Facilitated synthesis of peptaibols: alamethicin via enzymatic segment condensation.

We have used a combined chemical-enzymatic approach to facilitate the total synthesis of the 20-residue peptaibol, alamethicin. The 1-11 segment of alamethicin, having a C-terminal Gly, and the 12-20 segment, having an N-terminal Leu, were prepared by well-established chemical methods, and then coupled using papain to afford a 54% yield of alamethicin in straightforward fashion. In contrast to the reported chemical syntheses of alamethicin requiring side-chain protection at Glu,18 the papain-catalyzed coupling proceeded readily and selectively using a C-terminal segment having a free gamma-carboxyl group at this position. Several alamethicin partial sequences were obtained via enzymatic formation of the Gly11-Leu12 bond. The high efficiency of this route is illustrated by the enzymatic assembly of the 1-17 alamethicin fragment on a 400-mg scale in 62% yield. An alternative route to alamethicin through enzymatic formation of the Ala6-Gln7 bond was less successful because of a low yield in the final coupling.     

Characterization of adenylyl cyclase in heart sarcolemma in the absence or presence of alamethicin

Alamethicin is commonly used as an agent for unmasking the latent enzyme activities in vesicular membrane preparations; however, relatively little is known about the effect of this agent on the characteristics of adenylyl cyclase in heart sarcolemma. By employing rat heart sarcolemmal preparation, we observed 5 to 6 fold increase in adenylyl cyclase activity upon treatment with alamethicin. Kinetic experiments using various concentrations of MgATP revealed that the increase in adenylyl cyclase activity in alamethicin treated membranes was associated with an increase in V_max as well as affinity of the substrate for the enzyme. Dose-responses of the control and alamethicin-treated preparations to various activators of adenylyl cyclase revealed that the sensitivity of the enzyme to forskolin, NaF and GppNHp, was markedly increased upon treating sarcolemma with alamethicin. The activation of adenylyl cyclase by forskolin was also enhanced by increasing the concentration of alamethicin in the incubation medium. Furthermore, there was a greater increase in adenylyl cyclase activity with different concentrations of Mn²⁺ in the presence of alamethicin. These results suggest that alamethicin treatment alters the characteristics of adenylyl cyclase in addition to unmasking the enzyme activity in the purified sarcolemmal vesicular preparation.     

Alamethicin adsorption to a planar lipid bilayer.

The effect of alamethicin and its derivatives on the voltage-dependent capacitance of phosphatidylethanolamine (squalane) membranes was measured using two different methods: lock-in detection and voltage pulse. Alamethicin and its derivatives modulate the voltage-dependent capacitance at voltages lower than the voltage at which alamethicin-induced conductance is detected. The magnitude and sign of this alamethicin-induced capacitance change depends on the aqueous alamethicin concentration and the kind of alamethicin used. Our experimental data can be interpreted as a potential-dependent pseudocapacitance associated with adsorbed alamethicin. Pseudocapacitance is expressed as a function of alamethicin charge, its concentration in the bathing solution and the applied electric field. The theory describes the dependence of the capacitance on applied voltage and alamethicin concentration. When alamethicin is neutral the theory predicts no change of the voltage-dependent capacitance with either sign of applied voltage. Experimental data are consistent with the model in which alamethicin molecules interact with each other while being adsorbed to the membrane surface. The energy of this interaction depends on the alamethicin concentration.     

Voltage-dependent conductance for alamethicin in phospholipid vesicles. A test for the mechanism of gating.

The ion currents induced by alamethicin were investigated in unilamellar vesicles using electron paramagnetic resonance probe techniques. The peptide induced currents were examined as a function of the membrane bound peptide concentration, and as a function of the transmembrane electrical potential. Because of the favorable partitioning of alamethicin to membranes and the large membrane area to aqueous volume in vesicle suspensions, these measurements could be carried out under conditions where all the alamethicin was membrane bound. Over the concentration range examined, the peptide induced conductances increased approximately with the fourth power of the membrane bound peptide concentration, indicating a channel molecularity of four. When the alamethicin induced currents were examined as a function of voltage, they exhibited a superlinear behavior similar to that seen in planar bilayers. Evidence for the voltage-dependent conduction of alamethicin was also observed in the time dependence of vesicle depolarization. These observations indicate that the voltage-dependent behavior of alamethicin can occur in the absence of a voltage-dependent phase partitioning. That is, a voltage-dependent conformational rearrangement for membrane bound alamethicin leads to a voltage-dependent activity.     

Dipole moment of alamethicin as related to voltage-dependent conductance in lipid bilayers.

The dipole moment of alamethicin, which produces voltage-dependent conductance in lipid-bilayer membranes, was measured in mixed solvents of ethanol and dioxane. The value of the dipole moment was found to increase from 40 to 75 DU (Debye units), as the concentration of ethanol increased from 0 (pure dioxane) to 40%. The relaxation frequency of alamethicin also changes from 10 to 40 MHz, depending upon the concentration of ethanol in mixed solvents. The length of alamethicin was calculated by using the relaxation time and was found to range from approximately 40 to 20 A. The dipole moment was independently calculated from voltage-dependent conductance and compared with the measured value. The calculated value was found to be larger than the value of direct measurements, indicating that several alamethicin molecules are required to form a conducting pore and that their dipole moments are oriented parallel to each other.     

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.     

Template-free self-assembling fullerene and lipopeptide conjugates of alamethicin form voltage-dependent ion channels of remarkable stability and activity.

N- and C-terminally modified with fullerene or lipopeptide alamethicin molecules were designed for the formation of template-free, self-assembling, voltage-dependent ion conducting channels. The automated solid phase synthesis of the alamethicin-F30 sequence was performed by in situ fluoride activation on 2-chlorotritylchloride-polystyrene resin and the conjugation with fullerenes-C60 and -C70 was carried out in solution. Voltage-dependent bilayer experiments revealed preferred channel sizes for C-terminal alamethicin F30-fullerene-C60 and -C70 conjugates and higher activity compared with native alamethicin, whereas N-terminally linked fullerene balls destabilize pore formation. C-terminal alamethicin F30-fullerene-C70 conjugates show pore states with remarkably long lifetimes of seconds. C-terminal lipopeptide conjugates of alamethicin were prepared by coupling via short peptide spacers with synthetic tripalmitoyl-S-glyceryl-cysteine. which represents the strong membrane anchoring N-terminus of bacterial lipoprotein. Alamethicin-lipopeptide conjugates exhibit high channel forming activities, whereby they self-assemble and adopt preferred pore states with extremely long lifetimes. The novel membrane modifying peptaibol constructs are valuable lead compounds for developments in sensorics related to transmembrane ion conductance.     

Properties of the conductance induced in lecithin bilayer membranes by alamethicin

Current-voltage relations have been measured across lecithin bilayers doped with alamethicin molecules. The results show that there are two aspects of the induced conductances, a voltage-dependent and a voltage-independent conductance. Both have been characterized as a function of alamethicin and KCl concentration. The two aspects of the conductances do not show the same changes with those two variables. The voltage-independent conductance is affected very little by changes in KCl concentration, and its dependance on alamethicin concentration reveals that it is produced by two or three alamethicin molecules. The voltage-dependent conductance is shifted by the changes in KCl concentration only when the concentrations are greater than or equal to 100 mM; below 100 mM KCl the slope of the log conductance-voltage curve is also reduced. The effect of changing alamethicin concentration reveals that nine or ten molecules are involved for KCl concentrations larger than 100 mM; if the KCl concentration is less than 100 mM, the effect of changing the alamethicin concentration is reduced. Time-dependent measurements have also been performed; only one time constant was found and it is strongly voltage-dependent. Also a very slow voltage-dependent absorption process is found. These results can be explained if it is assumed that pores are formed of a mixture of charged and uncharged alamethicin molecules when a voltage is applied and that uncharged alamethicin can also form pores without applying a voltage, once the absorption process has been started by previously applied voltages. The voltage dependence of the time constant seems to indicate that the voltage-dependent pore formation is produced by aggregates of charged alamethicin rather than independent molecules.     

Distribution of alamethicin in lipid membranes and water

The concentration of alamethicin in aqueous solutions was quantitated using measurements of the spot area on thin-layer chromatograms. These data were utilized to measure a partition coefficient of 17 for alamethicin in a phospholipid membrane-water system under equilibrium dialysis conditions.     

Intrinsic rectification of ion flux in alamethicin channels: studies with an alamethicin dimer.

Covalent dimers of alamethicin form conducting structures with gating properties that permit measurement of current-voltage (I-V) relationships during the lifetime of a single channel. These I-V curves demonstrate that the alamethicin channel is a rectifier that passes current preferentially, with voltages of the same sign as that of the voltage that induced opening of the channel. The degree of rectification depends on the salt concentration; single-channel I-V relationships become almost linear in 3 M potassium chloride. These properties may be qualitatively understood by using Poisson-Nernst-Planck theory and a modeled structure of the alamethicin pore.     

Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles

A mechanism for the activating effect of alamethicin on membrane enzymes was investigated, using a purified preparation of cardiac sarcolemmal vesicles. (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities were measured. alamethicin increased ouabain-sensitive (Na+,K+)-ATPase activity of sarcolemmal vesicles 5- to 7-fold and adenylate cyclase activity 2.5- to 4-fold. Adenylate cyclase retained its sensitivity to the beta-adrenergic agonist isoproterenol after membranes were treated with alamethicin. Alamethicin caused a 4- to 6-fold increase in the number of detectable (Na+,K+)-ATPase enzymic sites, but no increase ws noted for the number of muscarinic-cholinergic receptor-binding sites. Phosphorylation of endogenous proteins of sarcolemmal vesicles by an intrinsic cAMP-dependent protein kinase activity was stimulated 5- to 7-fold by alamethicin. The regulatory subunit of the membrane-bound cAMP-dependent protein kinase was labeled with the photoaffinity probe 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP), and it migrated with an apparent molecular weight of 55,000 in sodium dodecyl sulfate polyacrylamide gels. Alamethicin stimulated autophosphorylation of the regulatory subunit by [gamma-32P]ATP 6-fold and incorporation of of 8-N3-[32P]cAMP into the subunit 2.6-fold. The results suggest that alamethicin disrupts membrane barriers of sarcolemmal vesicles, which are mostly right side out, giving substrates and activators access to enzymic sites in the interior of the vesicles, while preserving functional coupling of enzymes to their effectors.     

Molecular flexibility demonstrated by paramagnetic enhancements of nuclear relaxation. Application to alamethicin: a voltage-gated peptide channel.

A nitroxide spin label attached to the C-terminus of the channel forming peptide alamethicin produces an enhancement of the nuclear spin-lattice relaxation rates of peptide protons as a result of both intermolecular and intramolecular magnetic dipole-dipole interactions. The intermolecular contribution provides evidence that alamethicin monomers collide preferentially in a C-terminal-to-N-terminal configuration in methanol. From the intramolecular paramagnetic enhancement of nuclear spin-lattice relaxation times, effective distances between the unpaired electron on the nitroxide at the C-terminus of alamethicin and protons along the peptide backbone were calculated. These distances are much shorter than distances based on the reported crystal structure of alamethicin, and cannot be accounted for by motion in the bonds that attach the nitroxide to the peptide. In addition, the differences between distances deduced from the nuclear spin relaxation and the distances seen in the crystal structure increase toward the N-terminal end of the peptide. The simplest explanation for these data is that the alamethicin backbone suffers large structural fluctuations that yield shorter effective distances between the C-terminus and positions along the backbone. This finding can be interpreted in terms of a molecular mechanism for the voltage-gating of the alamethicin channel. When the distances between a paramagnetic center and a nucleus fluctuate, paramagnetic enhancements are expected to yield distances that are weighted by r-6, and distances calculated using the Solomon-Bloembergen equations may more nearly represent a distance of closest approach than a time average distance.(ABSTRACT TRUNCATED AT 250 WORDS)     

C-terminally shortened alamethicin on templates: influence of the linkers on conductances.

In order to test the influence of chemical modifications designed to allow covalent coupling of channel-forming peptide motifs into variable sized oligomers, a series of alamethicin derivatives was prepared. The building block encompassing the N-terminal 1-17 residues of alamethicin behaved normally in the conductance assay on planar lipid bilayers, albeit at higher concentration and with a slightly reduced voltage-dependence. A linker Ac-K-OCH(2)C(6)H(4)CH(3)p attached via the epsilon amino group of lysine to the C-terminus of alamethicin(1-17) increased membrane affinity. The latter was further enhanced in a dimer and a tetramer in which alamethicin(1-17) chains were tethered to di- or tetra-lysine linkers, respectively, but macroscopic current-voltage curves displayed much reduced voltage-dependencies and reversed hysteresis. An usual behaviour with high voltage-dependence was restored with the modified dimer of alamethicin(1-17) in which alanine separated the two consecutive lysine residues in the linker. Of special interest was the development of a 'negative resistance' branch in macroscopic current-voltage curves for low concentrations of this dimer with the more flexible linker. Single channel events displayed only one single open state with fast kinetics and whose conductance matches that of the alamethicin heptamer or octamer.     

Alamethicin channel permeation by Ca2+, Mn2+ and Ni2+ in bovine chromaffin cells.

Alamethicin causes a concentration-dependent increase of [Ca2+]i in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Cai2+ (234 +/- 37 nM; n = 4) increased to a maximum of 2347 +/- 791 nM (n = 3) with 100 micrograms/ml alamethicin. In the presence of 1 mM Cae2+ the increase reached a plateau within about 2-5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Cae2+ alamethicin did not modify [Ca2+]i. This contrasts with ionomycin (1 microM) which produced a Cai2+ transient even in the absence of Cae2+. Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2 fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 mM) addition. This suggests that Mn2+ is able to permeate voltage-dependent Ca2+ channels. Ni2+ uptake into either resting or K(+)-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.     

Alamethicin-induced current-voltage curve asymmetry in lipid bilayers.

We have examined the causes of the asymmetry of the current-voltage curve induced by addition of alamethicin to one side of a black lipid membrane. We find that the alamethicin-induced current-voltage (I-V) curve has an inherent asymmetry. If it were possible to confine all alamethicin molecules to one side of the membrane, the I-V curve would exhibit a positive branch (voltage being measured with respect to the side of the membrane trans to the alamethicin addition) of steeper logarithmic slope than the negative branch and at a lower absolute value of potential. This condition is not usually realized, however, because alamethicin can leak through the membrane, so that, except at very high alamethicin concentrations and in certain kinds of membranes, the positive branch of the current-voltage curve has the same logarithmic slope as the negative branch and appears to arise from alamethicin which diffuses from the cis to the trans side of the membrane. We develop simple quantitative models for these two cases.     

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.     

Antimicrobial peptides in toroidal and cylindrical pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ∼ 7, the net charge of alamethicin is − 1 whereas that of melittin is + 5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.     

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.     

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.