Mutational Analysis of the Absolutely Conserved B8Gly: Consequence on Foldability and Activity of Insulin

摘要： <正> B8Gly is absolutely conserved in insulins during evolution.Moreover,its correspondingposition is always occupied by a Gly residue in other members of insulin superfamily.Previous work showedthat Ala replacement of BSGly significantly decreased both the activity and the foldability of insulin.However.the effects of substitution are complicated,and different replacements sometimes cause significantly differentresults.To analyze the effects of B8 replacement by different amino acids,three new insulin/single-chaininsulin mutants with BSGly replaced by Ser,Thr or Leu were prepared by protein engineering,and both theirfoldability and activity were analyzed.In general,replacement of BSGly by other amino acids causes significantdetriment to the foldability of single-chain insulin:the conformations of the three B8 mutants are essentiallydifferent from that of wild-type molecules as revealed by circular dichroism;their disulfide stabilities inredox buffer are significantly decreased:their in vitro refolding efficiencies are decreased approximatelytwo 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 biologicalactivity is concerned.Ser replacement of B8Gly has only a moderate effect:its insulin receptor-bindingactivity 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 thatGly is likely the only applicable natural amino acid for the B8 position of insulin where both foldability andactivity are concerned. ... （共7頁(yè)）