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 regulation1. Artificial allosteric effectors enable remote manipulation of cell function2,3; their engineering, however, is hampered by our limited understanding of allosteric residue networks. Here, we introduce a phage-assisted evolution4 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 recombineering5 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 -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.