Intra-Molecular Chaperone Mediated Folding of a Peptide Hormone in Molecular Evolution

Abstract

Uroguanylin, an endogenous ligand of guanylyl cyclase C, consists of 16 amino acid residues and contains two intra-molecular disulfide bonds. The folding of uroguanylin is regulated by the assistance of the pro-peptide region that functions as an intra-molecular chaperone. Our previous studies suggested that a mis-bridged disulfide isomer of prouroguanylin is formed at an early stage of the refolding reaction and the molecule is then converted to the native conformation. Importantly, this folding mechanism involves a change in the secondary structure from an α-helix to a β-sheet structure at the processing site and at the mature region, respectively. However, eel-prouroguanylin possesses three Pro residues at the processing site, which inhibits α-helix formation during folding. To investigate the folding mechanism and quality control during molecular evolution, a cassette mutation was incorporated into eel and human prouroguanylin and its folding examined.The cassette mutants were expressed in E. coli cells using the T7-expression system, and produced as inclusion bodies. Therefore, the proteins were solubilized in 8 M urea containing dithiothreitol and purified by a reversed-phase high performance liquid chromatography as the reduced forms. The oxidative refolding reactions of the cassette mutants were then examined under the conditions of kinetic or thermodynamic control. The folding mechanism of the mutants and wild type prouroguanylins were compared among several species of prourogaunylin and the findings were evaluated from the standpoint of molecular evolution. The results are discussed in this paper.