Unfolding of human proinsulin. Intermediates and possible role of its C-peptide in folding/unfolding.
We have investigated the in vitro refolding process of human proinsulin (HPI) and an artificial mini-C derivative of HPI (porcine insulin precursor, PIP), and found that they have significantly different disulfide-formation pathways. HPI and PIP differ in their amino acid sequences due to the presence of the C-peptide linker found in HPI, therefore suggesting that the C-peptide linker may be responsible for the observed difference in folding behaviour. However, the manner in which the C-peptide contributes to this difference is still unknown. We have used both the disulfide scrambling method and a redox-equilibrium assay to assess the stability of the disulfide bridges. The results show that disulfide reshuffling is easier to induce in HPI than in PIP by the addition of thiol reagent. Thus, the C-peptide may affect the unique folding pathway of HPI by allowing the disulfide bonds of HPI to be easily accessible. The detailed processes of HPI unfolding by reduction of its disulfide bonds and by disulfide scrambling methods were also investigated. In the reductive unfolding process no accumulation of intermediates was detected. In the process of unfolding by disulfide scrambling, HPI gradually rearranged its disulfide bonds to form three major isomers G1, G2 and G3. The most abundant isomer, G1, contains the B7-B19 disulfide bridge. Based on far-UV CD spectra, native gel analysis and cleavage by endoproteinase V8, the G1 isomer has been shown to resemble the intermediate P4 found in the refolding process of HPI. Finally, the major isomer G1 is allowed to refold to native protein HPI by disulfide rearrangement, which indicates that a similar molecular mechanism may exist for the unfolding and refolding process of HPI.
Min
Cheng-Yin
CY
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Qiao
Zhi-Song
ZS
Feng
You-Min
YM
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011384:Proinsulin / Q000737:chemistry*; D017510:Protein Folding
D004220:Disulfides; D010446:Peptide Fragments; D011384:Proinsulin