Influence
of A7-B7 Disulfide Bond Deletion on the Refolding
and Structure of Proinsulin
LIU Ying, TANG Jian-Guo*
( National Laboratory
of Protein Engineering and Plant Genetic Engineering,
College of Life Science, Peking University, Beijing 100871, China )
Abstract
To probe the role of [A7-B7] disulfide bond in the structure and fold
ing of proinsulin, [A7, B7Ser]-proinsulin was prepared. The differences in
in vitro refolding, oxidation of free thiol groups, circular
dichroism (CD) spectra, antibody and receptor binding assays, and sensitivity
to tryptic digestion between the mutant and the wild type proteins were studied.
The deletion of [ A7-B7] disulfide bond in proinsulin resulted in a significant
decrease of &agr;-helix content of the molecule and a great increase in sensitivity
to tryptic dig estion. The [A7-B7] disulfide bond deleted proinsulin showed
a great loss of its receptor binding activity. The in vitro
refolding study indicated that the r ate of the oxidation of free thiol groups
in the mutant was a little slower at t he later stage as compared to the native
molecule, but the deletion of [A7-B7 ] disulfide bond had little effect on
the refolding yield. A possible proinsuli n folding pathway way was proposed.
During the folding, the intra-A chain disulfide bond forms first and very
fast. The formation of the [A20-B19] disulfide bond is more crucial than [A7-B7]
disulfide bond and forms very likely earlier than the latter.
Key words isulfide bond; in vitro refolding; proinsulin; secondary structure
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