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 1H 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.