Mutational analysis of the absolutely conserved B8Gly: consequence on foldability and activity of insulin.
B8Gly is absolutely conserved in insulins during evolution. Moreover, its corresponding position is always occupied by a Gly residue in other members of insulin superfamily. Previous work showed that Ala replacement of B8Gly significantly decreased both the activity and the foldability of insulin. However, the effects of substitution are complicated, and different replacements sometimes cause significantly different results. To analyze the effects of B8 replacement by different amino acids, three new insulin/single-chain insulin mutants with B8Gly replaced by Ser, Thr or Leu were prepared by protein engineering, and both their foldability and activity were analyzed. In general, replacement of B8Gly by other amino acids causes significant detriment to the foldability of single-chain insulin: the conformations of the three B8 mutants are essentially different from that of wild-type molecules as revealed by circular dichroism; their disulfide stabilities in redox buffer are significantly decreased; their in vitro refolding efficiencies are decreased approximately two folds; the structural stabilities of the mutants with Ser or Thr substitution are decreased significantly, while Leu substitution has little effect as measured by equilibrium guanidine denaturation. As far as biological activity is concerned, Ser replacement of B8Gly has only a moderate effect: its insulin receptor-binding activity is 23% of native insulin. But Thr or Leu replacement produces significant detriment: the receptor-binding potencies of the two mutants are less than 0.2% of native insulin. The present results suggest that Gly is likely the only applicable natural amino acid for the B8 position of insulin where both foldability and activity are concerned.
Guo
Zhan-Yun
ZY
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Zhang
Zhou
Z
Jia
Xiao-Yuan
XY
Tang
Yue-Hua
YH
Feng
You-Min
YM
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D005998:Glycine / Q000235:genetics*; D007328:Insulin / Q000737:chemistry / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011972:Receptor, Insulin / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D007328:Insulin; D011972:Receptor, Insulin; D005998:Glycine