Folding Analyses of a de novo Designed Prouroguanylin

Abstract

Peptide hormones are typically produced in the form of a prepro-peptide, precursor molecule, which is then processed into the biologically active mature form. However, the role of the pro-peptide region, especially its role in the correct folding which is absolutely required for biological activity, has not been explained in detail. Uroguanylin folds into its native conformation with the assistance of the pro-peptide region, which functions as an intra-molecular chaperone. We recently applied its chaperone function to construct a de novo designed peptide, which is able to fold into only a bioactive conformation via the fusion of the propeptide region of uroguanylin. In addition, our previous studies of the disulfide-coupled folding of a series of Gly mutants of prouroguanylin suggested that only a few amino acid residues of prouroguanylin showed critical effects in the formation of the native conformation. To further investigate the mechanism for the propeptide-mediated folding of peptide hormones, the several amino acid residues of the de novo fusion protein were mutated to Gly residues and the refolding reactions of these proteins were examined. The mutant proteins were prepared using an E. coli expression system. The mutant proteins were produced in the form of inclusion bodies and then solubilized in 8 M urea containing dithiothreitol. The reduced forms of the mutant proteins were purified by RP-HPLC and the structures confirmed by MALDI-TOF/MS analyses. The oxidative refolding reactions of the proteins were carried out in the presence or absence of glutathione. The results of our studies will be discussed in this paper.