Deep mutational scanning of a synthetic allosteric construct - DHFR/LOV2 fusion

Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mutations that tune allosteric function. It is unclear if such mutations are few or abundant; localized to the allosteric site or structurally distributed. To examine this, we conducted saturation mutagenesis of a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. Using a high-throughput assay wherein DHFR activity is coupled to E. coli growth, we assessed the allosteric impact of 1548 viable DHFR single mutations. We found that 5% of mutations had a statistically significant influence on regulation; with generally modest effect sizes of a few fold. Most allostery disrupting mutations were proximal to the LOV2 insertion site. In contrast, allostery enhancing mutations were structurally distributed and enriched on the protein surface, indicating that weakly conserved and solvent accessible positions play an important role in tuning allosteric dynamic range.