Discovery of Thermostable, Fluorescently Responsive Glucose Biosensors by Structure-Assisted Function Extrapolation.

Accurate assignment of protein function from sequence remains a fascinating and difficult challenge. The periplasmic-binding protein (PBP) superfamily present an interesting case of function prediction because they are both ubiquitous in prokaryotes and tend to diversify through gene duplication "explosions" that can lead to large numbers of paralogs in a genome. An engineered version of the moderately thermostable glucose-binding PBP from Escherichia coli has been used successfully as a reagentless fluorescent biosensor both in vitro and in vivo. To develop more robust sensors that meet the challenges of real-world applications, we report the discovery of thermostable homologues that retain a glucose-mediated conformationally coupled fluorescence response. Accurately identifying a glucose-binding PBP homologue among closely related paralogs is challenging. We demonstrate that a structure-based method that filters sequences by residues that bind glucose in an archetype structure is highly effective. Using fully sequenced bacterial genomes, we found that this filter reduced high paralog numbers to single hits in a genome, consistent with the accurate separation of glucose binding from other functions. We expressed engineered proteins for eight homologues, chosen to represent different degrees of sequence identity, and tested their glucose-mediated fluorescence responses. We accurately predicted the presence of glucose binding down to 31% sequence identity. We have also successfully identified suitable candidates for next-generation robust, fluorescent glucose sensors.