Interactions of the channel forming peptide alamethicin with artificial and natural membranes

Alamethicin and related α-aminoisobutyric acid peptides form transmembrane channels across lipid bilayers. This article briefly reviews studies on the effect of alamethicin on lipid phase transitions in lipid bilayers and on mitochondrial oxidative phosphorylation. Fluorescence polarization studies, employing 1,6-diphenyl-1,3,5-hexatriene as a probe, suggest that alamethicin fluidizes lipid bilayers below the phase transition t-emperature, but has little effect above the gel-liquid crystal transition point. Alamethicin is shown to function as an uncoupler of oxidative phosphorylation in rat liver mitochondria. The influence of alamethicin on mitochondrial respiration is modulated by the phosphate ion concentration in the medium. Classical uncoupling activity is evident at low phosphate levels while inhibitory effects set in at higher phosphate concentrations. Time-dependent changes in respiration rates following peptide addition are rationalized in terms of alamethicin interactions with mitochondrial membrane components.     

Effects of n-alkanes on the morphology of lipid bilayers A freeze-fracture and negative stain analysis

The effect of $\text{n-}\text{alkanes}$ on the ultrastructure of lipid bilayers has been investigated using freeze-fracture and negative stain electron microscopy. It has been found that the morphology of bilayers containing the long alkane tetradecane is quite different from bilayers containing the short alkane hexane. The smooth fracture faces of gel and liquid crystalline state bilayers are unmodified by tetradecane. However, hexane dramatically alters the hydrophobic bilayer interior, producing large (20 to 50 nm) mounds and depressions in the fracture faces. The fracture steps in these multilayer preparations containing hexane are variable in thickness and often considerably wider than the corresponding fracture steps in multilayers which contain tetradecane or are solvent-free. Alkanes also modify the structure of the P_β′ or ‘banded’ phase of phosphatidylcholine bilayers. The incorporation of tetradecane removes the banded structure from both the bilayer's hydrophilic surface, as viewed by negative staining, and the bilayer's hydrophobic interior, as viewed by the freeze-fracture technique. These results are consistent with X-ray diffraction data which imply that long alkanes are primarily located between adjacent lipid hydrocarbon chains in each monolayer of the bilayer, while short alkanes can partition into the geometric center of the bilayer between apposing monolayers.     

Interaction of alamethicin with ether-linked phospholipid bilayers: oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry studies

The arrangement of the antimicrobial peptide alamethicin was studied by oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry in ether-linked phospholipid bilayers composed of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC). The measurements were performed as a function of alamethicin concentration relative to the lipid concentration, and results were compared to those reported in the literature for ester-linked phospholipid bilayers. At ambient temperature, alamethicin incorporates into the hydrophobic core of DHPC bilayers but results in more lipid disorder than observed for ester-linked 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers. This orientational disorder appears to depend on lipid properties such as bilayer thickness. Moreover, the results suggest that alamethicin inserts into the hydrophobic core of the bilayers (at high peptide concentration) for both ether- and ester-linked lipids but using a different mechanism, namely toroidal for DHPC and barrel-stave for POPC.     

Structure of magainin and alamethicin in model membranes studied by x-ray reflectivity

We have investigated the structure of lipid bilayers containing varied molar ratios of different lipids and the antimicrobial peptides magainin and alamethicin. For this structural study, we have used x-ray reflectivity on highly aligned solid-supported multilamellar lipid membranes. The reflectivity curves have been analyzed by semi-kinematical reflectivity theory modeling the bilayer density profile rho(z). Model simulations of the reflectivity curves cover a large range of vertical momentum transfer q(z), and yield excellent agreement between data and theory. The structural changes observed as a function of the molar peptide/lipid concentration P/L are discussed in a comparative way.     

Single-Molecule Investigation of the Influence Played by Lipid Rafts on Ion Transport and Dynamic Features of the Pore-Forming Alamethicin Oligomer

In this experimental work we employed single-molecule electrical recordings on alamethicin oligomers inserted in lipid bilayers made of brain sphingomyelin (bSM), palmitoyloleoylphosphatidylcholine (POPC) and cholesterol (chol) to unravel novel aspects regarding lipid raft interactions with pore-forming peptides. We probed the effect of lipid rafts on electrical properties of inserted alamethicin oligomers, and our data convincingly prove that the single-channel electrical conductance of various subconductance states of the alamethicin oligomer (1) increases in the presence of raft-containing ternary lipid mixtures (POPC-chol-bSM) compared to cases when bilayers were made of POPC-chol and POPC and (2) decreases in the presence of raft-containing ternary lipid mixtures compared to nonraft ternary mixtures which favor the fluid and liquid ordered phases alone. Our data demonstrate that the presence of lipid rafts leads to a slower association kinetics of alamethicin oligomers, seemingly reflecting a slower lateral diffusion process of such peptide aggregates compared to the case of nonraft, binary lipid mixtures. Furthermore, we show that the electrical capacitance of ternary lipid mixtures (POPC-chol-bSM) decreases in the presence of raft domains by comparison to nonraft binary phases (POPC-chol) or POPC alone, and this could constitute an additional mechanism via which macroscopic electrical manifestations of eukaryotic cells are modulated by the coexistence of gel and fluid domains of the plasma membrane.     

Hydrogen bond stabilities in membrane-reconstituted alamethicin from amide-resolved hydrogen-exchange measurements.

Amide-resolved hydrogen-deuterium exchange-rate constants were measured for backbone amides of alamethicin reconstituted in dioleoylphosphatidylcholine vesicles by an exchange-trapping method combined with high-resolution nuclear magnetic resonance spectroscopy. In vesicles containing alamethicin at molar ratios between 1:20 and 1:100 relative to lipid, the exchange-rate constants increased with increasing volume of the D20 buffer in which the vesicles were suspended, indicating that exchange under these conditions is dominated by partitioning of the peptide into the aqueous phase. This was supported by observation of a linear relationship between the exchange-rate constants for amides in membrane-reconstituted alamethicin and those for amides in alamethicin dissolved directly into D2O buffer. Significant protection of amides from exchange with D2O buffer in membrane-reconstituted alamethicin is interpreted in terms of stabilization by helical hydrogen bonding. Under conditions in which amide exchange occurred by partitioning of the peptide into solution, only lower limits for hydrogen-bond stabilities in the membrane were determined; all the potentially hydrogen-bonded amides of alamethicin are at least 1000-fold exchange protected in the membrane-bound state. When partitioning of alamethicin into the aqueous phase was suppressed by hydration of reconstituted vesicles in a limiting volume of water [D2O:dioleoylphosphatidylcholine:alamethicin; 220:1:0.05; (M:M:M)], the exchange-protection factors exhibited helical periodicity with highly exchange-protected, and less well-protected, amides on the nonpolar and polar helix faces, respectively. The exchange data indicate that, under the conditions studied, alamethicin adopts a stable helical structure in DOPC bilayers in which all the potentially hydrogen-bonded amides are stabilized by helical hydrogen bonds. The protection factors define the orientation of the peptide helix with respect to an aqueous phase, which is either the bulk solution or water within parallel or antiparallel transmembrane arrays of reconstituted alamethicin.     

Calcium-induced inactivation of alamethicin in asymmetric lipid bilayers

This paper discusses a calcium-dependent inactivation of alamethicin- induced conductance in asymmetric lipid bilayers. The bilayers used were formed with one leaflet of phosphatidyl ethanolamine (PE) and one of phosphatidyl serine (PS). Calcium, initially confined to the neutral lipid (PE) side, can pass through the open alamethicin channel to the negative lipid (PS) side, where it can bind to the negative lipid and reduce the surface potential. Under appropriate circumstances, the voltage-dependent alamethicin conductance is thereby inactivated. We have formulated a model for this process based on the diffusion of calcium in the aqueous phases and we show that the model describes the kinetic properties of the alamethicin conductance under various circumstances. EGTA on the PS side of the membrane reduces the effects of calcium dramatically as predicted by the model.     

Structure of the alamethicin pore reconstructed by x-ray diffraction analysis

We reconstructed the electron density profile of the alamethicin-induced transmembrane pore by x-ray diffraction. We prepared fully hydrated multiple bilayers of alamethicin-lipid mixtures in a condition where pores were present, as established previously by neutron in-plane scattering in correlation with oriented circular dichroism. At dehydrated conditions, the interbilayer distance shortened and the interactions between bilayers caused the membrane pores to become long-ranged correlated and form a periodically ordered lattice of rhombohedral symmetry. To resolve the phase problem of diffraction, we used a brominated lipid and performed multiwavelength anomalous diffraction at the bromine K edge. The result unambiguously shows that the alamethicin pore is of the barrel-stave type consisting of eight alamethicin helices. This pore structure corresponds to the stable pores detected by neutron in-plane scattering in fully hydrated fluid bilayers at high peptide/lipid ratios, which are the conditions at which alamethicin was tested for its antibacterial activity.     

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 mus and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.     

Conformation of peptides in lipid membranes studied by x-ray grazing incidence scattering

Although the antimicrobial, fungal peptide alamethicin has been extensively studied, the conformation of the peptide and the interaction with lipid bilayers as well as the mechanism of channel gating are still not completely clear. As opposed to studies of the crystalline state, the polypeptide structures in the environment of fluid bilayers are difficult to probe. We have investigated the conformation of alamethicin in highly aligned stacks of model lipid membranes by synchrotron-based x-ray scattering. The (wide-angle) scattering distribution has been measured by reciprocal space mappings. A pronounced scattering signal is observed in samples of high molar peptide/lipid ratio which is distinctly different from the scattering distribution of an ideal helix in the transmembrane state. Beyond simple models of ideal helices, the data is analyzed in terms of models based on atomic coordinates from the Brookhaven Protein Data Bank, as well as from published molecular dynamics simulations. The results can be explained by assuming a wide distribution of helix tilt angles with respect to the membrane normal and a partial insertion of the N-terminus into the membrane.     

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 μs and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Micropatterned solid-supported membranes formed by micromolding in capillaries

The formation of individually addressable micropatterned solid-supported lipid bilayers has been accomplished by means of micromolding in capillaries. Small unilamellar vesicles were spread on glass slides to form planar supported membranes along microscopic capillaries molded as trenches into a polydimethylsiloxane (PDMS) elastomer. PDMS provides an elastic and transparent carrier for microcapillaries molded from silicon wafers displaying the desired inverse trenches. The so-called master structure has been conventionally etched into silicon by photolithography. The cured PDMS elastomer was briefly exposed to an oxygen plasma, rendering the surface hydrophilic, and subsequently attached to a glass surface in order to form hydrophilic capillaries equipped with flow-promoting pads on either side. One flowpad acts as a reservoir to be filled with the vesicle suspension, while the other one serves as a collector to ensure a sufficient capillary flow to cover the substrate completely. Formation of planar lipid bilayers on the glass slide along the capillaries was followed by imaging the flow and spreading of fluorescently labeled DMPC liposomes with confocal laser scanning microscopy. By means of scanning force microscopy in aqueous solution the formed lipid structures were identified and the height of the lipid bilayers was accurately determined. With both techniques, it was shown that the patterned bilayers remain separated and persist for several hours on the substrate in aqueous solution.     

Alamethicin influence on the membrane bending elasticity

We investigate the bending elasticity of lipid membranes with the increase of the alamethicin concentrations in the membrane via analysis of the thermally induced shape fluctuations of quasi-spherical giant vesicles. Our experimental results prove the strong influence of alamethicin molecules on the bending elasticity of diphytanoyl phosphatidylcholine and dilauroyl phosphatidylcholine membranes even in the range of very low peptide concentrations (less than 10⁻³ mol/mol in the membrane). The results presented in this work, testify to the peripheral orientation of alamethicin molecules at low peptide concentrations in the membrane for both types of lipid bilayers. An upper limit of the concentration of the peptide in the membrane is determined below which the system behaves as an ideal two-dimensional solution and the peptide molecules have a planar orientation in the membrane.     

Trichotoxin A-40, a new membrane-exciting peptide. Part B. Voltage-dependent pore formation in bilayer lipid membranes and comparison with other alamethicin analogues

Trichotoxin A-40 induces voltage-dependent pores in bilayer lipid membranes comparable to those formed by alamethicin and suzukacillin. The conductance values of the trichotoxin A-40 pores are of similar magnitude and show the same characteristic pattern sequence of non-integral multiples of a unit-conductance step as alamethicin and suzukacillin.However, voltage-jump current-relaxation experiments show significant differences between trichotoxin A-40 and alamethicin and suzukacillin. With trichotoxin A-40 three different relaxation processes could be observed, whereas with alamethicin and suzukacillin only two processes had been distinguished. The fast process in each case is related to pore state transitions and the slower (medium) process to the decay rate of pores. The third very slow process, which is not found with alamethicin and suzukacillin, could not clearly be assigned to a molecular mechanism. Whereas in the case of alamethicin and suzukacillin the relaxation amplitude of the slow process is considerably larger than the relaxation amplitude of the fast process, the reverse is true for trichotoxin A-40, where the largest relaxation amplitude is that of the fast process.Contrary to alamethicin and suzukacillin, trichotoxin A-40 is soluble in the lipid/decane membrane-forming solution, when added from an ethanolic stock solution. Its bilayer-modifying properties are not changed, whether the antibiotic is added to the aqueous salt solution or to the membrane-forming solution.Several different analogues of alamethicin, suzukacillin and trichotoxin A-40 have been investigated and compared with respect to the induced current-voltage characteristics in lipid bilayers. A suzukacillin A-derivative where phenylalaninol had been split off is active as well as trichotoxin A-40 which lacks the phenylalaninol group by nature. Different C-terminal groups like -COOH, -CONH₂, -COOCH₃ and -CO-Ala-Ala-OCH₃ cause qualitative changes but not the loss of the pore-formation property.     

Voltage-dependent insertion of alamethicin at phospholipid/water and octane/water interfaces

Understanding the binding and insertion of peptides in lipid bilayers is a prerequisite for understanding phenomena such as antimicrobial activity and membrane-protein folding. We describe molecular dynamics simulations of the antimicrobial peptide alamethicin in lipid/water and octane/water environments, taking into account an external electric field to mimic the membrane potential. At cis-positive potentials, alamethicin does not insert into a phospholipid bilayer in 10 ns of simulation, due to the slow dynamics of the peptide and lipids. However, in octane N-terminal insertion occurs at field strengths from 0.33 V/nm and higher, in simulations of up to 100 ns duration. Insertion of alamethicin occurs in two steps, corresponding to desolvation of the Gln7 side chain, and the backbone of Aib10 and Gly11. The proline induced helix kink angle does not change significantly during insertion. Polyalanine and alamethicin form stable helices both when inserted in octane and at the water/octane interface, where they partition in the same location. In water, both polyalanine and alamethicin partially unfold in multiple simulations. We present a detailed analysis of the insertion of alamethicin into the octane slab and the influence of the external field on the peptide structure. Our findings give new insight into the mechanism of channel formation by alamethicin and the structure and dynamics of membrane-associated helices.     

Antimicrobial peptide magainin I from Xenopus skin forms anion-permeable channels in planar lipid bilayers.

The ionophore properties of magainin I, an antimicrobial and amphipathic peptide from the skin of Xenopus, were investigated in planar lipid bilayers. Circular dichroism studies, performed comparatively with alamethicin, in small or large unilamellar phospholipidic vesicles, point to a smaller proportion of alpha-helical conformation in membranes. A weakly voltage-dependent macroscopic conductance which is anion-selective is developed when using large aqueous peptide concentration with lipid bilayer under high voltages. Single-channel experiments revealed two main conductance levels occurring independently in separate trials. Pre-aggregates lying on the membrane surface at rest and drawn into the bilayer upon voltage application are assumed to account for this behaviour contrasting with the classical multistates displayed by alamethicin.     

Barrel-stave model or toroidal model? A case study on melittin pores

Transmembrane pores induced by amphiphilic peptides, including melittin, are often modeled with the barrel-stave model after the alamethicin pore. We examine this assumption on melittin by using two methods, oriented circular dichroism (OCD) for detecting the orientation of melittin helix and neutron scattering for detecting transmembrane pores. OCD spectra of melittin were systematically measured. Melittin can orient either perpendicularly or parallel to a lipid bilayer, depending on the physical condition and the composition of the bilayer. Transmembrane pores were detected when the helices oriented perpendicularly to the plane of the bilayers, not when the helices oriented parallel to the bilayers. The evidence that led to the barrel-stave model for alamethicin and that to the toroidal model for magainin were reviewed. The properties of melittin pores are closely similar to that of magainin but unlike that of alamethicin. We conclude that, among naturally produced peptides that we have investigated, only alamethicin conforms to the barrel-stave model. Other peptides, including magainins, melittin and protegrins, all appear to induce transmembrane pores that conform to the toroidal model in which the lipid monolayer bends continuously through the pore so that the water core is lined by both the peptides and the lipid headgroups.     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: First, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 μF/cm² for DPhyPC bilayers and 0.75 and 0.77 μF/cm² for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 MΩ cm² were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.