Abstract
Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mutations that tune allostery. Are these mutations few or abundant, structurally localized or 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 catalytic activity is coupled to E. coli growth, we assessed the impact of 1548 viable DHFR single mutations on allostery. Despite most mutations being deleterious to activity, fewer than 5% of mutations had a statistically significant influence on allostery. 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. Combining several allostery enhancing mutations yielded near-additive improvements to dynamic range. Our results indicate a path toward optimizing allosteric function through variation at surface sites.
Highlights
In allosteric regulation, protein activity is modulated by an input effector signal spatially26 removed from the active site
66 synthetic allosteric fusion between Dihydrofolate reductase (DHFR) and LOV2 generated in earlier work [19, 20]
69 insertion site was guided by Statistical Coupling Analysis (SCA), an approach for analyzing
Summary
Protein activity is modulated by an input effector signal spatially26 removed from the active site. Protein activity is modulated by an input effector signal spatially. 27 sensitive, reversible, and rapid control of protein activity in response to diverse inputs [1,2,3]. 28 One common approach for achieving allosteric regulation in both engineered and evolved. 29 systems is through domain insertion: the transposition, recombination, or otherwise fusion of an. Domain insertions have been used to 33 generate fluorescent metabolite biosensors [7], sugar-regulated TEM-1 -lactamase variants. 34 [12], and a myriad of light controlled proteins including kinases, ion channels, guanosine. 36 insertion provides a powerful means to confer new regulation in a modular fashion. Naïvely created domain insertion chimeras sometimes exhibit relatively
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