posted on 2023-11-27, 16:14authored byPaul L. Blanchard, Brandon J. Knick, Sarah A. Whelan, Benjamin J. Hackel
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 Tms 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.