC 16:1 in the dC 18:1 +dC 22:1 and dC 16:1 +dC 24:1 bilayers, respectively. 3.5-μs molecular dynamics simulations show 66 ± 2% dC 16:1 in the first lipid shell around the channel in the dC 16:1 +dC 24:1 bilayer, but no significant redistribution (50 ± 4% dC 18:1 ) in the dC 18:1 +dC 22:1 bilayer; these simulated values are within the 95% confidence intervals of the experimental averages. The strong preference for the better matching lipid (dC 16:1 ) near the channel in the dC 16:1 +dC 24:1 mixture and lesser redistribution in the dC 18:1 +dC 22:1 mixture can be explained by the energetic cost associated with compressing the lipids to match the channel's hydrophobic length., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Specificity of small ions, the Hofmeister ranking, is long-known and has many applications including medicine. Yet it evades consistent theoretical description. Here we study the effect of Hofmeister anions on gramicidin A channels in lipid membranes. Counterintuitively, we find that conductance of this perfectly cation-selective channel increases about two-fold in the H 2 PO 4 - - ≈Br - ≈NO 3 - 4 - - series. Channel dissociation kinetics show even stronger dependence, with the dwell time increasing about 20-fold. While the conductance can be quantitatively explained by the changes in membrane surface potential due to exclusion of kosmotropes from (or accumulation of chaotropes at) the surface, the kinetics proved to be more difficult to treat. We estimate the effects of changes in the energetics at the bilayer surfaces on the channel dwell time, concluding that the change would have to be greater than typically observed for the Hofmeister effect outside the context of the lipid bilayer., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The effects of local anesthetics (LAs), including aminoamides and aminoesters, on the characteristics of single gramicidin A (GA) channels in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers were studied. Aminoamides, namely lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), and bupivacaine (BPV), reduced the conductance of GA channels. Aminoesters influenced the current fluctuations induced by GA differently; procaine (PC) did not affect the fluctuations, whereas tetracaine (TTC) distinctly reduced the conductance of single GA channels. Using electrophysiological technique, we estimated the changes in the membrane boundary potential at the adsorption of LAs; LDC, PLC, MPV, BPV, and TTC substantially increased, while PC did not affect it. To elucidate which component of the membrane boundary potential, the surface or dipole potential, is responsible for the observed effects of LAs, we employed a fluorescence assay. We found that TTC led to a significant increase in the membrane dipole potential, whereas the adsorption of LDC, PLC, MPV, BPV, and PC did not produce any changes in the membrane dipole potential. We concluded that aminoamides affected the surface potential of lipid bilayers. Together, these data suggest that the effects of LAs on the conductance of single GA channels are caused by their influence on membrane electrostatic potentials; the regulation of GA pores by aminoamides is associated with the surface potential of membranes, whereas TTC modulation of channel properties is predominantly due to changes in dipole potential of lipid bilayers. These data might provide some significant implications for voltage-gated ion channels of cell membranes.

Three unimolecular peptide channels have been designed and prepared by using the β-helical conformation of gramicidin A (gA). The new peptides bear one to three NH 3 + groups at the N-end and one to three CO 2 - groups at the C-end. These zwitterionic peptides were inserted into lipid bilayers in an orientation-selective manner. Conductance experiments on planar lipid bilayers showed that this orientation bias could lead to observable directional K + transport under multi-channel conditions. This directional transport behavior can further cause the generation of a current across a planar bilayer without applying a voltage. More importantly, in vesicles with identical external and internal KCl concentrations, the channels can pump K + across the lipid bilayer and cause a membrane potential., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that synthesize a myriad of diverse molecules. Tailoring domains have been co-opted into NRPSs to introduce further variety into nonribosomal peptide products. Linear gramicidin synthetase contains a unique formylation-tailoring domain in its initiation module (F-A-PCP). The structure of the F-A di-domain has previously been determined in a crystal form which had large solvent channels and no density for the minor A sub subdomain. An attempt was made to take advantage of this packing by removing the A sub subdomain from the construct (F-A Δsub ) in order to produce a crystal that could accommodate the PCP domain. In the resulting crystal the original packing network was still present, but a second network with the same packing and almost no contact with the original network took the place of the solvent channels and changed the space group of the crystal.

Host defense peptides (HDPs) are positively-charged and amphipathic components of the innate immune system that have demonstrated great potential to become the next generation of broad spectrum therapeutic agents effective against a vast array of pathogens and tumor. As such, many approaches have been taken to improve the therapeutic efficacy of HDPs. Amongst these methods, the incorporation of d-amino acids (d-AA) is an approach that has demonstrated consistent success in improving HDPs. Although, virtually all HDP review articles briefly mentioned about the role of d-AA, however it is rather surprising that no systematic review specifically dedicated to this topic exists. Given the impact that d-AA incorporation has on HDPs, this review aims to fill that void with a systematic discussion of the impact of d-AA on HDPs.

Classical molecular dynamic (MD) simulation of membrane proteins faces significant challenges in accurately reproducing and predicting experimental observables such as ion conductance and permeability due to its incapability of precisely describing the electronic interactions in heterogeneous systems. In this work, the free energy profiles of K(+) and Na(+) permeating through the gramicidin A channel are characterized by using the AMOEBA polarizable force field with a total sampling time of 1 μs. Our results indicated that by explicitly introducing the multipole terms and polarization into the electrostatic potentials, the permeation free energy barrier of K(+) through the gA channel is considerably reduced compared to the overestimated results obtained from the fixed-charge model. Moreover, the estimated maximum conductance, without any corrections, for both K(+) and Na(+) passing through the gA channel are much closer to the experimental results than any classical MD simulations, demonstrating the power of AMOEBA in investigating the membrane proteins.

Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations.

The impact of double bonds in fatty acyl tails of unsaturated lipids on the photodynamic inactivation of ion channels formed by the pentadecapeptide gramicidin A in a planar bilayer lipid membrane was studied. The presence of unsaturated acyl tails protected gramicidin A against photodynamic inactivation, with efficacy depending on the depth of a photosensitizer in the membrane. The protective effect of double bonds was maximal with membrane-embedded chlorin e6-monoethylenediamine monoamide dimethyl ester, and minimal - in the case of water-soluble tri-sulfonated aluminum phthalocyanine (AlPcS3) known to reside at the membrane surface. By contrast, the protective effect of the hydrophilic singlet oxygen scavenger ascorbate was maximal for AlPcS3 and minimal for amide of chlorin e6 dimethyl ester. The depth of photosensitizer position in the lipid bilayer was estimated from the quenching of photosensitizer fluorescence by iodide. Thus, the protective effect of a singlet oxygen scavenger against photodynamic inactivation of the membrane-inserted peptide is enhanced upon location of the photosensitizer and scavenger molecules in close vicinity to each other.

Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly (13)C/(15)N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided.

A new series of hydrogen-bonded helical aromatic hydrazide oligomers and polymer that bear phenylalanine tripeptide chains have been designed and synthesized. It was revealed that the helical structures could insert into lipid bilayers to form unimolecular channels. The longest oligomeric and polymeric helical channels exhibited an NH4(+)/K(+) selectivity that was higher than that of natural gramicidin A, whereas the transport of a short helical channel for Tl(+) could achieve an efficiency as high as that of gramicidin A.

A black lipid membrane (BLM) is a powerful platform for studying the electrophysiology of cell membranes as well as transmembrane proteins. However, BLMs have disadvantages in terms of stability, accessibility, and transportability, which preclude their industrial applications. To resolve these issues, frozen membrane precursor (MP) was devised to improve the transportability and storability of BLMs. As described previously, MP is a storable and transportable platform that can be delivered to the point-of-use, where BLMs are automatically formed upon thawing at room temperature. However, MP has an inconsistent thinning-out time, ranging from 30 min to 24 h, as well as a low success rate of BLM formation (~27%), which make it undesirable for practical use. In our study, polydimethylsiloxane (PDMS) was introduced as a replacement for conventionally used Teflon film to control thinning-out time. As such, we used a PDMS thin-film, a porous-structured hydrophobic polymer, and squalene, a high viscosity solvent, to facilitate membrane formation, whereas the absorption rates of solvents were controlled to achieve consistent BLM formation time. We successfully reduced thinning-out time down to <1 h as well as enhanced the success rate of BLM formation to greater than 80%. Moreover, we demonstrated the feasibility of our platform for use in drug screening using gramicidin A and guanidine.

The self-assembly of different classes of peptide, including cyclic peptides, amyloid peptides and surfactant-like peptides into nanotube structures is reviewed. The modes of self-assembly are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The interaction of membranes with peptides and proteins is largely determined by their amphiphilic character. Hydrophobic moments of helical segments are commonly derived from their two-dimensional helical wheel projections, and the same is true for β-sheets. However, to the best of our knowledge, there exists no method to describe structures in three dimensions or molecules with irregular shape. Here, we define the hydrophobic moment of a molecule as a vector in three dimensions by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule, e.g., partial unfolding, bending, and side-chain torsion angles. Extended all-atom molecular dynamics simulations are then used to calculate the equilibrium hydrophobic moments for two antimicrobial peptides, gramicidin S and PGLa, under different conditions. We show that their effective hydrophobic moment vectors reflect the distribution of polar and nonpolar patches on the molecular surface and the calculated electrostatic surface potential. A comparison of simulations in solution and in lipid membranes shows how the peptides undergo internal conformational rearrangement upon binding to the bilayer surface. A good correlation with solid-state NMR data indicates that the hydrophobic moment vector can be used to predict the membrane binding geometry of peptides. This method is available as a web application on http://www.ibg.kit.edu/HM/., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The mechanism by which amyloid-β (Aβ) produces brain dysfunction in patients with Alzheimer's disease is largely unknown. According to previous studies, Aβ might share perforating properties with gramicidin, a well-accepted membrane-disrupting peptide. Therefore, we hypothesize that the key steps leading to synaptotoxicity by Aβ and gramicidin involve peptide aggregation, pore formation, and calcium dysregulation. Here, we show that Aβ and gramicidin form aggregates enriched in β-sheet structures using electron microscopy, and Thioflavin and Congo Red staining techniques. Also, we found that Aβ and gramicidin display fairly similar actions in hippocampal cell membranes, i.e. inducing Ca(2+) entry and synaptoxicity characterized by the loss of synaptic proteins and a decrease in neuronal viability. These effects were not observed in a Ca(2+) free solution, indicating that both Aβ and gramicidin induce neurotoxicity by a Ca(2+)-dependent mechanism. Using combined perforated patch clamp and imaging recordings, we found that only Aβ produced a perforation that progressed from a small (Cl(-)-selective pore) to a larger perforation that allowed the entry of fluorescent molecules. Therefore, based on these results, we propose that the perforation at the plasma membrane by Aβ is a dynamic process that is critical in producing neurotoxicity similar to that found in the brains of AD patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pore-forming compounds are attracting much attention due to the signal transduction ability for the development of highly sensitive biosensing. In this review, we describe an overview of the recent advances made by our group in the design of molecular sensing interfaces of spherical and planar lipid bilayers and natural bilayers. The potential uses of pore-forming compounds, such as gramicidin and MCM-41, in lipid bilayers and natural glutamate receptor channels in biomembrane are presented.

In Alzheimer's disease, amyloid-β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β-sheet conformation, binds to the Aβ peptide hairpin-stacked fibril through β-sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four-times higher than that of the natural product was identified., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na(+) and organic cations through gramicidin channels and detecting the Cl(-)-channel functions of the (α5β2γ2) GABAA receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.

Biological ion channels present unique ionic properties. They can be highly permeable to ions, while selecting only one type of ions, without external energy supply. An important research field has been developed to transfer these properties to solid state nanoporous membranes in order to develop artificial biomimetic nanofilters. One of the promising ways to develop biomimetic structures is based on the direct insertion of the gramicidin A, i.e. an ionic channel, inside a nanopore. Experiments have recently proved the feasibility of such a hybrid membrane with very interesting results regarding the ionic selectivity. Here, we propose to interpret these experiments using theoretical molecular dynamic simulations which allow us to analyze more profoundly the structures of the proteins confined inside the nanopore and the relation between their conformation and the observed ionic properties.

A parallel finite element simulator, ichannel, is developed for ion transport through three-dimensional ion channel systems that consist of protein and membrane. The coordinates of heavy atoms of the protein are taken from the Protein Data Bank and the membrane is represented as a slab. The simulator contains two components: a parallel adaptive finite element solver for a set of Poisson-Nernst-Planck (PNP) equations that describe the electrodiffusion process of ion transport, and a mesh generation tool chain for ion channel systems, which is an essential component for the finite element computations. The finite element method has advantages in modeling irregular geometries and complex boundary conditions. We have built a tool chain to get the surface and volume mesh for ion channel systems, which consists of a set of mesh generation tools. The adaptive finite element solver in our simulator is implemented using the parallel adaptive finite element package Parallel Hierarchical Grid (PHG) developed by one of the authors, which provides the capability of doing large scale parallel computations with high parallel efficiency and the flexibility of choosing high order elements to achieve high order accuracy. The simulator is applied to a real transmembrane protein, the gramicidin A (gA) channel protein, to calculate the electrostatic potential, ion concentrations and I - V curve, with which both primitive and transformed PNP equations are studied and their numerical performances are compared. To further validate the method, we also apply the simulator to two other ion channel systems, the voltage dependent anion channel (VDAC) and α-Hemolysin (α-HL). The simulation results agree well with Brownian dynamics (BD) simulation results and experimental results. Moreover, because ionic finite size effects can be included in PNP model now, we also perform simulations using a size-modified PNP (SMPNP) model on VDAC and α-HL. It is shown that the size effects in SMPNP can effectively lead to reduced current in the channel, and the results are closer to BD simulation results., (Copyright © 2013 Wiley Periodicals, Inc.)