ANTIFUNGAL ACTIVITY OF SYNTHETIC 15-MER PEPTIDES BASED ON THE RS-AFP2(RAPHANUS-SATIVUS ANTIFUNGAL PROTEIN-2) SEQUENCE

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A s eries of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptid es 6,7,8 and 9, comprising the region from cystein 27 to cystein 47 of Rs-AFP2, showed substantial antifungal activity against several funga l species (minimal inhibitory concentrations of 30-60 mu g/mL), but no activity towards bacteria (except peptide 6 at 100 mu g/mL). The acti ve peptides were shown to be sensitive to the presence of cations in t he medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 mu g/mL), peptides 1,7,8 and 10 pot entiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptides on the structure of Rs-AFP 2 as determined by nuclear magnetic resonance, the active region of th e antifungal protein appears to involve beta-strands 2 and 3 in combin ation with the loop connecting those strands. A cyclized synthetic mim ic of the loop, cysteine 36 to cysteine 45, was shown to have antifung al activity. Substitution of tyrosine 38 by alanine in the cyclic pept ide substantially reduced the antifungal activity, indicating the impo rtance of this residue for the activity of Rs-AFP2 as demonstrated ear lier by mutational analysis.