Alamethicin F50

The PELDOR technique was used to obtain the spectra of distances between spin labels for mono and double TOAC substituted analogues of [Glu(OMe)(7,18,19)] alamethicin F50/5 (Alm') peptaibiotic on the surface of the organic sorbent Oasis HLB and in ethanol solution at 77 K. For the double-labeled Alm', the free radical probes are at positions 1 and 16 (Alm'1,16). The intra- and intermolecular contributions to the PELDOR time traces were separated, with regard to the fractality of the system studied. We established that on HLB the labeled Alm' molecules are prone to aggregation. The distance spectra for Alm'1,16 show that, in both adsorbed state and in ethanol solution, the peptaibiotic is predominantly folded in the α-helix conformation. We assign the asymmetry of the distance spectrum in both cases to the occurrence of an admixture of more elongated α/3(10)-helical conformers. The portion of these conformers is higher for the peptide adsorbed on HLB. We speculate that both the broadening of the basic spectrum line at r(max) = 2.0 nm and the increase in the contribution of elongated conformers might be associated with the spread of the peptaibiotic adsorption sites on HLB as compared with the more uniform Alm'1,16 trap structure in frozen ethanol solution. The aggregates of mono-labeled Alm'1 and Alm'16 also studied. The intermolecular distance spectrum for Alm'1 on HLB is shifted toward longer distances as compared with those of Alm'16. This result suggests that in the aggregates Alm' molecules are preferentially oriented with their C-terminal regions in the vicinity.

Peptaibol antibiotics are membrane-active linear peptides of fungal origin that are characterized by a high population of the C(α) -tetrasubstituted, strongly helicogenic, α-amino acid, α-aminoisobutyric acid, an N-terminal acetyl group, and a C-terminal 1,2-amino alcohol. Alamethicins (Alms), among the longest peptaibiotics, are a group of closely sequence-related peptides composed of 19 amino acid residues. [TOAC(1) , Glu(OMe)(7, 18, 19) ] Alm and [TOAC(16) , Glu(OMe)(7, 18, 19) ] Alm are synthetic, nitroxide free-radical labeled analogs of [Glu(OMe)(7, 18, 19) ] Alm F50/5. In this work, nitroxide to peptide NH proton distance information obtained from paramagnetic relaxation enhancement (PRE) studies on [TOAC(1) , Glu(OMe)(7, 18, 19) ] Alm is compared with distances derived from an X-ray diffraction-based model. The methodology for PRE determination, as well as the generation of the X-ray diffraction-based model three-dimensional structures, is discussed. The distances obtained from PRE measurements are in close agreement with the information derived from the X-ray diffraction-based model. This finding suggests that this type of information could be implemented as long-range distance restraints in NMR-based structure determination.

The antimicrobial action of the peptide antibiotic alamethicin (Alm) is commonly related to peptide self-assembling resulting in the formation of voltage-dependent channels in bacterial membranes, which induces ion permeation. To obtain a deeper insight into the mechanism of channel formation, it is useful to know the dependence of self-assembling on peptide concentration. With this aim, we studied Alm F50/5 spin-labeled analogs in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, for peptide-to-lipid (P/L) ratios varying between 1/1500 and 1/100. Pulsed electron-electron double resonance (PELDOR) spectroscopy reveals that even at the lowest concentration investigated, the Alm molecules assemble into dimers. Moreover, under these conditions, electron spin echo envelope modulation (ESEEM) spectroscopy of D2O-hydrated membranes shows an abrupt change from the in-plane to the trans-membrane orientation of the peptide. Therefore, we hypothesize that dimer formation and peptide reorientation are concurrent processes and represent the initial step of peptide self-assembling. By increasing peptide concentration, higher oligomers are formed. A simple kinetic model of equilibrium among monomers, dimers, and pentamers allows for satisfactorily describing the experimental PELDOR data. The inter-label distances in the oligomers obtained from PELDOR experiments become better resolved with increasing P/L ratio, thus suggesting that the supramolecular organization of the higher-order oligomers becomes more defined.