As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.Most organisms produce membrane-active peptides that exhibit antibiotic, fungicidal, hemolytic, virucidal, and tumoricidal activities (1, 2). It is becoming clear through many studies that antimicrobial peptides are an important component of the innate defenses of all species of life (3-8). Antimicrobial peptides function by interacting with the cell membrane (1-6). In particular, cationic, linear, helical peptides, which are the most well characterized group of antimicrobial peptides, function by the "barrel-stave" and/or "carpet-like" mechanism, leading to bacterial membrane permeation (1, 5, 9 -13). In membrane environments, the peptides generally adopt an amphipathic helical structure, positioning the hydrophobic residues on one side and the hydrophilic residues on the other side of the helical axis, whereas they assume a random coil conformation in aqueous solutions.Recently, antimicrobial peptides have become a potential source of new antibiotics to combat the increasing emergence of drug-resistant bacteria (4, 5). Several antimicrobial peptides such as magainin, a 23-residue antimicrobial peptide, have been successful in pharmaceutical and commercial development (5). Particularly, from the first discovery of bombinins in Bombina variegata (7), the skin of anurans (frogs and toads) has proven to be a rich source of peptides with ...

Gaegurin 4 (GGN4), a 37-residue antimicrobial peptide, consists of two amphipathic a helices (residues 2-10 and 16-32) connected by a flexible loop region (residues 11-15). As part of an effort to develop new peptide antibiotics with low molecular mass, the activities of C-terminally truncated GGN4 analogues were tested. D 24)37 GGN4, a peptide analogue with 14 residues truncated from the C-terminus of GGN4, showed a complete loss of antimicrobial activity. However, the single substitution of aspartic acid 16 by tryptophan (D16W) in the D 24)37 GGN4 completely restored the antimicrobial activity, without any significant hemolytic activity. In contrast, neither the D16F nor K15W substitution of the D 24)37 GGN4 allowed such a dramatic recovery of activity. In addition, the D16W substitution of the native GGN4 significantly enhanced the hemolytic activity as well as the antimicrobial activity. The structural effect of the D16W substitution in the D 24)37 GGN4 was investigated by CD, NMR, and fluorescence spectroscopy. The results showed that the single tryptophanyl substitution at position 16 of the D 24)37 GGN4 induced an a helical conformation in the previously flexible loop region in intact GGN4, thereby forming an entirely amphipathic a helix. In addition, the substituted tryptophan itself plays an important role in the membrane-interaction of the peptide.