Formation and characterization of a single Trp-Trp cross-link in indolicidin that confers protease stability without altering antimicrobial activity

Indolicidin is a 13-residue cationic, antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils. The unique composition of indolicidin distinguishes it from alpha-helical and P-structured cation ic peptides, because five of indolicidin's 13 residues are tryptophans: H-I le-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2. Solid phase synthes is of indolicidin gave rise to a minor byproduct that possessed unusual flu orescence and UV absorbance properties compared with authentic indolicidin, The byproduct was purified by combined ion exchange and reversed phase hig h pressure liquid chromatography steps and was shown be identical to authen tic indolicidin in its microbicidal activity against Staphylococcus aureus, Escherichia coli, Candida albicans, and Cryptococcus neoformans. Mass anal ysis of the byproduct revealed a a-atomic mass unit reduction compared with indolicidin, suggesting the deprotonation of two indole side chains to for m an intrachain delta(1),delta(1)'-ditryptophan derivative. We confirmed th e nature of the cross-linked byproduct, termed X-indolicidin, by absorbance and fluorescence spectroscopy, peptide mapping, and sequence analysis. Edm an degradation revealed that Trp-g and Trp-9 were covalently cross-linked. Compared with indolicidin, X-indolicidin was partially resistant to digesti on with trypsin and chymotrypsin, suggesting that the ditryptophan stabiliz es a subset of molecular conformations that are protease resistant but that are absent in the native structure.