ALANINE SCANNING MUTAGENESIS OF INSULIN

Alanine scanning mutagenesis has been used to identify specific side c hains of insulin which strongly influence binding to the insulin recep tor. A total of 21 new insulin analog constructs were made, and in add ition 7 high pressure liquid chromatography-purified analogs were test ed, 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, A 12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for bin ding 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 p ositions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala in sulin retains high affinity binding to the receptor. GlyB20 is conserv ed in insulins from different species, and in the structure of the B-c hain it appears to be essential for the shift from the alpha-helix B8- B19 to the beta-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.