Disulfide-Coupled Folding of Prouroguanylin on Molecular Evolution

Abstract

The folding of uroguanylin is assisted by the pro-peptide region, which functions as an intra-molecular chaperone. Our previous study of the disulfide-coupled folding of prouroguanylin suggested that a mis-bridged disulfide isomer is produced at the early stage of folding and the molecule is then shifted to the native conformation, accompanied by a secondary structural change from an alpha-helix to a beta-sheet structure at the processing site and the C-terminal region, respectively. Thus, the necessity of a folding intermediate being produced suggests that the mechanism responsible for the chaperone-assisted folding of prouroguanylin includes quality control in constructing the native conformation expressly through the formation of a non-native disulfide isomer as the folding intermediate on molecular evolution. To validate this hypothesis, the folding of eel uroguanylin and prouroguanylin was compared to that for human uroguanylin and prouroguanylin, respectively. For this purpose, a series of N-terminal extended eel uroguanylin analogs was chemically synthesized. And a recombinant eel prouroguanylin was expressed as a thioredoxin-fused protein in E. coli cells because the expression efficiency of the recombinant eel prouroguanylin was quite low. The fusion protein was purified by Ni-affinity chromatography and treated with PreScission protease to release the eel prouroguanylin. The refolding reactions of eel uroguanylin analogs and eel prouroguanylin were then examined and the folding mechanism of eel prouroguanylin was compared to that for human prouroguanylin.