Magainin 2, an antimicrobial peptide from the Xenopus skin, kills bacteria by permeabilizing the cell membranes. We have proposed that the peptide preferentially interacts with acidic phospholipids to form a peptide-lipid supramolecular complex pore, which allows mutually coupled transbilayer traffic of ions, lipids, and peptides, thus simultaneously dissipating transmembrane potential and lipid asymmetry [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In this paper, we examined the effect of membrane curvature strain on pore formation. Magainin effectively forms the pore only in phosphatidylglycerol bilayers at low peptide-to-lipid ratios, well below 1/100. In contrast, the permeabilization of phosphatidylserine, phosphatidic acid, or cardiolipin bilayers occurred at much higher peptide-to-lipid ratios (1/50 to 1/10) with some morphological change of the vesicles. The latter three classes of phospholipids are known to form hexagonal II structures under conditions of reduced interlipid electrostatic repulsions. Incorporation of phosphatidylethanolamine also inhibited the magainin-induced pore formation in the inhibitory order of dioleoylphosphatidylethanolamine > dielaidoylphosphatidylethanolamine. Addition of a small amount of palmitoyllysophosphatidylcholine enhanced the peptide-induced permeabilization of phosphatidylglycerol bilayers. Magainin greatly raised the bilayer to hexagonal II phase transition temperature of dipalmitoleoylphosphatidylethanolamine. These results suggest that the peptide imposes positive curvature strain, facilitating the formation of a torus-type pore, and that the presence of negative curvature-inducing lipids inhibits pore formation.

Magainin peptides, isolated from Xenopus skin, kill bacteria by permeabilizing their cell membranes whereas they do not lyse erythrocytes. To elucidate the rationale for this membrane selectivity, we compared the effects of the membrane lipid composition and the transmembrane potential on the membrane-lytic power of magainin 2 with that of hemolytic melittin. The activity of magainin to zwitterionic phospholipids constituting the erythrocyte surface was extremely weak compared with that of melittin, and acidic phospholipids are necessary for effective action. The presence of sterols reduced the susceptibility of the membrane to magainin. The generation of an inside-negative transmembrane potential enhanced magainin-induced hemolysis. We can conclude that the absence of any acidic phospholipids on the outer monolayer and the abundant presence of cholesterol, combined with the lack of the transmembrane potential, contribute to the protection of erythrocytes from magainin's attack.

Magainin peptides, isolated from Xenopus skin, have broad spectra of antimicrobial activity and low toxicities to normal eukaryotic cells, thus being good candidates for therapeutic agents. The mechanism of action is considered to be the permeabilization of bacterial membranes. A number of studies using lipid vesicles have elucidated its molecular detail. However, their interactions with bacteria are not yet well understood. In this paper, we synthesized several magainin analogs with different charges (0 to +6) and hydrophobicities, and systematically studied their interactions with the outer and inner membranes of three species of Gram-negative bacteria (Escherichia coli, Acinetobacter calcoaceticus, Proteus vulgaris). The treatment of the E. coli cells with native magainin 2 (+4) immediately induced the efflux of the intracellular K+ ions and the cell death. A number of blebs were formed on the bacterial surface and the outer membrane became leaky. An increase in the peptide's positive charge enhanced the outer membrane permeabilization and the bactericidal activity. The cationic peptides also effectively permeabilized the inner membranes rich in acidic phospholipids, indicating the importance of electrostatic interactions. Substitution of Trp for Phe simultaneously increased the bactericidal activity and the hemolytic activity. A strategy to develop potent antimicrobial peptides was discussed on the basis of these results.

Magainin 2, an antimicrobial peptide from Xenopus skin, assumes an amphiphilic helix when bound to acidic phospholipids, forming a pore composed of a dynamic, peptide-lipid supramolecular complex [Matsuzaki et al. (1996) Biochemistry 35, 11361-11368]. Upon the disintegration of the pore, a fraction of the peptide molecules stochastically translocates across the bilayer (Matsuzaki, et al., 1995). In order to investigate the effects of peptide charge on the magainin 2-lipid bilayer interactions, we synthesized four magainin 2 analogs with different charges (0-6+). MG0: K10E, K11E, F12W-magainin 2. MG2+: K10E, F12W-magainin 2. MG4+: F12W-magainin 2. MG6+: F12W, E19Q-magainin 2 amide. An increase in charge resulted in a stronger binding of the peptide to the negatively charged membranes, suggesting that electrostatic attractions play a crucial role in the binding process. The helical stability in a trifluoroethanol/buffer mixture was decreased with increasing positive charge because of electrostatic repulsions between the closely spaced positive side chains, whereas the helicity in the lipid bilayer was much higher and appeared to be independent of the peptide charge. However, enhanced repulsions between the highly positively charged helices destabilized the pore. Therefore, the efficiency of the most basic peptide (MG6+) to translocate across the bilayer was the greatest by virtue of the short life span of its pore and the very tight membrane binding. The charge distribution of wild-type magainin 2 was found to be so designed as to exhibit the maximal lytic activity by simultaneously achieving a strong binding and a moderate pore stability.