Alanine scanning mutagenesis of insulin

Claus Kristensen*, Thomas Kjeldsen, Finn C. Wiberg, Lauge Schäffer, Morten Hach, Svend Havelund, Joseph Bass, Donald F. Steiner, Asser S. Andersen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

166 Scopus citations


Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B- chain it appears to be essential for the shift from the α-helix BS-B19 to the β-turn B20-B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.

Original languageEnglish (US)
Pages (from-to)12978-12983
Number of pages6
JournalJournal of Biological Chemistry
Issue number20
StatePublished - May 16 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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