Putative disulfide-forming pathway of porcine insulin precursor during its refolding in vitro.
Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time points, and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. An intra-A disulfide bridge was found in the former but not in the latter. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins first with the formation of the A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11, and CysB7, until the native disulfide bond A6-A11 or A7-B7 is formed to complete the refolding of PIP. The proposed refolding behavior of PIP is compared with that of IGF-I and discussed.
Qiao
Z S
ZS
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China.
Guo
Z Y
ZY
Feng
Y M
YM
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D000837:Anion Exchange Resins; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D019803:Glutathione Disulfide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000737:chemistry; D019807:Iodoacetic Acid / Q000737:chemistry; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000737:chemistry* / Q000302:isolation & purification; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019163:Reducing Agents / Q000737:chemistry; D012117:Resins, Synthetic; D013552:Swine
D000837:Anion Exchange Resins; D004220:Disulfides; D011994:Recombinant Proteins; D019163:Reducing Agents; D012117:Resins, Synthetic; D007334:Insulin-Like Growth Factor I; C042855:Mono Q; D011384:Proinsulin; D005978:Glutathione; D019803:Glutathione Disulfide; D019807:Iodoacetic Acid