title: 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.
authors.affiliation: Institute of Chemical Kinetics and Combustion, Novosibirsk, 630090, Russian Federation.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Milov
authors.forename: Yuri D
authors.initials: YD
authors.lastname: Tsvetkov
authors.forename: Marco
authors.initials: M
authors.lastname: Bortolus
authors.forename: Anna Lisa
authors.initials: AL
authors.lastname: Maniero
authors.forename: Marina
authors.initials: M
authors.lastname: Gobbo
authors.forename: Claudio
authors.initials: C
authors.lastname: Toniolo
authors.forename: Fernando
authors.initials: F
authors.lastname: Formaggio
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D002942:Circular Dichroism; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005452:Fluoresceins / Q000378:metabolism; D054848:Peptaibols; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D014364:Tryptophan / Q000378:metabolism
chemical_list: D003497:Cyclic N-Oxides; D005452:Fluoresceins; D054848:Peptaibols; D010455:Peptides; D013113:Spin Labels; C109004:ampullosporin; C024098:6-carboxyfluorescein; D014364:Tryptophan; C037109:2,2,6,6-tetramethylpiperidine-N-oxide-4-amino-4-carboxylic acid
keywords: ampullosporin; circular dichroism; conformation; electron paramagnetic resonance; peptaibiotic synthesis