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The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered via a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes. The working principle of the sensor exploits the ability of Hg 2+ ions to complex mismatching thymine-thymine sequences between the PNA receptor moiety on Alm oligomers and free, thymine-based, single-stranded DNAs (ssDNAs) in solution, thus creating a stable base pair at the oligomer entrance. This generates a transducing mechanism which converts the metal ion complexation into a specific electrical signature of the self-assembled Alm oligomers, enabling selective Hg 2+ ion detection. The platform is programmable, whereby the simple exchange of the PNA sequence and its ssDNA counterpart in solution rendered the system selective for Cu 2+ ion detection. With further optimization, the presented solution has the potential to translate into miniaturized, cost-effective biosensors suitable for the real-time, label-free and continuous detection of metal ions or other biomolecules.

Membrane active peptides are known to porate lipid bilayers, but their exact permeabilization mechanism and the structure of the nanoaggregates they form in membranes have often been difficult to determine experimentally. For many sequences at lower peptide concentrations, transient leakage is observed in experiments, suggesting the existence of transient pores. For two well-know peptides, alamethicin and melittin, we show here that molecular mechanics simulations i) can directly distinguish equilibrium poration and non-equilibrium transient leakage processes, and ii) can be used to observe the detailed pore structures and mechanism of permeabilization in both cases. Our results are in very high agreement with numerous experimental evidence for these two peptides. This suggests that molecular simulations can capture key membrane poration phenomena directly and in the future may develop to be a useful tool that can assist experimental peptide design., (© 2024. The Author(s).)

Alamethicin, a peptide consisted of 20 amino acid residues, has been known to function as an antibiotic. The peptides self-associate in biological membranes, form an ion channel, and then induce cell death by leaking intracellular contents through a transmembrane pore of an ion channel. We investigated conformation and its thermal stability of alamethicin-A6 and -U6 in ethanol using proton nuclear magnetic resonance (NMR) spectroscopy; alamethicin-A6 and -U6 have the amino acid sequences of UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, respectively, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated by the under bars in the sequences, only the residue 6 differs between the alamethicins. We show that the alamethicins in ethanol form helix conformation in the region of the residues 2-11 and a non-regular conformation in the regions of the N- and C-termini, and that the helices are maintained up to 66 °C at least. Conformations in the region of the residues 12-18 of the alamethicins, however, are not well identified due to the lack of NMR data. In addition, we demonstrate that the amide proton chemical shift temperature coefficients' method, which is known as an indicator for intramolecular hydrogen bonds in peptides and proteins in aqueous solutions, can be also applied to the alamethicins in ethanol. Further, we show that the conformation around the C-terminus of alamethicin-A6 is restrained by intramolecular hydrogen bonds, whereas that of alamethicin-U6 is either restrained or unrestrained by intramolecular hydrogen bonds; the alamethicin-U6 molecules having the restrained and unrestrained conformations coexist in ethanol. We discuss the two types of conformations using a model chain consisting of particles linked by rigid bonds called as the free jointed chain., (© 2024. European Biophysical Societies' Association.)

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, C α -tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol-membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure-activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes., (© 2023 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Transmembrane assemblies of the peptaibol alamethicin (ALM) are among the most extensively studied ion channels not only because of their antimicrobial activity but also as models for channel structure and aggregation. In this study, several oligomeric states of ALM are investigated with molecular dynamics simulations to establish properties of the channel and obtain free energy profiles for ion transport and the corresponding values of conductance. The hexamer, heptamer, and octamer of ALM in phospholipid membrane are found to be stable but highly dynamic in barrel-stave structures, with calculated conductance equal to 18, 195, and 1270 pS, respectively, in 1 M KCl ion solution. The corresponding free energy profiles, reported for the first time, are reconstructed from simulations at applied voltage of 200 mV with the aid of the electrodiffusion model both with and without the knowledge of diffusivity. The calculated free energy barriers are equal to 2.5, 1.5, and 0.5 kcal/mol for K + and 4.0, 2.2, and 1.5 kcal/mol for Cl - , for hexamer, heptamer, and octamer, respectively. The calculated conductance and the ratio between conductance in consecutive states are in good agreement with those measured experimentally. This suggests that the hexamer is the lowest conducting state, with measured conductance equal to 19 pS. The selectivity of K + over Cl - is calculated as 1.5 and 2.3 for the octameric and heptameric channels, close to the selectivity measured for high-conductance states. Selectivity increases to 13 in the hexameric channel in which the narrowest Gln7 site has a pore radius of only ∼1.6 Å, again in accord with experiment. A good agreement found between calculated and measured conductance through a hexamer templated on cyclodextrin lands additional support for the results of our simulations, and the comparison with ALM reveals the dependence of conductance on the nature of phospholipid membrane., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The inhibition effect of amiloride on alamethicin ion channels was studied in a model zwitterionic floating bilayer lipid membrane (fBLM). The EIS studies indicated that amiloride prevents the transport of ions through the alamethicin channels leading to an overall increase in membrane resistance. The PM-IRRAS data demonstrated that amiloride has no influence on the secondary structure of alamethicin but restricts the insertion of the peptides into the bilayer and blocks ion transport through preformed alamethicin channels. The effect of amiloride on ion channel formation in the floating bilayer formed by a zwitterionic lipid was compared to those of previous studies involving negatively charged fBLMs and tethered zwitterionic lipid bilayers. The findings from these studies show that the effects of amiloride on ion channel formation strongly depend on the mobility and charge of the membrane lipids.

The structures of many membrane-bound proteins and polypeptides depend on the membrane potential. However, spectroscopically studying their structures under an applied field is challenging, because a potential is difficult to generate across more than a few bilayers. We study the voltage-dependent structures of the membrane-bound polypeptide, alamethicin, using a spectroelectrochemical cell coated with a rough, gold film to create surface plasmons. The plasmons sufficiently enhance the 2D IR signal to measure a single bilayer. The film is also thick enough to conduct current and thereby apply a potential. The 2D IR spectra resolve features from both 3 10 - and α-helical structures and cross-peaks connecting the two. We observe changes in the peak intensity, not their frequencies, upon applying a voltage. A similar change occurs with pH, which is known to alter the angle of alamethicin relative to the surface normal. The spectra are modeled using a vibrational exciton Hamiltonian, and the voltage-dependent spectra are consistent with a change in angle of the 3 10 - and α-helices in the membrane from 55 to 44°and from 31 to 60°, respectively. The 3 10 - and α-helices are coupled by approximately 10 cm -1 . These experiments provide new structural information about alamethicin under a potential difference and demonstrate a technique that might be applied to voltage-gated membrane proteins and compared to molecular dynamics structures.

Alamethicin is an antibiotic peptide comprising 20 amino acid residues and functions as an ion channel in biological membranes. Natural alamethicins have a variety of amino acid sequences. Two of them, used as a mixed sample in this study, are: UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated, only the amino acid at position six differs, and the two alamethicins are referred to as alamethicin-A6 and -U6, respectively. The conformation and thermal stability of alamethicin-A6 and -U6 in methanol were examined using proton nuclear magnetic resonance (NMR) spectroscopy. Both alamethicins form an α-helix between the 2nd and 11th residues. The N-terminal, 19th and C-terminal residues take a non-helical conformation. The structure between the 12th and 18th residues has not been well determined due to the absence of cross peaks in the two-dimensional NMR data. The α-helices are maintained up to 54 °C at least. In contrast to these similarities, it has been found that the length of the α-helix of alamethicin-U6 is somewhat shorter than that of alamethicin-A6, the intra-molecular hydrogen bonds formed by the amide proton of the seventh residue is much more thermally stable for alamethicin-U6 than for alamethicin-A6, and the C-terminal residue of alamethicin-U6 has higher mobility than that of alamethicin-A6. The mobility of the N- and C-terminal residues is discussed on the basis of a model chain which consists of particles connected by rigid links, and the physiological significance of the mobility is emphasized.

The droplet interface bilayer (DIB) method offers simple control over initial leaflet compositions in model membranes, enabling an experimental path to filling gaps in our knowledge about the interplay between compositional lipid asymmetry, membrane properties, and the behaviors of membrane-active species. Yet, the stability of lipid leaflet asymmetry in DIBs has received very little attention, particularly in the presence of peptides and ion channels that are often studied in DIBs. Herein, we demonstrate for the first time parallel, capacitance-based measurements of intramembrane potential with arrays of asymmetric DIBs assembled in a microfluidic device to characterize the stability of leaflet asymmetry over many hours in the presence and absence of membrane-active peptides. DIBs assembled from opposing monolayers of the ester (DPhPC) and ether (DOPhPC) forms of diphytanoyl-phosphatidylcholine yielded asymmetric bilayers with leaflet compositions that were stable for at least 18 h as indicated by a stable |137 mV| intramembrane potential. In contrast, the addition of surface-bound alamethicin peptides caused a gradual, concentration-dependent decrease in the magnitude of the dipole potential difference. Intermittent current-voltage measurements revealed that alamethicin in asymmetric DIBs also shifts the threshold voltage required to drive peptide insertion and ion channel formation. These outcomes take place over the course of 1 to 5 h after membrane formation, and suggest that alamethicin peptides promote lipid flip-flop, even in the un-inserted, surface-bound state, by disordering lipids in the monolayer to which they bind. Moreover, this methodology establishes the use of parallel electrophysiology for efficiently studying membrane asymmetry in arrays of DIBs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-β-d-glucose (β-Tg). The EIS data showed that the presence of alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 ± 0.2 nm. The pore formation by alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes.

Niosomes are used in studies for drug delivery or gene transfer. However, their physical properties and features relative to liposomes are not well documented. To characterize and more rationally optimize niosome formulations, the properties of these vesicle systems are compared to those of liposomes composed of phosphatidylcholine and phosphatidylethanolamine lipids plus cholesterol. Niosomes are highly stable and only slightly more leaky than liposomes as assayed by calcein leakage; the permeability for ions (KCl) is higher than that of liposomes. Contrary to liposomes, the size of niosomes decreases substantially upon freezing in liquid nitrogen and subsequent thawing, as shown by cryo-EM and dynamic light scattering. The packing of niosomal membranes was determined by laurdan fluorescence and is slightly lower than that of liposomes. We did not succeed in the functional reconstitution of the L-arginine/L-ornithine antiporter ArcD2 in niosomes, which we attribute to the non-ionic nature of the surfactants. The antimicrobial peptides alamethicin and melittin act similarly on niosomes and liposomes composed of unsaturated components, whereas both niosomes and liposomes are unaffected when saturated amphiphiles are used. In conclusion, in terms of stability and permeability for drug-size molecules niosomes are comparable to liposomes and they may offer an excellent, inexpensive alternative for delivery purposes.

All-atom molecular dynamics (MD) simulations are performed to examine the stabilities of a variety of binding configurations of alamethicin, a 20-amino-acid amphipathic peptide, in the bilayers of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine (DMPC). The binding free energy of alamethicin is calculated through a combination of MD simulation and the energy-representation theory of solutions, and it is seen that the transmembrane configuration is stable in both membranes. A surface-bound state is also found to be stable due to the balance between the attractive and repulsive interactions of the peptide with lipid and water, and the key role of water is pointed out for the stability in the interfacial region. A difference between the POPC and DMPC systems is noted when the polar C-terminal domain is buried in the hydrophobic region of the membrane. In POPC, the peptide is unfavorably located with that configuration due to the loss of electrostatic interaction between the peptide and lipid.

Antimicrobial peptides (AMPs) are the first line of defense after contact of an infectious invader, for example, bacterium or virus, with a host and an integral part of the innate immune system of humans. Their broad spectrum of biological functions ranges from cell membrane disruption over facilitation of chemotaxis to interaction with membrane-bound or intracellular receptors, thus providing novel strategies to overcome bacterial resistances. Especially, the clarification of the mechanisms and dynamics of AMP incorporation into bacterial membranes is of high interest, and different mechanistic models are still under discussion. In this work, we studied the incorporation of the peptaibol alamethicin (ALM) into tethered bilayer lipid membranes on electrodes in combination with surface-enhanced infrared absorption (SEIRA) spectroscopy. This approach allows monitoring the spontaneous and potential-induced ion channel formation of ALM in situ. The complex incorporation kinetics revealed a multistep mechanism that points to peptide-peptide interactions prior to penetrating the membrane and adopting the transmembrane configuration. On the basis of the anisotropy of the backbone amide I and II infrared absorptions determined by density functional theory calculations, we employed a mathematical model to evaluate ALM reorientations monitored by SEIRA spectroscopy. Accordingly, ALM was found to adopt inclination angles of ca. 69°-78° and 21° in its interfacially adsorbed and transmembrane incorporated states, respectively. These orientations can be stabilized efficiently by the dipolar interaction with lipid head groups or by the application of a potential gradient. The presented potential-controlled mechanistic study suggests an N-terminal integration of ALM into membranes as monomers or parallel oligomers to form ion channels composed of parallel-oriented helices, whereas antiparallel oligomers are barred from intrusion.

The antimicrobial action of the peptide antibiotic alamethicin (Alm) is commonly related to peptide self-assembling resulting in the formation of voltage-dependent channels in bacterial membranes, which induces ion permeation. To obtain a deeper insight into the mechanism of channel formation, it is useful to know the dependence of self-assembling on peptide concentration. With this aim, we studied Alm F50/5 spin-labeled analogs in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, for peptide-to-lipid (P/L) ratios varying between 1/1500 and 1/100. Pulsed electron-electron double resonance (PELDOR) spectroscopy reveals that even at the lowest concentration investigated, the Alm molecules assemble into dimers. Moreover, under these conditions, electron spin echo envelope modulation (ESEEM) spectroscopy of D 2 O-hydrated membranes shows an abrupt change from the in-plane to the trans-membrane orientation of the peptide. Therefore, we hypothesize that dimer formation and peptide reorientation are concurrent processes and represent the initial step of peptide self-assembling. By increasing peptide concentration, higher oligomers are formed. A simple kinetic model of equilibrium among monomers, dimers, and pentamers allows for satisfactorily describing the experimental PELDOR data. The inter-label distances in the oligomers obtained from PELDOR experiments become better resolved with increasing P/L ratio, thus suggesting that the supramolecular organization of the higher-order oligomers becomes more defined.

Alamethicin (Alm) is a 19-mer antimicrobial peptide produced by fungus Trichoderma viride. Above a threshold concentration, Alm forms pores across the membrane, providing a mechanism of its antimicrobial action. Here we show that at a small concentration which is below the threshold value, Alm participates in formation of nanoscale lipid-mediated clusters of guest lipid-like molecules in the membrane. These results are obtained by electron spin echo (ESE) technique-a pulsed version of electron paramagnetic resonance-on spin-labeled stearic acid in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer with Alm added at 1/200 peptide-to-lipid ratio. ESE decay measurements are interpreted assuming that stearic acid molecules in the membrane are assembling around the Alm molecule. One may suggest that this Alm capturing effect on the guest lipid-like molecules could be important for the peptide antimicrobial action.

Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C 19 F 3 -labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19 F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19 F- 19 F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In biological systems, intercellular communication is mediated by membrane proteins and ion channels that regulate traffic of ions and small molecules across cell membranes. A bioelectronic device with ion channels that control ionic flow across a supported lipid bilayer (SLB) should therefore be ideal for interfacing with biological systems. Here, we demonstrate a biotic-abiotic bioprotonic device with Pd contacts that regulates proton (H + ) flow across an SLB incorporating the ion channels Gramicidin A (gA) and Alamethicin (ALM). We model the device characteristics using the Goldman-Hodgkin-Katz (GHK) solution to the Nernst-Planck equation for transport across the membrane. We derive the permeability for an SLB integrating gA and ALM and demonstrate pH control as a function of applied voltage and membrane permeability. This work opens the door to integrating more complex H + channels at the Pd contact interface to produce responsive biotic-abiotic devices with increased functionality.

An all-atom molecular dynamics simulation of the archetype barrel-stave alamethicin (alm) pore in a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer at 313 K indicates that ∼7 μs is required for equilibration of a preformed 6-peptide pore; the pore remains stable for the duration of the remaining 7 μs of the trajectory, and the structure factors agree well with experiment. A 5 μs simulation of 10 surface-bound alm peptides shows significant peptide unfolding and some unbinding, but no insertion. Simulations at 363 and 413 K with a -0.2 V electric field yield peptide insertion in 1 μs. Insertion is initiated by the folding of residues 3-11 into an α-helix, and mediated by membrane water or by previously inserted peptides. The stability of five alm pore peptides at 413 K with a -0.2 V electric field demonstrates a significant preference for a transmembrane orientation. Hence, and in contrast to the cationic antimicrobial peptide described in the following article, alm shows a strong preference for the inserted over the surface-bound state., (Published by Elsevier Inc.)

Alamethicin is a well-studied antimicrobial peptide (AMP) that kills Gram-positive bacteria. It forms narrow, barrel-stave pores in planar lipid bilayers. We present a detailed, time-resolved microscopy study of the sequence of events during the attack of alamethicin on individual, live Bacillus subtilis cells expressing GFP in the cytoplasm. At the minimum inhibitory concentration (MIC), the first observed symptom is the halting of growth, as judged by the plateau in measured cell length vs time. The data strongly suggest that this growth-halting event occurs prior to membrane permeabilization. Gradual degradation of the proton-motive force, inferred from a decrease in pH-dependent GFP fluorescence intensity, evidently begins minutes later and continues over about 5 min. There follows an abrupt permeabilization of the cytoplasmic membrane to the DNA stain Sytox Orange and concomitant loss of small osmolytes, causing observable cell shrinkage, presumably due to decreased turgor pressure. This permeabilization of the cytoplasmic membrane occurs uniformly across the entire membrane, not locally, on a timescale of 5s or less. GFP gradually leaks out of the cell envelope, evidently impeded by the shrunken peptidoglycan layer. Disruption of the cell envelope by alamethicin occurs in stages, with larger and larger species permeating the envelope as time evolves over tens of minutes. Some of the observed symptoms are consistent with the formation of barrel-stave pores, but the data do not rule out "chaotic pore" or "carpet" mechanisms. We contrast the effects of alamethicin and the human cathelicidin LL-37 on B. subtilis., (Copyright © 2016 Elsevier B.V. All rights reserved)

The structure, topology and orientation of membrane-bound antibiotic alamethicin were studied using solid state nuclear magnetic resonance (NMR) spectroscopy. (13)C chemical shift interaction was observed in [1-(13)C]-labeled alamethicin. The isotropic chemical shift values indicated that alamethicin forms a helical structure in the entire region. The chemical shift anisotropy of the carbonyl carbon of isotopically labeled alamethicin was also analyzed with the assumption that alamethicin molecules rotate rapidly about the bilayer normal of the phospholipid bilayers. It is considered that the adjacent peptide planes form an angle of 100° or 120° when it forms α-helix or 310-helix, respectively. These properties lead to an oscillation of the chemical shift anisotropy with respect to the phase angle of the peptide plane. Anisotropic data were acquired for the 4 and 7 sites of the N- and C-termini, respectively. The results indicated that the helical axes for the N- and C-termini were tilted 17° and 32° to the bilayer normal, respectively. The chemical shift oscillation curves indicate that the N- and C-termini form the α-helix and 310-helix, respectively. The C-terminal 310-helix of alamethicin in the bilayer was experimentally observed and the unique bending structure of alamethicin was further confirmed by measuring the internuclear distances of [1-(13)C] and [(15)N] doubly-labeled alamethicin. Molecular dynamics simulation of alamethicin embedded into dimyristoyl phophatidylcholine (DMPC) bilayers indicates that the helical axes for α-helical N- and 310-helical C-termini are tilted 12° and 32° to the bilayer normal, respectively, which is in good agreement with the solid state NMR results., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A concise synthetic pathway yielding to hydrophobic α,α-disubstituted disilylated amino acids based on a hydrosilylation reaction is described. As a first example of utilization in solid-phase peptide synthesis, TESDpg was introduced in replacement of Aib in an alamethicin sequence, leading to analogues with modified physicochemical properties and conserved helical structures. This study highlights the potential of these new amino acids as tools for peptide modulation.

Total syntheses and complete characterizations of singly substituted PheCN -based analogs of alamethicin AlaP, which is active on model and natural membranes, and the TM peptide, which inserts in a transmembrane orientation in lipid bilayers, are reported. The syntheses of the AlaP analogs were performed in solution, while those of TM and its analogs were carried out by solid phase. Using the cyanophenyl fluorescence and infrared (IR) absorption probe, an in-depth investigation of the self-association, membrane-interacting, permeabilizing, and orientation properties of these peptides were conducted. The aromatic residue incorporated induces only a negligible modification to the properties of the parent peptides. The PheCN IR absorption band was located between 2228 and 2230 cm(-1) for all peptides, irrespective of the position of labeling. By contrast, as the width of this band varied significantly with the depth of probe insertion in the bilayer, it could represent a good marker of the PheCN position in phospholipid membranes., (© 2015 Wiley Periodicals, Inc.)

Interaction between peptide and lipid membrane plays a major role in biological activity of membrane-active peptide. We describe here a new biocompatible polymeric assembly to support membrane peptide. Specifically, chitosan-graft-poly(sulfobetaine methacrylate) (CS-g-PSBMA) was synthesized for alamethicin assembly by controlled polymerization under γ-ray irradiation. The graft copolymer could self-assemble into micelles in distilled water for supporting alamethicin. The assembly of alamethicin with CS-g-PSBMA micelles in aqueous solutions was related with the ratio of alamethicin/CS-g-PSBMA: the more alamethicin, the smaller sizes of the hybrid complex. Moreover, alamethicin penetrated into the hydrophobic cores of CS-g-PSBMA micelles while displayed secondary helical conformation in the complex. The results indicate that CS-g-PSBMA assemblies can be used to support membrane peptide., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Many antimicrobial peptides (AMPs) kill bacteria by disrupting the lipid bilayer structure of their inner membrane. However, there is only limited quantitative information in the literature to differentiate between AMPs of differing molecular properties, in terms of how they interact with the membrane. In this study, we have used quartz crystal microbalance with dissipation monitoring (QCM-D) to probe the interactions between a supported bilayer membrane of egg phosphatidylcholine (egg PC) and four structurally different AMPs: alamethicin, chrysophsin-3, indolicidin, and sheep myeloid antimicrobial peptide (SMAP-29). Multiple signatures from the QCM-D measurements were extracted, differentiating the AMPs, that provide information on peptide addition to and lipid removal from the membrane, the dynamics of peptide-membrane interactions and the rates at which the peptide actions are initiated. The mechanistic variations in peptide action were related to the fundamental structural properties of the peptides including the hydrophobicity, hydrophobic moment, and the probability of α-helical secondary structures., (Published by Elsevier B.V.)

Cell membranes are complex mixtures of lipids, proteins, and other molecules that serve as active, semipermeable barriers between cells, as well as between their internal organelles, and the surrounding medium. Their compositions and structures are tightly regulated to ensure proper function. Cholesterol is a key component in mammalian cellular membranes, where it serves to maintain membrane fluidity and permeability. Here, the interaction of alamethicin, a 20 amino acid residue peptide that creates transmembrane pores in lipid bilayer membranes in a concentration-dependent manner, with bilayer membranes composed of dimyristoyl phosphatidylcholine (DMPC) and cholesterol (Chol) was studied. Small-angle neutron scattering (SANS) data demonstrate that a low concentration of alamethicin (peptide-to-lipid ratio of 1/200) disrupts a lateral inhomogeneity seen in peptide-free DMPC:Chol vesicles, which analysis of the SANS data indicates are Chol-rich and Chol-poor phases having different thicknesses. Alamethicin disrupts this structure, producing laterally homogeneous bilayers that are thinner than either phase of the peptide-free bilayers, and possess a strong asymmetry in the Chol content of the inner and outer bilayer leaflets. The results suggest that a secondary membrane disruption mechanism exists in parallel with the well-understood cytotoxic membrane permeabilization that results when alamethcin forms transmembrane pores. Specifically, the peptide can disrupt laterally organized lipidic structures in cell membranes, as well as significantly perturb the compositions of the inner and outer leaflets of the membrane. The existence of a secondary mechanism of action against cellular membranes for alamethicin raises the possibility that other membrane-active peptides function similarly.

In this work, we investigate the structure, dynamic and thermodynamic properties of noncanonical disubstituted amino acids (α,α-dialkyl glycines), also known as non-natural amino acids, in the peptaibol Alamethicin. The amino acids under study are Aib (α-amino isobutyric acid or α-methyl alanine), Deg (α,α-diethyl glycine), Dpg (α,α-dipropyl glycine), Dibg (α,α-di-isobutyl glycine), Dhg (α,α-dihexyl glycine), DΦg (α,α-diphenyl glycine), Dbzg (α,α-dibenzyl glycine), Ac6c (α,α-cyclohexyl glycine), and Dmg (α,α-dihydroxymethyl glycine). It is hypothesized that these amino acids are able to induce well-defined secondary structure in peptidomimetics. To test this hypothesis, new peptidomimetics of Alamethicin were constructed by replacing the native Aib positions of Alamethicin by one or more new α,α-dialkyl glycines. Dhg and Ac6c demonstrated the capacity to induce well-defined α-helical structures. Dhg and Ac6c also promote the thermodynamic stabilization of these peptides in a POPC model membrane and are better alternatives to the Aib in Alamethicin. These noncanonical amino acids also improved secondary structure properties, revealing preorganization in water and maintenance of α helical structure in POPC. We show that it is possible to optimize the helicity and thermodynamic properties of native Alamethicin, and we suggest that these amino acids could be incorporated in other peptides with similar structural effect.

We prepared, by solution-phase methods, and fully characterized three analogs of the membrane-active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N-terminus, at position 9 (central region) or at position 19 (C-terminus), and with the three Gln at positions 7, 18, and 19 replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a γ-trifluoroacetylated α,γ-diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val(9) or Glu(OMe)(19) amino acid. We performed a detailed conformational analysis of the three analogs (using FT-IR absorption, CD, 2D-NMR, and X-ray diffraction), which clearly showed that Tfa labeling does not introduce any dramatic backbone modification in the predominantly α-helical structure of the parent peptaibiotic. The results of an initial solid-state (19)F-NMR study on one of the analogs favor the conclusion that the Tfa group is a very promising reporter for the analysis of peptaibioticmembrane interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs depend on the position of the Tfa label in the peptide sequence., (Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.)

Peptides confer interesting properties to materials, supramolecular assemblies and to lipid membranes and are used in analytical devices or within delivery vehicles. Their relative ease of production combined with a high degree of versatility make them attractive candidates to design new such products. Here, we review and demonstrate how CD- and solid-state NMR spectroscopic approaches can be used to follow the reconstitution of peptides into membranes and to describe some of their fundamental characteristics. Whereas CD spectroscopy is used to monitor secondary structure in different solvent systems and thereby aggregation properties of the highly hydrophobic domain of p24, a protein involved in vesicle trafficking, solid-state NMR spectroscopy was used to deduce structural information and the membrane topology of a variety of peptide sequences found in nature or designed. (15)N chemical shift solid-state NMR spectroscopy indicates that the hydrophobic domain of p24 as well as a designed sequence of 19 hydrophobic amino acid residues adopt transmembrane alignments in phosphatidylcholine membranes. In contrast, the amphipathic antimicrobial peptide magainin 2 and the designed sequence LK15 align parallel to the bilayer surface. Additional angular information is obtained from deuterium solid-state NMR spectra of peptide sites labelled with (2)H3-alanine, whereas (31)P and (2)H solid-state NMR spectra of the lipids furnish valuable information on the macroscopic order and phase properties of the lipid matrix. Using these approaches, peptides and reconstitution protocols can be elaborated in a rational manner, and the analysis of a great number of peptide sequences is reviewed. Finally, a number of polypeptides with membrane topologies that are sensitive to a variety of environmental conditions such as pH, lipid composition and peptide-to-lipid ratio will be presented., (Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.)

Bicelles are model membrane systems that can be macroscopically oriented in a magnetic field at physiological temperature. The macroscopic orientation of bicelles allows to detect, by means of magnetic resonance spectroscopies, small changes in the order of the bilayer caused by solutes interacting with the membrane. These changes would be hardly detectable in isotropic systems such as vesicles or micelles. The aim of this work is to show that bicelles represent a convenient tool to investigate the behavior of antimicrobial peptides (AMPs) interacting with membranes, using electron paramagnetic resonance (EPR) spectroscopy. We performed the EPR experiments on spin-labeled bicelles using various AMPs of different length, charge, and amphipathicity: alamethicin, trichogin GA IV, magainin 2, HP(2-20), and HPA3. We evaluated the changes in the order parameter of the spin-labeled lipids as a function of the peptide-to-lipid ratio. We show that bicelles labeled at position 5 of the lipid chains are very sensitive to the perturbation induced by the AMPs even at low peptide concentrations. Our study indicates that peptides that are known to disrupt the membrane by different mechanisms (i.e., alamethicin vs magainin 2) show very distinct trends of the order parameter as a function of peptide concentration. Therefore, spin-labeled bicelles proved to be a good system to evaluate the membrane disruption mechanism of new AMPs., (Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.)

The PELDOR technique was used to obtain the spectra of distances between spin labels for mono and double TOAC substituted analogues of [Glu(OMe)(7,18,19)] alamethicin F50/5 (Alm') peptaibiotic on the surface of the organic sorbent Oasis HLB and in ethanol solution at 77 K. For the double-labeled Alm', the free radical probes are at positions 1 and 16 (Alm'1,16). The intra- and intermolecular contributions to the PELDOR time traces were separated, with regard to the fractality of the system studied. We established that on HLB the labeled Alm' molecules are prone to aggregation. The distance spectra for Alm'1,16 show that, in both adsorbed state and in ethanol solution, the peptaibiotic is predominantly folded in the α-helix conformation. We assign the asymmetry of the distance spectrum in both cases to the occurrence of an admixture of more elongated α/3(10)-helical conformers. The portion of these conformers is higher for the peptide adsorbed on HLB. We speculate that both the broadening of the basic spectrum line at r(max) = 2.0 nm and the increase in the contribution of elongated conformers might be associated with the spread of the peptaibiotic adsorption sites on HLB as compared with the more uniform Alm'1,16 trap structure in frozen ethanol solution. The aggregates of mono-labeled Alm'1 and Alm'16 also studied. The intermolecular distance spectrum for Alm'1 on HLB is shifted toward longer distances as compared with those of Alm'16. This result suggests that in the aggregates Alm' molecules are preferentially oriented with their C-terminal regions in the vicinity.

Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents.

We present a method to calculate (31)P solid-state NMR spectra based on the dynamic input from extended molecular dynamics (MD) simulations. The dynamic information confered by MD simulations is much more comprehensive than the information provided by traditional NMR dynamics models based on, for example, order parameters. Therefore, valuable insight into the dynamics of biomolecules may be achieved by the present method. We have applied this method to study the dynamics of lipid bilayers containing the antimicrobial peptide alamethicin, and we show that the calculated (31)P spectra obtained with input from MD simulations are in agreement with experiments under a large range of different sample conditions, including vesicles and oriented samples with and without peptides. We find that the changes in the (31)P spectra upon addition of peptide stem from lipids with reduced diffusion due to peptide-lipid interactions.

The structure and energetics of alamethicin Rf30 monomer to nonamer in cylindrical pores of 5 to 11Å radius are investigated using molecular dynamics simulations in an implicit membrane model that includes the free energy cost of acyl chain hydrophobic area exposure. Stable, low energy pores are obtained for certain combinations of radius and oligomeric number. The trimer and the tetramer formed 6Å pores that appear closed while the larger oligomers formed open pores at their optimal radius. The hexamer in an 8Å pore and the octamer in an 11Å pore give the lowest effective energy per monomer. However, all oligomers beyond the pentamer have comparable energies, consistent with the observation of multiple conductance levels. The results are consistent with the widely accepted "barrel-stave" model. The N terminal portion of the molecule exhibits smaller tilt with respect to the membrane normal than the C terminal portion, resulting in a pore shape that is a hybrid between a funnel and an hourglass. Transmembrane voltage has little effect on the structure of the oligomers but enhances or decreases their stability depending on its orientation. Antiparallel bundles are lower in energy than the commonly accepted parallel ones and could be present under certain experimental conditions. Dry aggregates (without an aqueous pore) have lower average effective energy than the corresponding aggregates in a pore, suggesting that alamethicin pores may be excited states that are stabilized in part by voltage and in part by the ion flow itself.

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., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Alamethicin is a 20-amino-acid, α-helical antimicrobial peptide that is believed to kill bacteria through pore formation in the inner membranes. We used quartz crystal microbalance with dissipation monitoring (QCM-D) to explore the interactions of alamethicin with a supported lipid bilayer. Changes in frequency (Δf) and dissipation (ΔD) measured at different overtones as a function of peptide concentration were used to infer peptide-induced changes in the mass and rigidity of the membrane as well as the orientation of the peptide in the bilayer. The measured Δf were positive, corresponding to a net mass loss from the bilayer, with substantial mass losses at 5 μM and 10 μM alamethicin. The measured Δf at various overtones were equal, indicating that the mass change in the membrane was homogeneous at all depths consistent with a vertical peptide insertion. Such an orientation coupled to the net mass loss was in agreement with cylindrical pore formation and the negligibly small ΔD suggested that the peptide walls of the pores stabilized the surrounding lipid organization. Dynamics of the interactions examined through Δf vs. ΔD plots suggested that the peptides initially inserted into the membrane and caused disordering of the lipids. Subsequently, lipids were removed from the bilayer to create pores and alamethicin caused the remaining lipids to reorder and stabilize within the membrane. Based on model calculations, we concluded that the QCM-D data cannot confirm or rule out whether peptide clusters coexist with pores in the bilayer. We have also proposed a way to calculate the peptide-to-lipid ratio (P/L) in the bilayer from QCM-D data and found the calculated P/L as a function of the peptide concentration to be similar to the literature data for vesicle membranes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A diisopropylcarbodiimide/Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) coupling cocktail was successfully incorporated into the automated microwave-assisted synthesis of two peptaibols and one analog, whose previously reported syntheses were complicated by steric hindrance. This method utilizes commercially available reagents and affords alamethicin F50/5 and bergofungin D in high yields and purities along with an appreciable reduction of synthesis time and cost when compared to previously reported methods.

Ion channels are specialized proteins that enable the movement of charges through otherwise impermeable lipidic membranes. Their action is essential in living organisms facilitating electric signaling, muscle contraction or osmotic stress response among other effects. The protein and the lipid charges configure a polarized interface that yields local ionic concentrations and electric potentials that are very different from those of the bulk electrolyte. The combined effect of gradients of ionic concentration and electric potential causes the transport of ions through channels. Here we analyze charge regulation effects in different protein-lipid conformations, stressing how important is the role of electrostatic interactions in the ion channel function that traditionally has been rationalized paying attention mainly to changes in pore size. Tuning lipid charge combined with conductance and selectivity measurements is shown to be a complementary method to evidence lipid involvement in the structure of a biological ion channel.

The structure and energetics of alamethicin Rf30 monomer to nonamer in cylindrical pores of 5 to 11Å radius are investigated using molecular dynamics simulations in an implicit membrane model that includes the free energy cost of acyl chain hydrophobic area exposure. Stable, low energy pores are obtained for certain combinations of radius and oligomeric number. The trimer and the tetramer formed 6Å pores that appear closed while the larger oligomers formed open pores at their optimal radius. The hexamer in an 8Å pore and the octamer in an 11Å pore give the lowest effective energy per monomer. However, all oligomers beyond the pentamer have comparable energies, consistent with the observation of multiple conductance levels. The results are consistent with the widely accepted "barrel-stave" model. The N terminal portion of the molecule exhibits smaller tilt with respect to the membrane normal than the C terminal portion, resulting in a pore shape that is a hybrid between a funnel and an hourglass. Transmembrane voltage has little effect on the structure of the oligomers but enhances or decreases their stability depending on its orientation. Antiparallel bundles are lower in energy than the commonly accepted parallel ones and could be present under certain experimental conditions. Dry aggregates (without an aqueous pore) have lower average effective energy than the corresponding aggregates in a pore, suggesting that alamethicin pores may be excited states that are stabilized in part by voltage and in part by the ion flow itself., (© 2013.)

Ion channels allow the influx and efflux of specific ions through a plasma membrane. Many ion channels can sense, for example, the membrane potential (the voltage gaps between the inside and the outside of the membrane), specific ligands such as neurotransmitters, and mechanical tension within the membrane. They modulate cell function in response to these stimuli. Researchers have focused on developing peptide- and non-peptide-based model systems to elucidate ion-channel protein functions and to create artificial sensing systems. In this Account, we employed a typical peptide that forms ion channels,alamethicin, as a model to evaluate our methodologies for controlling the assembly states of channel-forming molecules in membranes. As alamethicin self-assembles in membranes, it prompts channel formation, but number of peptide molecules in these channels is not constant. Using planar-lipid bilayer methods, we monitored the association states of alamethicin in real time. Many ligand-gated, natural-ion channel proteins have large extramembrane domains. As these proteins interact with specific ligands, those conformational alterations in the extramembrane domains are transmitted to the transmembrane, pore-forming domains to open and close the channels. We hypothesized that if we conjugated suitable extramembrane segments to alamethicin, ligand binding to the extramembrane segments could alter the structure of the extramembrane domains and influence the association states or association numbers of alamethicin in the membranes. We could then assess those changes by using single-channel current recording. We found that we could modulate channel assembly and eventual ion flux with attached leucine-zipper extramembrane peptide segments. Using conformationally switchable leucine-zipper extramembrane segments that respond to Fe(3+), we fabricated an artificial Fe(3+)-sensitive ion channel; a decrease in the helical content of the extramembrane segment led to an increase in the channel current. When we added a calmodulin C-terminus segment, we formed a channel that was sensitive to Ca(2+). This result demonstrated that we could prepare artificial channels that were sensitive to specific ligands by adding appropriate extramembrane segments from natural protein motifs that respond to external stimuli. In conclusion, our research points to the possibility of creating tailored sensor or signal transduction systems through the conjugation of a conformationally switchable extramembrane peptide/protein segment to a suitable transmembrane peptide segment.

Alamethicin is a 19-amino-acid residue hydrophobic peptide of the peptaibol family that has been the object of numerous studies for its ability to produce voltage-dependent ion channels in membranes. In this work, for the first time electron paramagnetic resonance spectroscopy was applied to study the interaction of alamethicin with oriented bicelles. We highlighted the effects of increasing peptide concentrations on both the peptide and the membrane in identical conditions, by adopting a twofold spin labeling approach, placing a nitroxide moiety either on the peptide or on the phospholipids. The employment of bicelles affords additional spectral resolution, thanks to the formation of a macroscopically oriented phase that allows to gain information on alamethicin orientation and dynamics. Moreover, the high viscosity of the bicellar solution permits the investigation of the peptide aggregation properties at physiological temperature. We observed that, at 35°C, alamethicin adopts a transmembrane orientation with the peptide axis forming an average angle of 25° with respect to the bilayer normal. Moreover, alamethicin maintains its dynamics and helical tilt constant at all concentrations studied. On the other hand, by increasing the peptide concentration, the bilayer experiences an exponential decrease of the order parameter, but does not undergo micellization, even at the highest peptide to lipid ratio studied (1:20). Finally, the aggregation of the peptide at physiological temperature shows that the peptide is monomeric at peptide to lipid ratios lower than 1:50, then it aggregates with a rather broad distribution in the number of peptides (from 6 to 8) per oligomer., (© 2013.)

We address the problem of antimicrobial peptides that create pores in lipid bilayers, focusing on voltage-temperature dependence of pore opening. Two novel experiments (voltage clamp with alamethicin as an emblematic representative of these peptides and neutron reflectivity of lipid monolayer at solid-water interface under electric field) serve to revise the only current theoretical model. We introduce a general contribution of peptide adsorption and electric field as being responsible for an unbalanced tension of the two bilayer leaflets and we claim that the main entropy cost of one pore opening is due to the corresponding excluded area for lipid translation.

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

Implicit membrane models usually treat the membrane as a hydrophobic slab and neglect lateral pressure/curvature stress effects. As a result, they cannot distinguish, for example, PE from PC lipids. Here, the implicit membrane model IMM1 is extended to include these effects using a combination of classical thermodynamics and membrane elasticity theory. The proposed model is tested by molecular dynamics simulation of the peptides alamethicin, melittin, cyclotide kalata B1, 18A, and KKpL15. The lateral pressure term stabilizes interfacial binding due to the negative pressure at the hydrocarbon-water interface. In agreement with experiment, increase in the peptide/lipid molar ratio shifts the equilibrium from the interfacial to the transmembrane orientation. Simulations of mixed DOPC/DOPE bilayers show that increase of the DOPE mole fraction in general stabilizes interfacial orientations and destabilizes transmembrane orientations. The extent of the stabilization or destabilization varies depending on the exact position of the peptides. The computational results are in good agreement with experiments., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bilayer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer associates closely with peptide concentration in solution and saturates at ~4%, compared to the ~8% maximum thinning observed for the correspondingly premixed peptide-MLM bilayers. Linearly scaling the thinning of peptide-ULV bilayers to that of the corresponding peptide-MLM of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant of melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy-Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC share a same thinning constant upon binding of a hydrophobic peptide alamethicin; this result supports the linear scaling approach used in the melittin-ULV bilayer thinning for thermodynamic binding parameters of water-soluble peptides., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We present direct visualization of pores formed by alamethicin (Alm) in a matrix of phospholipids using electrochemical scanning tunneling microscopy (EC-STM). High-resolution EC-STM images show individual peptide molecules forming channels. The channels are not dispersed randomly in the monolayer but agglomerate forming 2D nanocrystals with a hexagonal lattice in which the average channel-channel distance is 1.90 ± 0.1 nm. The STM images suggest that each Alm is shared between the two adjacent channels. Every channel consists of six Alm molecules. Three or four of these molecules have the hydrophilic group oriented toward the center of the channel allowing for water column formation inside the channel. The dimensions of the central pore in the images are consistent with the dimension of the water column in a model of hexameric pore proposed in the literature. The images obtained in this work validate the barrel-stave model of the pore formed in phospholipid membranes by amphiphatic peptides. They also provide direct evidence for cluster formation by such pores.

Antimicrobial peptides usually kill bacteria by making their membranes permeable. Two main models (barrel-stave and Shai-Matsuzaki-Huang) have been proposed to describe the peptide-induced pores. Although several experimental tests can be exploited to discriminate between these two models, the dependence of peptide activity on lipid properties (intrinsic curvature and membrane thickness) is routinely used for this purpose. Here, we show that, contrary to what is currently accepted, this criterion is unreliable.

Bacterial resistance to classical antibiotics is a serious medical problem, which continues to grow. Small antimicrobial peptides represent a potential solution and are increasingly being developed as novel therapeutic agents. Many of these peptides owe their antibacterial activity to the formation of trans-membrane ion-channels resulting in cell lysis. However, to further develop the field of peptide antibiotics, a thorough understanding of their mechanism of action is needed. Alamethicin belongs to a class of peptides called peptaibols and represents one of these antimicrobial peptides. To examine the dynamics of assembly and to facilitate a thorough structural evaluation of the alamethicin ion-channels, we have applied click chemistry for the synthesis of templated alamethicin multimers covalently attached to cyclodextrin-scaffolds. Using oriented circular dichroism, calcein release assays, and single-channel current measurements, the α-helices of the templated multimers were demonstrated to insert into lipid bilayers forming highly efficient and remarkably stable ion-channels.

Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analog MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136ns to 216ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melittin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its configuration diverges from that of melittin and it is probably metastable., (Copyright © 2012 Elsevier B.V. All rights reserved.)