Figure 3. Experimental characterization of selected designed binders from the affinity improving round.

(a-d) Nanomolar affinity CHD binders CHD_d1 to d4 (full list in Fig S9), (e) nanomolar binder for AMA (see Fig S12); (f) micromolar methotrexate binder, (g-h) two nanomolar T44 binders (full list at Fig S14). For each panel, from left to right, the design model, zoom in on the sidechain-ligand interactions, FP (or SPR in the case of AMA) binding measurements, and SEC traces. Kd values and error bars are from two independent experiments. Interacting side chains and ligands are shown in sticks, with oxygen, nitrogen, iodine, and polar hydrogen colored in red, blue, purple, and white, respectively. Key interactions are indicated by gray dashed lines. The cartoon of and sticks from helixes, sheets, and loops are colored in teal, magenta, and dark blue, respectively.