Fig. 3: High-frequency alanine pairs observed in the affinity selection show high tolerance for double mutations.
a (i, i + 4) pairwise alanine substitution frequency extracted from Fig. 2c. b Binding affinity of (i, i + 4) pairwise alanine-substituted peptides. c Binding affinity of (i, i + 4) perfluoroaryl stapled peptides. The three most potent stapled peptides correlate with the three highest frequency pairwise alanine substitutions. Therefore, the (i, i + 4) pairwise alanine substitution frequencies accurately indicate retention of high affinity in peptide binders substituted at the corresponding positions. Binding dissociation constants (KD) were determined by a competition assay using BLI. d Representative chemical structures of an (i, i + 4) pairwise alanine-substituted peptide (1) and an (i, i + 4) perfluoroaryl-stapled peptide (9).
