Zhao Zhuangyu, Zhou Mingyang, Zemerov Serge D, Marmorstein Ronen, Dmochowski Ivan J
Department of Chemistry, University of Pennsylvania Philadelphia Pennsylvania 19104-6323 USA
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania Philadelphia Pennsylvania 19104-6323 USA.
Chem Sci. 2023 Mar 13;14(14):3809-3815. doi: 10.1039/d3sc00437f. eCollection 2023 Apr 5.
Elucidating the biochemical roles of the essential metal ion, Zn, motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn, we converted the MBP salt bridge into a Zn binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 μM achieved 'turn-on' detection of Zn with pronounced hyper-CEST contrast. This made it possible to determine different Zn levels in a biological fluid hyper-CEST. ZS was responsive to low-micromolar Zn, only modestly responsive to Cu, 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 Zn ion using anomalous scattering: Zn was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, Xe NMR chemical shift, and exchange rate are tunable parameters protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.
阐明必需金属离子锌的生化作用，推动了在生物系统中具有高灵敏度、高选择性、定量性且微创的检测策略的发展。荧光探针已用于识别细胞中的锌，但缺乏采用核磁共振（NMR）的互补方法。最近利用超灵敏氙核磁共振光谱对麦芽糖结合蛋白（MBP）的研究发现了一种可切换的盐桥，该盐桥导致氙的缓慢交换并引发强烈的超极化氙化学交换饱和转移（hyper-CEST）核磁共振信号。为了构建首个用于锌的基因编码的、具有核磁共振活性的传感器，我们将MBP盐桥转化为锌结合位点，同时保留特定的氙结合腔。仅1 μM的锌传感器（ZS）实现了对锌的“开启”检测，并具有明显的hyper-CEST信号。这使得通过hyper-CEST能够测定生物流体中的不同锌水平。根据hyper-CEST核磁共振光谱和等温滴定量热法（ITC），ZS对低微摩尔浓度的锌有反应，但对铜只有适度反应，对其他生物重要金属离子无反应。蛋白质X射线晶体学利用反常散射证实了结合的锌离子的身份：锌与两个组氨酸侧链和三个水分子配位。五配位的锌形成了一个氢键介导的门控结构，控制着氙的交换速率。金属离子结合亲和力、氙核磁共振化学位移和交换速率是通过蛋白质工程可调节的参数，这突出了开发蛋白质作为核磁共振光谱和成像的选择性金属离子传感器的潜力。
