Flexibility exists in the region of [A6-A11, A7-B7] disulfide bonds during insulin precursor folding.
To investigate the possible similarity of the proinsulin folding process with insulin-like growth factor I, two swap-like human proinsulin mutant proteins [A7,A11Ser]-HPI and [A11Ser,A12Cys]-HPI were prepared. Their in vitro refolding yields, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion were studied and compared with both native HPI and [A6,A11Ser]-HPI. The results indicate that the shift mutation in the disulfide bond caused more conformational change and a greater decrease in biological activity than the deletion mutation on the proinsulin molecule. However, the shift of the intra-A chain disulfide bond had little effect on the refolding rate of the molecule. In vitro refolding yields of HPI analogues with shift or deletion mutations in the region of the [A6-A11,A7-B7] disulfide bonds were almost as high as that of wild type HPI suggesting that the region of the [A6-A11,A7-B7] disulfide bonds possesses some flexibility as is found in the corresponding region of insulin-like growth factor I.
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, 100871, People's Republic of China.
Ying
Y
Liu
Zhao-Hui
ZH
Wang
Jian-Guo
JG
Tang
D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000737:chemistry; D018583:Pliability; D011384:Proinsulin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding
D004220:Disulfides; D007328:Insulin; D007334:Insulin-Like Growth Factor I; D011384:Proinsulin