Phage-Assisted Evolution of Allosteric Protein Switches

Allostery, the transmission of locally induced conformational changes to distant functional sites, is a key mechanism for protein regulation ¹ . Artificial allosteric effectors enable remote manipulation of cell function ^2,3 ; their engineering, however, is hampered by our limited understanding of allosteric residue networks. Here, we introduce a phage-assisted evolution ⁴ platform for in vivo optimization of allosteric proteins. It applies opposing selection pressures to enhance activity and switchability of phage-encoded effectors and leverages retron-based recombineering ⁵ to broadly explore fitness landscapes, introducing point mutations, insertions, and deletions. Applying this framework to the transcription factor AraC yielded near-binary optogenetic switches, with light-controlled activity spanning ∼1,000-fold dynamic range. Long-read sequencing across selection cycles enabled high-resolution tracking of evolving variant pools, revealing adaptive trajectories and context-dependent residue interactions. Mechanistically, we found that linker mutations promoting α-helix extension at the sensor-effector junction enhance conformational coupling between LOV2 and AraC. These variants emerged consistently across independently evolved pools, underscoring their functional relevance. Together, we developed a framework for the directed evolution of programmable allosteric switches in vivo . By coupling dynamic selection with deep mutational scanning and temporal sequencing, it enables both functional optimization and mechanistic insight into allosteric networks.