Rational design of a genetically encoded NMR zinc sensor

Zhao, Zhuangyu; Zhou, Mingyang; Zemerov, Serge D.; Marmorstein, Ronen; Dmochowski, Ivan J.

doi:10.1039/d3sc00437f

Rational design of a genetically encoded NMR zinc sensor

Journal Article · Mon Mar 13 00:00:00 EDT 2023 · Chemical Science

DOI:https://doi.org/10.1039/d3sc00437f· OSTI ID:2423577

^[1]; ^[1]; Zemerov, Serge D. ^[1]; ^[1]; ^[1]

Univ. of Pennsylvania, Philadelphia, PA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-06CH11357; SC0012704

OSTI ID:: 2423577

Journal Information:: Chemical Science, Journal Name: Chemical Science Journal Issue: 14 Vol. 14; ISSN 2041-6520

Publisher:: Royal Society of ChemistryCopyright Statement

Country of Publication:: United States

Language:: English

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