Rational 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 via 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 via protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.