Bombinin H2 and H4 are peptides isolated from the skin of the frog Bombina variegata that exhibit antimicrobial activity against Leishmania as well as bacteria. H4 is an isomer of H2 that has d-allo-Ile at position 2 from the N-terminus. Although H4 exhibits higher antimicrobial activity than that of H2, the molecular mechanism has remained unclear. In this study, we tried to reveal the molecular mechanism in terms of lipid membrane disruption through pore formation, using electrophysiological measurements. Based on our experiments, we estimated the pore-forming structure, pore size, and the kinetics in a bacteria model membrane. Stochastic analysis of the current data indicated that peptide isomerization enables us to accelerate the pore formation owing to the higher affinity between the peptide and lipid membrane. Additionally, the H2/H4 mixture was studied with 31P NMR and cross-linking experiment with mass spectrometry. It was found that heterogeneous pore formation with H2 and H4 was indicated. This electrophysiological approach will likely be promising as a useful tool for analyzing the molecular mechanism of pore-forming peptides.
Keywords: antimicrobial peptide; bombinin; electrophysiology; isomer; lipid bilayer; solid-state NMR.