Gérard Spach

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.

A family of double helices of alternating poly(γ-benzyl-d-l-glutamate), a stereochemical model for gramicidin A

Abstract Poly(γ-benzyl- d - l -glutamate) with strict alternation of l and d residues is found to exist, in addition to the αDL and πDL4.4 helical structures already described (Heitz et al., 1975a), in four more helical structures. Models based on double helices made of antiparallel strands are proposed for all four structures, based on infrared, X-ray and electron diffraction data. These double helices are, like the single-stranded πDL helices, specific to polypeptides with a strict stereosequence of alternating l and d residues. The diameter of the helical core of three of these helices appears to depend on the dimensions of the solvent molecules. Conformational angles (located in the β regions) and atomic co-ordinates determined by conformational energy analysis are given for the four structures. Experimental conditions used to obtain these helices, and to induce transconformations between the various helical structures of PB d - l G are described. The present investigations on PB d - l G help to make more precise the structure and geometry of models proposed (Veatch et al., 1974) for the antibiotic gramicidin A.

Single channels of 9, 11, 13, 15-destryptophyl-phenylalanyl-gramicidin A.

Analysis of the single-channel behavior of an analogue of gramicidin A in which all four tryptophyl residues are substituted by phenylalanyl suggests that the nature of the side chains may play an important role in the ion translocation process. Indeed, while infrared spectroscopy indicates that both peptides have very similar backbone conformations, they have different single-channel characteristics. The unit conductance of the analogue is much smaller than that of the natural product. Moreover, contrary to gramicidin A, it is voltage dependent.

β-structures of polypeptides with L- and D-residues

SummaryA series of five alternating poly(leucyl-lysyl) samples with varying amounts of L- and D-residues randomly distributed along the chain, but evenly shared out amongst leucyl and lysyl residues were synthesized by condensation of a mixture of the four diastereoisomeric dipeptidep-nitro-phenylesters. Their behavior in aqueous solution at various ionic strengths was studied by infrared spectroscopy which allowed measurement of the total amount ofβ-structures, and by circular dichroism which gives the excess of L-residues over D-residues in the same structures. Comparison with the properties of the all L-poly(Lys-Leu-Lys-Leu) shows that incorporation of a few D-residues in a L-chain seems to reduce the width of theβ-sheets obtained in presence of salt. Higher proportions of D-isomers prevent the coil →β transition from occurring when the ionic strength is increased except for segments containing at least 6 to 7 adjacent residues of the same configuration.

Beta-Structures of polypeptides with L- and D-residues. Part III. Experimental evidences for enrichment in enantiomer.

SummaryIt was previously shown that nuclei ofβ-sheets surrounded by unordered segments are formed in polypeptide chains built up with alternating hydrophobic and hydrophilic residues and containing both L- and D-enantiomers. It was also established that segments of residues having the same configuration tend to segregate in these nuclei when the starting composition of stereomonomers departs from the racemic mixture.Soft acidic hydrolysis of these polymers has been studied. Kinetic measurements show two pseudo first order rate constants, in agreement with the existence of two conformational species. The unordered part of the chains is hydrolyzed more rapidly, allowing the isolation of aβ-fraction enriched in one enantiomer. Thus, a plausible process of enrichment in enantiomer during prebiotic evolution has been described, which however does not explain the preference of one enantiomer over the other one.

Voltage-dependent and multi-state ionic channels induced by trichorzianines, anti-fungal peptides related to alamethicin

The ionophore properties of two peptaibols of the trichorzianine family have been investigated in planar lipid bilayers and compared to those of alamethicin. Macroscopic conductance experiments reveal voltage‐dependent channels only in the thinnest membranes and a greater efficiency of the neutral analog. In single‐channel experiments, a multi‐state behaviour, consistent with the usual barrel‐stave model, is disclosed but the discrete current fluctuations are much more rapid than for alamethicin. The results indicate a stringent requirement for the helix length/bilayer thickness match in agreement with a previous model and suggest the design of new synthetic peptides.

Beta-structures of polypeptides with L-and D-residues. Part II. Statistical analysis and enrichment in enantiomer.

SummaryThe possible formation ofβ-structures from polypeptide chains with L-and D-residues randomly distributed was statistically analyzed within the frame of two hypotheses. Firstly, only those segments containing residues of identical chirality can associate to form antiparallelβ-structures, and secondly these segments must have a minimum length. The influence of different factors was examined: initial ratio of the L-and D-monomer, minimum length required for the segments to be incorporated intoβ-sheets, average length of the peptide molecules, and stereoselectivity in the course of the polymerization process.The results show that in all cases nuclei ofβ-sheets surrounded by random coil segments are formed, the optical activity of which very rapidly increases to purity when the initial ratio of monomers deviates from the racemic mixture. This suggests experiments to enrich the system in one enantiomer. Comparison is made with the corresponding behavior and properties of the a-helical structure.

Conductance properties of des-Aib-Leu-des-Pheol-Phe-alamethicin in planar lipid bilayers

Abstract An alamethicin analogue in which all the amino-isobutyric acid and the C-terminal Pheol residues were replaced with Leu and an amidated Phe, respectively, has been synthesized. The purpose was to remove the ambiguity of a partial 3 10 helical character in alamethicin and thus, to study the conductance properties of a virtually full α helical rod modelling the natural voltage-dependent ionic channels. Macroscopic and single-channel experiments are consistent with the ‘barrel-staves’ model. The sequence of the conductance ratios of the sub-levels is very similar to the alamethicin one. The main difference lies in the very short-lived fluctuations displayed by the new analogue and is discussed in terms of helical conformation and length.

The influence of the trichorzianin C-terminal residues on the ion channel conductance in lipid bilayers

Four natural trichorzianin analogues, channel-forming peptaibols, differing in their C-terminal residues (Gln or Glu, Trpol or Pheol) were tested for their macroscopic and single-channel conductances in planar lipid bilayers. The results indicate that, as regards to the voltage threshold, the most efficient analogue is the charged Trpol-bearing one. In addition, Trpol brings about a drastic lengthening of the open channel life-times. This behaviour is attributed to the dipole moment of the end residues and to the bulkiness and hydrogen bonding ability of Trpol.

Synthetic analogues of alamethicin: effect of C-terminal residue substitutions and chain length on the ion channel lifetimes.

In a previous study, a synthetic analogue of the peptaibol alamethicin, in the sequence of which all alpha-aminoisobutyric acid (Aib) were substituted by leucine residues and the C-terminal residue modified, was shown to display the same single-channel behaviour as alamethicin in planar lipid bilayer, except that the sublevel lifetimes were much reduced. New analogues differing in their C-terminal residue (Phe-NH2, Pheol, Trp-NH2) have now been tested for their single channel properties in neutral lipid bilayers. The conductance amplitudes and open channel lifetimes do not differ significantly from the previous analogue. Thus, the nature of the last residue, which may be located near the membrane interface, does not seem to play an important role in the destabilisation of the conducting aggregate observed after the Aib substitution by Leu. Since the deletion of one residue (Glu18) in the 14-20 moiety induces a slight decrease of the increment between the conductance levels, but has no effect upon the channel lifetimes, this residue and the length of this segment do not interfer much with the channel lifetime of peptaibols. In conclusion the factors influencing the aggregate stability may be sought in the helix-helix interactions.