Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C-terminally truncated gaegurin 4.

Gaegurin 4 (GGN4), a 37-residue antimicrobial peptide, consists of two amphipathic alpha 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. Delta24-37 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 Delta24-37 GGN4 completely restored the antimicrobial activity, without any significant hemolytic activity. In contrast, neither the D16F nor K15W substitution of the Delta24-37 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 Delta24-37 GGN4 was investigated by CD, NMR, and fluorescence spectroscopy. The results showed that the single tryptophanyl substitution at position 16 of the Delta24-37 GGN4 induced an alpha helical conformation in the previously flexible loop region in intact GGN4, thereby forming an entirely amphipathic alpha helix. In addition, the substituted tryptophan itself plays an important role in the membrane-interaction of the peptide.