Fig. 4: Multiple alanine-substituted peptides exhibit nanomolar binding affinity.
a A subset of alanine-substituted peptides were identified and resynthesized and validated after increasing the selection stringency. Dissociation constants (KD) were determined by a competition assay using BLI. *Ala4 is not considered. **Theoretical KD (KD’) was calculated by adding the residue-specific energy contributions of individual alanine mutations (detailed calculation described in Supplementary Fig. 4). ***The reported KD of PMI25. b A 13-mer peptide 34 with seven alanine substitutions was identified (KD = 4.7 ± 2.5 nM). Molecular docking results of (c) PMI-K (gray) and (d) peptide 34 (yellow) bound to MDM2 (cyan). e Paired t-test of theoretical KD’ and measured KD. The left plot shows the KD and KD’ values. The right plot denotes the mean of difference calculated from the paired t-test. Error bar denotes 95% confidence level.
