Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part B. Pore formation in black lipid films

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete conductance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation tion starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger α-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of “inactivation”.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part A. Sequence and conformation

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin.A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalaninol have l-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular ¹³C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially α-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous media due to a longer α-helical part and higher number of aliphatic residues. A part of the α-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high α-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.     

Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin. Part A. Sequence and conformation.

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A is isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin. A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalainol have L-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular 13C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially alpha-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous medis due to a longer alpha-helical part and higher number of aliphatic residues. A part of the alpha-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high alpha-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.     

Activation of the calcium/calmodulin-dependent guanylate cyclase from Paramecium by polypeptide antibiotics and melittin

The activity of the calcium/calmodulin-regulated guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from Paramecium was stimulated by several polypeptides. The most potent activator was melittin (6-fold at 30 μM), followed by alamethicin, suzukacillin, trichotoxin and gramicidin S. Marginal effects were seen with herbicolin A and polymyxin B, whereas the following compounds had no effect: ionophore A23187, actinomycin C₁, destomycin A, gramicidin A, iturin A, nigericin, nonactin, Tü 1718B, valinomycin and synthetic peptide analogues of alamethicin. Guanylate cyclase activation was not related to ion-transport capacity or to the length of the α-helical segments. Rather, the degree of amphiphilicity seemed to be an important criterion. No difference in activation was seen between native guanylate cyclase and the reconstituted enzyme. Thus, in all likelihood, polypeptide stimulation requires the presence of the guanylate cyclase/calmodulin holo-enzyme. Guanylate cyclase activation was permanent. Enzyme kinetics, such as Michaelis-Menten behavior and non-cooperativity, were retained. Incubation with polypeptides at 37°C prior to substrate addition decreased enzyme stimulation. Activation of cGMP formation as enhanced at elevated incubation temperatures. The activation energy for hemolysis of erythrocytes favorably correlated with the extent of guanylate cyclase activation (r = 0.98), suggesting a similar mechanism of interaction with membrane constituents for both processes.     

Trichotoxin A-40, a new membrane-exciting peptide. Part A. Isolation,characterization and conformation

The new polypeptide antibiotic trichotoxin A-40 is isolated by chloroform/methanol extraction from the dry mycelium of Trichoderma viride NRRL 5242. The lipophilic peptide is purified by chromatography on Kieselgel H-60 and reverse-phase chromatography on Lichrosorb RP-8. The new antibiotic differs in amino acid composition and various chemical and physicochemical properties from similar peptides such as trichotoxin A, the suzukacillins or alamethicins. The amino acid composition is (Pro)₁ (Gly)₁ (Ala)₂ (Leu)₂ (Aib)₁₀ (Glx)₂. (Aib, α-aminoisobutyric acid.) The antibiotic has a carboxyl group which can be esterified by diazomethane, which results in slightly enhanced membrane-modifying activities.The peptide exhibits a right-handed α-helical conformation increasing about two-fold from aqueous to lipophilic media as shown by solvent-dependent circular dichroism measurements. Most of the ¹³C-NMR resonances can be assigned unequivocally and amino acids situated in the α-helical part show characteristic shift differences from those in the non-helical regions. No β-phenylalaninol residue could be identified by ¹³C-NMR and ultraviolet spectroscopy, as can be for alamethicins and suzukacillins. A pronounced hemolytic action is found on human erythrocytes, which develops at micromolar concentrations. Trichotoxin A-40 induces a voltage-dependent ionic conductance in bilayer lipid membranes and it can serve as a new pore-forming model system for structure/activity studies in membrane excitation by peptides.     

Trichotoxin A40. Purification by counter-current distribution and sequencing of isolated fragments

The isolation of the membrane-modifying polypeptide antibiotics from the mycelium of Trichoderma viride 5242 was optimized via extraction with dichloromethane and chromatography on Sephadex LH-20. The components trichotoxin A40 and A50 were separated from each other and purified by multiplicative counter-current distribution. The sequence of proteinase-resistant trichotoxin A40 was determined by combined gas chromatography and mass spectrometry of three isolated N-acetylated dodecapeptides and two N-prolylhexapeptides obtained after selective trifluoroacetolysis. Including amino acid exchanges due to natural microheterogeneity, the sequence is Ac-Aib-Gly(LAla)-Aib-LLeu-Aib-LGln-Aib-Aib-Aib(LAla )-LAla-Aib-Aib-LPro-LLeu -Aib-DIva(Aib)-LGlu-LValol. In contrast to the eicosapeptide alamethicin, trichotoxin A40 contains only 18 residues, with a higher proportion of alpha-aminoisobutyric acid (Aib), C-terminal L-valinol (Vol), one D-isovaline (Iva) and no proline at the N-terminal part.     

Fluctuation and relaxation analysis of monazomycin-induced conductance in black lipid membranes

Summary Fluctuation and relaxation analyses were performed on monazomycin-induced conductance of lipid bilayer membranes. With both methods a slow (sec) and a fast (msec) current component are apparent; however, the amplitude of the slow, voltage-dependent process is greater than that of the fast component in the step relaxation experiment and less in the fluctuation experiment. The fluctuation analysis showed principally a rapid voltage-dependent process which appears to be related to the multistate character of the conducting channel. The experimental results are interpreted in terms of a simplified kinetic model which is used to calculate relaxation and noise amplitudes.     

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.     

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.     

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.     

Potential-dependent conductances in lipid membranes containing alamethicin.

This article is concerned primarily with the mechanism of the potential-dependent conductance induced in artificial lipid membranes by the cyclic polypeptide andibiotic alamethicin. It has already been shown from studies of the fluctuations that can be detected in very small membrane currents that alamethicin forms transient pores of some 0.6 nm in diameter and that, for small inorganic ions, these are poorly selective. The origin of these pores, their spatial distribution and interaction are discussed. It is demonstrated that the sensitivity of the membrane conductance to the applied potential arises only to a slight extent from the current-voltage relations for the individual pores, and that the main effect stems from the influence of the potential on the frequency of opening of the pores. From the properties of lipid membranes containing alamethicin in a wide variety of electrolytes, and from other evidence, it is concluded that the polypeptide reacts to the electric field more probably because it has dipole moment than because it binds ions. It is proposed that the conducting complex is capable of functioning in either of two orientations, and that it is these two possibilities that give rise to certain differences in the single channel characteristics for the two directions of the field.     

Molecular structure of t-butyloxycarbonyl-Leu-Aib-Pro-Val-Aib-methyl ester,a fragment of alamethicin and suzukacillin: A 310-helical pentapeptide

The pentapeptide Boc-Leu-Aib-Pro-Val-Aib-OMe, a fragment of alamethicin and suzukacillin, crystallizes in the space group P2₁, with a = 11.034 (2), b = 10.894 (2), c = 15.483 (2) Å, β = 104.80 (2)° and Z = 2. The crystal structure has been solved by direct methods and refined to an R value of 0.069. The peptide backbone folds into a right-handed 3₁₀-helical conformation, stabilized by two intramolecular 4 → 1 hydrogen bonds between the Leu(1) CO and Val(4) NH and Aib(2) CO and Aib(5) NH groups. The solid-state conformation is consistent with results of spectroscopic analysis in solution.     

Peptaibiotics and Peptaibols: An Alternative to Classical Antibiotics?

The development and antimicrobial properties of peptaibiotics and peptaibols are discussed. Also, the role of emerging peptaibol analogues, of alamethicin, e.g., harzianins HC, trichotoxin, and antiamoebin, is outlined.     

The ion-channel activity of longibrachins LGA I and LGB II: effects of pro-2/Ala and gln-18/Glu substitutions on the alamethicin voltage-gated membrane channels.

Longibrachins LGA I (Ac Aib Ala Aib Ala Aib(5) Ala Gln Aib Val Aib(10) Gly Leu Aib Pro Val(15) Aib Aib Gln Gln Pheol(20), with Aib: alpha-aminoisobutyric acid, pheol: phenylalaninol) and LGB II are two homologous 20-residue long-sequence peptaibols isolated from the fungus Trichoderma longibrachiatum that differ between them by a Gln-18/Glu substitution. They distinguish from alamethicin by a Pro-2 for Ala replacement, which allowed to examine for the first time with natural Aib-containing analogues, the effect of Pro-2 on the ion-channel properties exhibited by alamethicin. The influence of these structural modifications on the voltage-gated ion-channel forming activity of the peptides in planar lipid bilayers were analysed. The general 'barrel-stave' model of ion-channel activity, already described for alamethicin, was preserved with both longibrachins. The negatively charged LGB II promoted higher oligomerisation levels, which could presumably dilute the repulsive effect of the negative Glu ring near the entrance of the channel and resulted in lower lifetimes of the substates, confirming the strong anchor of the peptide C-terminus at the cis-interface. Reduction of the channel lifetimes was observed for the longibrachins, compared to alamethicin. This argues for a better stabilisation of the channels formed by peptaibols having a proline at position 2, which results in better anchoring of the peptide monomer N-terminus at the trans-bilayer interface. Qualitative assays of the temperature dependence on the neutral longibrachin channel properties demonstrated a high increase of channel lifetimes and a markedly reduced voltage-sensitivity when the temperature was decreased, showing that such conditions may allow to study the channel-forming properties of peptides leading to fast current fluctuations.     

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.     

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.     

Quasielastic light-scattering study of polyadenylic acid solutions. II

Quasielastic light scattering is used to study saline solutions of polyadenylic acid with varying polymer concentrations and molecular masses. These experiments clearly show the existence of two relaxation times. For dilute solutions, when the chains are mutually independent, the fast mode is due to the free diffusion of the polymer chains. For concentrations above the overlap concentration C*, the fast mode is due to the propagation of collective excitations of the pseudolattice of polymer chains. The slow modes are observed when the polymer concentration is in the vicinity of the overlap concentration C*. A series of experiments shows that both their relaxation time and amplitude depend only on the polymer concentration and not on the polymer molecular mass. This result rules out any previous explanation based on individual chain motion. Furthermore, since the amplitudes depend on the time elapsed from the preparation of the solution, the slow modes are due to the diffusion of concentration inhomogeneities in the pseudolattice.     

Functional analysis of myosin mutations that cause familial hypertrophic cardiomyopathy.

Two approaches employing nuclear magnetic resonance (NMR) were used to investigate the transmembrane migration rate of the C-terminal end of native alamethicin and a more hydrophobic analog called L1. Native alamethicin exhibits a very slow transmembrane migration rate when bound to phosphatidylcholine vesicles, which is no greater than 1 x 10(-4) min(-1). This rate is much slower than expected, based on the hydrophobic partition energies of the amino acid side chains and the backbone of the exposed C-terminal end of alamethicin. The alamethicin analog L1 exhibits crossing rates that are at least 1000 times faster than that of native alamethicin. A comparison of the equilibrium positions of these two peptides shows that L1 sits approximately 3-4 A deeper in the membrane than does native alamethicin (Barranger-Mathys and Cafiso. 1996. Biochemistry. 35:489). The slow rate of alamethicin crossing can be explained if the peptide helix is irregular at its C-terminus and hydrogen bonded to solvent or lipid. We postulate that L1 does not experience as large a barrier to transport because its C-terminus is already buried within the membrane interface. This difference is most easily explained by conformational differences between L1 and alamethicin rather than differences in hydrophobicity. The results obtained here demonstrate that side-chain hydrophobicity alone cannot account for the energy barriers to peptide and protein transport across membranes.