The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite.
The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite.
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DOI：
10.1021/jacs.6b01285
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发表时间：
2016-04-13
影响因子：
15
通讯作者：
Ai HW
中科院分区：
文献类型：
--
作者：
Chen ZJ;Tian Z;Kallio K;Oleson AL;Ji A;Borchardt D;Jiang DE;Remington SJ;Ai HW
硼酸及其酯类化合物已被广泛用于分子识别和化学传感。我们最近报道了一种基因编码的过氧亚硝酸盐(ONOO−)特异性荧光传感器pnGFP，它基于将硼酸部分掺入到循环置换的绿色荧光蛋白(CpGFP)中，随后进行蛋白质定向进化。与典型的芳基硼酸和酯不同，pnGFP的生色团不受毫摩尔浓度的过氧化氢(H_2O_2)的影响。本研究的重点是通过定点突变、X射线结晶学、11B核磁共振和计算分析来探索pnGFP对过氧亚硝酸根而不是过氧化氢的异常化学选择性的机制。我们的数据共同支持，蛋白质支架上的His残基极化水分子，诱导生色团中SP3杂化的硼的形成，从而调节pnGFP与各种活性氧和氮物种(ROS/RNS)的反应活性。我们的研究展示了折叠非天然蛋白质中可调的硼化学的第一个例子，这为设计选择性化学探针提供了广泛的意义。
Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO−)-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, 11B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp3-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.