title: A peptide model of insulin folding intermediate with one disulfide.
abstract: Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20-B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.
authors.affiliation: Department of Bioengineering, Xi'an Jiaotong University, Xi'an 710049, China.
authors.forename: Han
authors.initials: H
authors.lastname: Yan
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
authors.forename: Xiao-Wen
authors.initials: XW
authors.lastname: Gong
authors.forename: Dan
authors.initials: D
authors.lastname: Xi
authors.forename: You-Min
authors.initials: YM
authors.lastname: Feng
mesh_terms: D000818:Animals; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008958:Models, Molecular; D017510:Protein Folding
chemical_list: D004220:Disulfides; D007328:Insulin