Synthesis and conformational properties of a TOAC doubly spin‐labeled analog of the medium‐length, membrane active peptaibiotic ampullosporin a as revealed by cd, fluorescence, and EPR spectroscopies

Abstract

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.

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs

“…In the spectra shown in Figure , the R values are 0.86 for MeOH and 0.96 for POPC vesicles. As typical R values for a fully developed α‐helix are close to 1.0 and for a fully developed 3 10 ‐helix are equal to or below 0.4, these results clearly point to a largely predominant α‐helix (over the 3 10 ‐helix) structure for this 14‐mer peptaibiotic under these experimental conditions, in agreement with our published data on the closely related compounds (Table ) tylopeptin B and ampullosporin A …”

“…For this compound, the calculated value (see Ref. for details) of the excitation parameter p B was found to be , which is quite close to the experimental one, .…”

“…More information on the mode of chalciporin A interaction with liposomes was obtained from further study of depth‐dependent Trp fluorescence quenching with lipids characterized by a nitroxide radical (doxyl) moiety at five diverging distances from the bilayer center . Rather surprisingly, the results obtained (not shown) indicate the degree of fluorescence quenching as almost independent from the quencher position in the lipid chain, with a slightly more pronounced effect (but within experimental error) when the quencher is positioned near the bilayer surface.…”

“…The distance distribution function was obtained by two methods: (i) using the PELDOR Analysis 2006 software, in which the regularization parameter was chosen in the L‐curve to avoid over‐smoothing at short distances, and (ii) Monte Carlo simulations of the averaged curve . The results are given in Figure .…”

“…In the last few years, we have been particularly attracted by the medium‐length compounds (ampullosporin, tylopeptin, and heptaibin) mainly because only this subclass of peptaibiotics has been poorly studied. A more in‐depth inspection of their sequences revealed that some of them exhibit a useful property, namely the presence of a Trp residue at (or very near) the N‐terminus.…”