Determining the mechanisms of the allosteric architecture of DHFR

Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mechanisms by which mutations tune allostery. To examine this, we investigated the effects of allosteric residues in a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. In prior work using saturation mutagenesis, we showed that less than 5% of mutations had a statistically significant influence on allostery. While allostery disrupting mutations were located near the LOV2 insertion site, we found that allostery enhancing mutations were widely dispersed and enriched on the protein surface. However, these experiments did not reveal the mechanism by which the allosteric signal is propagated or how those mutations tune it. To understand these effects, we are characterizing the light-dependent changes in conformational dynamics for the chimeric enzyme by solution nuclear magnetic resonance spectroscopy and analysis of correlations between residue pairs in long timescale molecular dynamics. We have also measured the entropic and enthalpic contribution of select mutations towards the allosteric effect through kinetics assays. From these data, we aim to understand the mechanisms by which the global map of allosteric contributions inside the enzyme operates and provide insight into how allostery can be evolutionarily optimized.