A competing salt-bridge suppresses helix formation by the isolated C-peptide carboxylate of ribonuclease A

Abstract The C-peptide of ribonuclease A (residues 1 to 13) is obtained by cyanogen bromide cleavage at Met13, which converts methionine to a mixture of homoserine lactone (giving C-peptide lactone) and homoserine carboxylate (giving C-peptide carboxylate). The helix-forming properties of C-peptide lactone have been reported. The helix is formed intramolecularly in aqueous solution, is stabilized at low temperatures (0 to 20 °C) and also by a pH-dependent interaction between sidechains. The C-peptide lactone helix is about 1000-fold more stable than expected from “host-guest” data for helix formation in synthetic polypeptides. Here we report the failure of C-peptide carboxylate to form an α-helix in comparable conditions. Formation of a salt-bridge between the α-COO − group and the imidazolium ring of His12 + appears to be responsible for the suppression of helix formation. The presence of the Hse13-COO − … His12 + salt-bridge in C-peptide carboxylate is shown by 1 H nuclear magnetic resonance titration of the amide proton resonances of His12 and Hse13, and is expected from model peptide studies. The most probable reason why C-peptide carboxylate does not form an α-helix is that the Hse13-COO − … His12 + salt-bridge competes successfully with a helix stabilizing salt-bridge (Glu9 − … His12 + ). S-peptide (residues 1 to 20 of ribonuclease A) does form an α-helix with properties similar to those of the C-peptide (lactone) helix, which shows that the lactone ring of C-peptide lactone is not needed for helix formation. These results support the hypothesis that a Glu9 − … His12 + salt-bridge stabilizes the C-peptide (lactone) helix, and they show that specific interactions between side-chains can be important in preventing as well as in promoting α-helix formation.