Self-assembling and membrane modifying properties of a lipopeptaibol studied by CW-ESR and PELDOR spectroscopies.
Trichogin GA IV is a short lipopeptaibol antibiotic that is capable of enhancing the transport of small cations through the phospholipid double layer of the membrane. The antibiotic activity of the undecapeptide is thought to be based on either its self-assembling or membrane-modifying property. The chemical equilibrium between self-aggregated and non-aggregated molecular states was studied by CW-ESR spectroscopy using solutions of TOAC nitroxide spin-labelled trichogin analogues in an apolar solvent to mimic the membrane bound state. At room temperature the two different sets of signals observed in the spectrum were attributed to the presence of both monomers and aggregates in the sample. The ESR spectra of the monomeric and aggregated forms were separated and the dependence of the fraction of monomeric peptide molecules on concentration was obtained over the range 5 x 10(-6) to 7 x 10(-4) M. A two-step aggregation mechanism is proposed: dimerization of peptide molecules followed by aggregation of dimers to assemblies of four peptide molecules per aggregate. The equilibrium constants were estimated for both steps. In addition, the lower lifetime limit was determined for dimers and tetramers. It is shown that when the peptide concentration exceeds 10(-5) M. the major part of the peptide molecules in solution has the form of tetrameric aggregates. Independently, the PELDOR technique was used to investigate the concentration dependence of the parameters of the dipole-dipole interaction between spin labels in frozen (77 K] glassy solutions of aggregates of mono-labelled TOAC analogues. The number of molecules in aggregates as well as the frequency and amplitude of PELDOR signal oscillations were found to be concentration independent in the range 5 x 10(-4) to 8 x 10(-3) M. In the frozen glassy solution state, the number of peptide molecules per aggregate was determined to be close to four, which is in agreement with the value obtained for spin-labelled trichogin at room temperature. The present data provide experimental evidence in favour of a self-assembling rather than a membrane-modifying ion conduction mechanism.
Milov
A D
AD
Institute of Chemical Kinetics and Combustion, Novosibirsk, 630090 Russian Federation.
Tsvetkov
Yu D
YD
Formaggio
F
F
Crisma
M
M
Toniolo
C
C
Raap
J
J
D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry* / Q000494:pharmacology; D006020:Glycopeptides; D007476:Ionophores / Q000737:chemistry; D007700:Kinetics; D055666:Lipopeptides; D008956:Models, Chemical; D010455:Peptides; D044367:Phase Transition; D013113:Spin Labels
D000900:Anti-Bacterial Agents; D005656:Fungal Proteins; D006020:Glycopeptides; D007476:Ionophores; D055666:Lipopeptides; D010455:Peptides; D013113:Spin Labels; C116419:trichogin GA IV