Peptide models of four possible insulin folding intermediates with two disulfides.
The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Xiao-Yuan
XY
Jia
Zhan-Yun
ZY
Guo
Yao
Y
Wang
Ye
Y
Xu
Shun-Shan
SS
Duan
You-Min
YM
Feng
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding
D004220:Disulfides; D007328:Insulin; D010455:Peptides; D007334:Insulin-Like Growth Factor I