The de novo designed protein ABLE exhibits promiscuous binding to diverse small molecules comparable to natural proteins, and this generalist binding capability can be computationally evolved into highly specialized functions including a specific fluorophore-binding protein (KD = 27.8 μM) and a Kemp eliminase with near-diffusion-limited catalytic efficiency (kcat/KM = 2,200,000 M⁻¹s⁻¹), demonstrating that simultaneous exploration of protein sequence space and chemical structure space enables the emergence of new binding and catalytic functions in designed proteins through mechanisms analogous to natural protein evolution.

The evolution of binding and catalysis played a central role in the emergence of life. While natural proteins have finely tuned affinities for their primary ligands, they also bind weakly and promiscuously to other molecules, which serve as starting points for stepwise, incremental evolution of entirely new specificities. Thus, modern proteins emerged from the joint exploration of sequence and structural space. The ability of natural proteins to bind small molecule fragments in well-defined geometries has been widely evaluated using methods including crystallographic fragment screening. However, this approach had not been applied to de novo proteins. Here, we apply this method to explore the binding specificity of a de novo small molecule-binding protein ABLE. As in Nature, we found ABLE was capable of forming weak complexes, which were excellent starting points for designing entirely new functions, including a binder of a turn-on fluorophore and a highly efficient Kemp eliminase enzyme (kcat/KM = 2,200,000 M-1s-1) approaching the diffusion limit. This work illustrates how simultaneous consideration of both sequence and chemical structure diversity can guide the emergence of new function in designed proteins.