title: Intra-A chain disulphide bond forms first during insulin precursor folding.
abstract: In this study, we investigated the folding pathway of insulin precursor and compared it with that of insulin-like growth factor I (IGF-I). The intra-A chain disulphide bond was found to form early in insulin precursor folding, whereas the corresponding disulphide bond in IGF-I formed late. Intra-A chain disulphide-bond deleted [A6, A11-Ser] proteins, including proinsulin, insulin, and A chain, were employed for this investigation. Under the same conditions the recombination yield of insulin from S-sulphonates of native A and B chains was 22%, while the yield of [A6, A11-Ser] insulin from S-sulphonates of [A6, A11-Ser] A chain and native B chains was only approx. 7%. This indicated that the intra-A chain disulphide bond may serve to stabilize the A chain folding intermediate so as to facilitate the correct recognition and pairing with the B chain. Time courses of oxidation of reduced insulin A chains, reduced A and B chains, and reduced proinsulins showed that the intra-A chain disulphide bond formed first during insulin precursor folding. The formation of intra-A chain disulphide bond further accelerated the formation of the other two inter-chain disulphide bonds. The time course of helix structure formation of insulin A chains also indicated that the intra-A chain disulphide bond formed first, and could stabilize partially folded A chain helix structure. The rate of intra-A chain disulphide bond formation was almost the same as that for both helix structure formation and insulin molecule formation, indicating that the formation of the intra-A chain disulphide bond was the rate limiting step for the folding of insulin precursor.
authors.affiliation: National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.
authors.forename: Y
authors.initials: Y
authors.lastname: Yuan
authors.forename: Z H
authors.initials: ZH
authors.lastname: Wang
authors.forename: J G
authors.initials: JG
authors.lastname: Tang
mesh_terms: D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D007328:Insulin / Q000737:chemistry*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry; D010084:Oxidation-Reduction; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry*; D017433:Protein Structure, Secondary
chemical_list: D004220:Disulfides; D007328:Insulin; D011498:Protein Precursors; D007334:Insulin-Like Growth Factor I