emerimicins and 6-carboxyfluorescein

We describe the challenging solid-phase synthesis of the medium-length (14 amino-acid residues) peptaibiotic ampullosporin A, originally extracted from the fungus Sepedonium ampullosporum, and an analog doubly spin labeled (at positions 3 and 13) with the stable nitroxyl free-radical 4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid (TOAC). The results of a circular dichrosim investigation in methanol strongly support the view that both peptides are essentially right-handed helical, in particular endowed with a large population of α-helical conformers. We also observed a significant quenching effect from the TOAC(3) nitroxyl radical on the fluorescence of Trp(1), compatible with that expected when both residues are closely located on the same helix segment. Combined continuous wave and pulsed electron-electron double resonance electron paramagnetic resonance methodologies converge on the conclusion obtained from the other aforementioned spectroscopies, namely, that the [TOAC(3,13)] ampullosporin A analog is mostly folded in the α-helical conformation. A liposome leakage assay demonstrated that the membrane-modifying properties of this bis-labeled analog are remarkable and even slightly superior to those of the natural peptaibiotic itself.

Topics: Cell Membrane; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Circular Dichroism; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Fluoresceins; Peptaibols; Peptides; Protein Conformation; Spectrometry, Fluorescence; Spin Labels; Tryptophan

Trichorzianines A (TA) and B (TB) are microheterogeneous mixtures of antibiotic nonadecapeptides of the peptaibol class which interact with lipidic membranes and modify their permeability properties. The TB differ from the TA by replacement of the Gln-18 by a Glu, giving rise to a C-terminal negative charge at neutral pH. The role of this charge on the trichorzianine-lipid interaction was investigated with model membranes by fluorescence spectroscopy and the results were correlated with the biological activity toward the amoeba Dictyostelium discoideum. The interaction of the acidic trichorzianine TB IIIc (Glu-18) with phospholipid bilayers and the subsequent induced permeability were weaker than that exhibited by the uncharged TA IIIc (Gln-18) and MeTB IIIc (TB IIIc monomethyl ester). The unfavourable effect of the negative charge in TB IIIc was strongly enhanced by incorporation of cholesterol in the bilayer. Similarly, TA IIIc as well as MeTB IIIc induced growth inhibition and lysis of the amoeba Dictyostelium at four times lower concentrations than TB IIIc. The results suggested that the interaction of trichorzianines with the phospholipid bilayer and the subsequent modifications of permeability were involved in the inhibitory properties and cell lysis induced by trichorzianines toward Dictyostelium.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cell Membrane Permeability; Cholesterol; Dictyostelium; Fluoresceins; Fungal Proteins; Kinetics; Lipid Bilayers; Liposomes; Membrane Lipids; Molecular Sequence Data; Peptaibols; Peptides; Spectrometry, Fluorescence