Hyperstable Synthetic Mini-Proteins as Effective Ligand Scaffolds

Small, single-domain protein scaffolds are compelling sources of molecular binding ligands with the potential for efficient physiological transport, modularity, and manufacturing. Yet, mini-proteins require a balance between biophysical robustness and diversity to enable new functions. We tested the developability and evolvability of millions of variants of 43 designed libraries of synthetic 40-amino acid βαββ proteins with diversified sheet, loop, or helix paratopes. We discovered a scaffold library that yielded hundreds of binders to seven targets while exhibiting high stability and soluble expression. Binder discovery yielded 6-122 nM affinities without affinity maturation and T_ms averaging ≥78 °C. Broader βαββ libraries exhibited varied developability and evolvability. Sheet paratopes were the most consistently developable, and framework 1 was the most evolvable. Paratope evolvability was dependent on target, though several libraries were evolvable across many targets while exhibiting high stability and soluble expression. Select βαββ proteins are strong starting points for engineering performant binders.