Phage-assisted evolution of allosteric protein switches

Area of Science:

• Molecular Biology
• Protein Engineering
• Synthetic Biology

Background:

• Allostery is crucial for protein regulation, enabling distant functional site modulation via local conformational changes.
• Engineering artificial allosteric effectors is challenging due to limited understanding of allosteric residue networks.

Purpose of the Study:

• To develop a phage-assisted evolution platform for in vivo optimization of allosteric proteins.
• To create novel, programmable allosteric switches with enhanced activity and switchability.

Main Methods:

• Phage-assisted evolution with opposing selection pressures.
• Retron-based recombineering for exploring protein fitness landscapes.
• Long-read sequencing for tracking evolving variant pools and adaptive trajectories.

Main Results:

• Engineered AraC transcription factor into near-binary optogenetic switches with a ~1000-fold dynamic range.
• Identified linker mutations enhancing α-helix extension, improving conformational coupling between LOV2 and AraC.
• Revealed context-dependent residue interactions and adaptive trajectories during evolution.

Conclusions:

• The developed framework enables in vivo directed evolution of programmable allosteric switches.
• Coupling dynamic selection with deep mutational scanning and temporal sequencing provides both functional optimization and mechanistic insights into allosteric networks.