Metal-Responsive Regulation of Enzyme Catalysis using Genetically Encoded Chemical Switches

The design of allosteric regulation in proteins to dynamically control function is a challenge in synthetic biology. To address this challenge, we developed an integrated computational and experimental workflow to incorporate a metal-responsive chemical switch into proteins. Pairs of bipyridinylalanine (BpyAla) residues were genetically encoded into two structurally distinct enzymes, a serine protease and firefly luciferase, so that metal coordination would bias the conformations of these enzymes, leading to reversible control of activity. MD-simulations guided rational BpyAla placement, significantly reducing experimental workload, and cell-free protein synthesis coupled with high-throughput experimentation enabled rapid prototyping of variants. Ultimately, this strategy yielded enzymes with a robust 20-fold dynamic range in response to divalent metals over 24 on/off switches, demonstrating the potential of this approach. We envision that this strategy of genetically encoding chemical switches into enzymes will complement other protein engineering and synthetic biology efforts, enabling new opportunities for applications where precise regulation of protein function is critical.