Journal of Peptide Research Volume 57 Issue 5 Page 409  - May 2001 doi:10.1034/j.1399-3011.2001.00842.x

Synthetic peptides derived from the ß2-ß3 loop of Raphanus sativus antifungal protein 2 that mimic the active site

W. M. M. Schaaper, G.A. Posthuma and R.H. Meloen, H.H. Plasman, L. Sijtsma and A. van Amerongen, F. Fant and F. A. M. Borremans, K. Thevissen and W.F. Broekaert,

Abstract: Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three ß-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure ßfunction relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the ß2-ß3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire ß2-ß3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.