Characterization and structural role of disulfide bonds in a highly knotted thionin from Pyrularia pubera.

Disulfide bonds play a crucial role in the stabilization of the amphipathic folding of the diverse families of cysteine-rich antimicrobial peptides. The determination of cysteine pairings in these peptides has largely depended on sequence homology criteria, since the classical methods of disulfide bond characterization, which usually require proteolysis as a first step, encounter serious drawbacks derived from the tight folding and the presence of vicinal cysteines. We have chosen the Pyrularia pubera thionin, a 47-residue peptide with four internal disulfides and a remarkable resistance to most proteases, as a representative member of this type of cysteine-rich peptides and have shown that a combination of partial reduction and cyanylation readily allows the determination of its disulfide bonds. We have also studied by molecular dynamics and a combination of partial reduction and proteolysis the role of disulfide bonds in the stabilization of the tridimensional structure of this thionin and found a good agreement with our partial reduction data, suggesting that removal of only one disulfide bond is enough to significantly alter the folding of the peptide.