Figure 2.

Disulfide stabilization of de novo designed mini‐protein binders. (A) The designed structure (cartoon representation) for the binder Bot.728.3 (the nondisulfide parent of Bot.2110.4, see ‘C’). (B) Effect on function of introduced disulfides on BoNT. X‐axis, the Rosetta monomer energy (kcal·mol⁻¹ per‐residue). Y‐axis, the number of hotspot residues between the two cysteines forming the disulfide, green, disulfide designs with improved binding (or stability), red, disulfide designs with decreased binding, grey, designs with no change in binding. Disulfide designs with lower energy and more hotspots enclosed are more likely to be improved. (C) Co‐crystal structure of binder Bot.2110.4 (cartoon representation) bound to H_CB (surface representation). Two copies of Bot.2110.4 in the asymmetric unit are shown in different shades of green, the computational design is in pink. The disulfide (sticks representation) is very similar in the design model and crystal structure.