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 they 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 homologs that retain a glucose-mediated conformationally coupled fluorescence response. Accurately identifying a glucose-binding PBP homolog among closely related paralogs is challenging. We demonstrate that a structure-based method that filters sequences by residues that bind glucose in an archetype structur...