Genetically Encoded Glucosensor for Multiplexed 129Xe NMR

Hyperpolarized 129Xe NMR provides a non-optical platform for molecular, multiplexed detection. Genetically encoded protein reporters are attractive for biological sensing, but creating new 129Xe NMR sensors remains challenging because productive xenon binding is difficult to engineer into protein scaffolds. Here, we use glucose/galactose-binding protein (GGBP) as a ligand-responsive model protein to examine how molecular dynamics (MD)-derived cavity descriptors can guide the design of a xenon-binding protein sensor. Direct transfer of cavity-forming mutation from a related periplasmic binding protein (PBP) did not produce a well-resolved 129Xe response, motivating a broader analysis of candidate cavities by MD simulations. Among the GGBP variants examined, I24F/F90A combined an appropriate cavity volume, high pocket persistence, solvent exclusion, and high hydrophobicity density within the mutant series. The resulting glucosensor, GS, bound glucose with low-micromolar affinity and produced a distinct glucose-dependent 129Xe@GS hyper-CEST resonance shifted 30 ppm upfield from the aqueous xenon signal. This upfield resonance expands the chemical-shift window for genetically encoded protein-based 129Xe sensors to 125 ppm, highlighting an additional cavity-engineering route for modulating 129Xe NMR chemical shifts. GS enabled quantification of micromolar glucose concentrations in E. coli lysate, with results comparable to an enzymatic glucose assay. The upfield-shifted 129Xe NMR signal also allowed 129Xe@GS to be spectrally resolved from other PBP-based 129Xe sensors, enabling simultaneous detection of glucose-, ribose-, and maltose-responsive signals in a single hyper-CEST experiment. Finally, fusion of GS to the "always-on" 129Xe NMR contrast agent TEM-1 β-lactamase generated a ratiometric sensor. These results establish GGBP as a new scaffold for ligand-responsive 129Xe biosensing and introduce an MD-informed strategy for developing multiplexed and self-referenced genetically encoded 129Xe NMR sensors.