biostudies-literatureZhao ZNational Institute of General Medical SciencesNational Science Foundation3809-3815https://www.ebi.ac.uk/biostudies/studies/S-EPMC10074429biostudies-literatureUnknown14(14)Elucidating the biochemical roles of the essential metal ion, Zn²⁺, motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn²⁺ in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive ¹²⁹Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized ¹²⁹Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn²⁺, we converted the MBP salt bridge into a Zn²⁺ binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 μM achieved 'turn-on' detection of Zn²⁺ with pronounced hyper-CEST contrast. This made it possible to determine different Zn²⁺ levels in a biological fluid <i>via</i> hyper-CEST. ZS was responsive to low-micromolar Zn²⁺, only modestly responsive to Cu²⁺, and nonresponsive to other biologically important metal ions, according to hyper-CEST NMR spectroscopy and isothermal titration calorimetry (ITC). Protein X-ray crystallography confirmed the identity of the bound Zn²⁺ ion using anomalous scattering: Zn²⁺ was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn²⁺ forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, ¹²⁹Xe NMR chemical shift, and exchange rate are tunable parameters <i>via</i> protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.Chemical scienceRational design of a genetically encoded NMR zinc sensor.PMC10074429R35-GM-131907ACI-1928147ACI-1548562Zhou MDmochowski IJZemerov SDZhao ZMarmorstein RfalseRational design of a genetically encoded NMR zinc sensor.Elucidating the biochemical roles of the essential metal ion, Zn²⁺, motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn²⁺ in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive ¹²⁹Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized ¹²⁹Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn²⁺, we converted the MBP salt bridge into a Zn²⁺ binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 μM achieved 'turn-on' detection of Zn²⁺ with pronounced hyper-CEST contrast. This made it possible to determine different Zn²⁺ levels in a biological fluid <i>via</i> hyper-CEST. ZS was responsive to low-micromolar Zn²⁺, only modestly responsive to Cu²⁺, and nonresponsive to other biologically important metal ions, according to hyper-CEST NMR spectroscopy and isothermal titration calorimetry (ITC). Protein X-ray crystallography confirmed the identity of the bound Zn²⁺ ion using anomalous scattering: Zn²⁺ was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn²⁺ forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, ¹²⁹Xe NMR chemical shift, and exchange rate are tunable parameters <i>via</i> protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.2023-01-01T00:00:00Z2023 Apr2026-05-29T07:58:32.691Z2025-02-19T01:32:36.741ZS-EPMC100744293703569910.1039/d3sc00437f