Quantifying the evolvability of allostery at different protein surfaces.

Abstract

Allosteric regulation requires the cooperative action of multiple amino acid residues to relay a signal from a distal surface site to the protein active site. It is an open problem to understand how new regulation evolves through a Darwinian process of single mutation and selection. Yet a better understanding of how allostery emerges is critical to our ability to engineer new custom biosensors. Are some protein surfaces more prone to evolve into allosteric sites than others? Or is the capacity to evolve allostery homogenously distributed across a protein's entire surface? Recent work has identified that allosteric regulation is more easily engineered at certain protein surfaces termed “allosteric hotspots.” These hotspots occur at surface positions that co-evolve with enzyme active sites. Allosteric hotspots have been used to engineer new allosteric regulation of dihydrofolate reductase (DHFR) by the light-oxygen-voltage-sensing domain (LOV2). When LOV2 is fused to allosteric hotspots on DHFR, DHFR activity is weakly regulated by light (1.3-fold). However, fusing LOV2 to non-hotspot positions does not result in allosteric control. The low level of regulation observed in DHFR/LOV2 fusions presents an opportunity to study how allostery can be optimized by evolution. In my work, I am using random mutagenesis combined with a high-throughput screen to evolve new allosteric regulation. I am performing forward evolution of allostery for a weakly allosteric, hotspot-connected fusion, and for a non-allosteric, non-hotspot-connected fusion. I am monitoring the distribution of mutational effects on allostery for each construct over rounds of selection. With this approach, I will quantify the evolvability of allosteric regulation at different protein surface sites. I hypothesize that allosteric hotspots will have a higher prevalence of allostery-tuning mutations, and that these mutations will result in higher levels of allosteric regulation.