Influence of A7-B7 disulfide bond deletion on the refolding and structure of proinsulin.
To probe the role of [A7-B7] disulfide bond in the structure and folding 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 alpha- helix content of the molecule and a great increase in sensitivity to tryptic digestion. 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 proinsulin 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.
Liu
Ying
Y
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Science, Peking University, Beijing 100871, China.
Tang
Jian-Guo
JG
D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007328:Insulin / Q000378:metabolism; D010084:Oxidation-Reduction; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011972:Receptor, Insulin / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D007328:Insulin; D013438:Sulfhydryl Compounds; D012694:Serine; D011384:Proinsulin; D011972:Receptor, Insulin; D014357:Trypsin; D003545:Cysteine