Interactions of the novel antimicrobial peptide buforin 2 with lipid bilayers: Proline as a translocation promoting factor

Buforin 2 is an antimicrobial peptide discovered in the stomach tissue of t he Asian toad Bufo bufo gargarizans. The 21-residue peptide with +6 net cha rge shows antimicrobial activity an order of magnitude higher than that of magainin 2, a membrane-permeabilizing antimicrobial peptide from Xenopus la evis [Park, C. B., Kim, M. S., and Kim, S. C. (1996) Biochem. Biophys. Res. Commun. 218, 408-413]. In this study, we investigated the interactions of buforin 2 with phospholipid bilayers in comparison with magainin 2 to obtai n insight into the mechanism of action of buforin 2. Equipotent Trp-substit uted peptides were used to fluorometrically monitor peptide-lipid interacti ons. Circular dichroism measurements showed that buforin 2 selectively boun d to liposomes composed of acidic phospholipids, assuming a secondary struc ture similar to that in trifluoroethanol/water, which is an amphipathic hel ix distorted around Pro(11) with a flexible N-terminal region [Yi, G. S., P ark, C. B., Kim, S. C., and Cheong, C. (1996) FEES Lett. 398, 87-90]. Magai nin 2 induced the leakage of a fluorescent dye entrapped within lipid vesic les coupled to Lipid flip-flop. These results have been interpreted as the formation of a peptide-lipid supramolecular complex pore [Matsuzaki, K. (19 98) Biochim. Biophys. Acta 1376, 391-400]. Buforin 2 exhibited much weaker membrane permeabilization activity despite its higher antimicrobial activit y. In contrast, buforin 2 was more efficiently translocated across lipid bi layers than magainin 2. These results suggested that the ultimate target of buforin 2 is not the membrane but intracellular components. Furthermore, b uforin 2 induced no lipid flip-flop, indicating that the mechanism of trans location of buforin 2 is different from that of magainin 2. The role of Pro was investigated by use of a P11A derivative of buforin 2. The derivation caused a change to magainin 2-like secondary structure and membrane behavio r. Pro(11) was found to be a very important structural factor for the uniqu e properties of buforin 2.