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

19 August 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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.


allosteric regulation
non-canonical amino acid
cell-free protein synthesis
protein engineering
artificial metalloenzyme

Supplementary materials

SUPPORTING INFORMATION: Metal-Responsive Regulation of Enzyme Catalysis using Genetically Encoded Chemical Switches
Supplementary figures, experimental methods, and characterization for all compounds and engineered proteins.


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.