Effects of deletion and shift of the A20-B19 disulfide bond on the structure, activity, and refolding of proinsulin.
To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the alpha-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [Delta A19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, 100871, People's Republic of China.
Zhao-Hui
ZH
Wang
Ying
Y
Liu
Jian-Guo
JG
Ji
Jian-Guo
JG
Tang
D001666:Binding Sites, Antibody; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011972:Receptor, Insulin / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship
D004220:Disulfides; D011384:Proinsulin; D011972:Receptor, Insulin