Regulation of Disulfide Coupled Folding of De Novo Designed Precursor Protein

Abstract

Uroguanylin, a peptide consisting of 16 amino acid residues and 2 disulfide bonds, participates in salt and water homeostasis in mammals and the correct disulfide pairings are absolutely required for its biological activity. However, uroguanylin is not able to fold into its native conformation by itself, and its disulfide-coupled folding is mediated by the pro-peptide region in the precursor protein. Thus, the pro-peptide region of uroguanylin serves as an intra-molecular chaperone.We previously proposed that a relationship existed between the partial folding of the bioactive conformation of the mature peptide hormone and the disulfide-coupled folding of the precursor protein. X-ray crystallography data for prouroguanylin revealed that the Asp72 interacts with the receptor-binding site (Asn79-Val80-Ala81). This implies that the Asp72 residue plays an important role in the folding of prouroguanylin by stabilizing the bioactive site. To address this issue, we studied the roles of the Asp72 residue in precursor proteins, prouroguanylin and a de novo designed protein (pro-NDD-hybrid protein).To investigate the role of hydrogen bonding between the Asp72 and Asn79 residue for the precursor folding, a series of mutant proteins was prepared using an E. coli expression system and the folding of these molecules was examined using the typical glutathione redox system.The replacement of the Asp72 residue with Gly had no effect on the folding of prouroguanylin but dramatically influenced the folding of the pro-NDD-hybrid protein. The results revealed that the Asp72 residue is important in stabilizing the native conformation. Thus the Asp72 residue plays an important role in the intra-molecular chaperone function of the pro-peptide region. Furthermore, to evaluate the role of the Asp72 residue in in vivo folding, mutant proteins were also expressed in human 293T cells. The results will be discussed in this paper.