Fig. 3. Affinity matured 16E6-binding peptide is endowed with improved reactivity. (A) Main binding interface of 16E6 and E6AP. Cys58 of 16E6 is highlighted in brown and targeted by an electrophile substituted at Gly14. (B) Sequence table of E6AP-mimicking 13 and 13-3L3A (control peptide). Apparent Ki is determined by BLI (n.b., non-binding). Peptides were in competition with immobilized 1-biotin following a 30 min incubation with 16E6. Dha: dehydroalanine. Structure of 13, Dha (green), tri-leucine (orange). (C) BLI competition assay measurement of 13 and 13-3L3A estimated apparent Ki in nM. (D) Apparent kinetic constant Kapp was calculated from a kinetic crosslinking study, where 10 nM of MBP–16E6 was incubated with different concentrations of 13. (E) Kapp was plotted against the corresponding concentrations of 13 to estimate kinact and Ki.
