Mapping the evolution of computationally designed protein binders

Computational protein design enables the generation of binders that target specific epitopes on proteins. However, current approaches often require substantial screening from which hits require further affinity maturation. Methods for experimentally improving designed proteins and exploring their sequence-affinity landscapes could therefore streamline the development of high-affinity binders and inform future design strategies. Here, we use OrthoRep, a system for continuous hypermutation in vivo, to drive the evolution of computationally designed mini protein binders ("minibinders") that target a mammalian receptor. Despite their small sizes (59-72 amino acids), we successfully affinity matured multiple minibinders through strong selection for improved binding and also sampled new regions of minibinder fitness landscapes through extensive neutral drift. One evolved minibinder variant was used to construct a combinatorially complete sequence-affinity map for its six affinity increasing mutations, which revealed nearly full additivity in their contributions to binding. Another minibinder was subjected to both deep mutational scanning and extensive evolution under weak selection, resulting in an evolutionarily diverged collection of binder sequences that revealed non-additive relationships among mutations. Our results highlight that the affinity of computationally designed binders can be rapidly increased through evolution and provide a scalable approach for the evolutionary exploration and subsequent mapping of sequence-affinity landscapes. We suggest that this work will complement protein binder design both as a reliable experimental optimization process and as a vehicle for generating new training data.