Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, Virginia 22908, United States.
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States.
ACS Chem Biol. 2023 Jun 16;18(6):1388-1397. doi: 10.1021/acschembio.3c00139. Epub 2023 May 15.
Boronic acid-containing fluorescent molecules have been widely used to sense hydrogen peroxide and peroxynitrite, which are important reactive oxygen and nitrogen species in biological systems. However, it has been challenging to gain specificity. Our previous studies developed genetically encoded, green fluorescent peroxynitrite biosensors by genetically incorporating a boronic acid-containing noncanonical amino acid (ncAA), -boronophenylalanine (BoF), into the chromophore of circularly permuted green fluorescent proteins (cpGFPs). In this work, we introduced BoF to amino acid residues spatially close to the chromophore of an enhanced circularly permuted red fluorescent protein (ecpApple). Our effort has resulted in two responsive ecpApple mutants: one bestows reactivity toward both peroxynitrite and hydrogen peroxide, while the other, namely, pnRFP, is a selective red fluorescent peroxynitrite biosensor. We characterized pnRFP and in live mammalian cells. We further studied the structure and sensing mechanism of pnRFP using X-ray crystallography, B-NMR, and computational methods. The boron atom in pnRFP adopts an sp-hybridization geometry in a hydrophobic pocket, and the reaction of pnRFP with peroxynitrite generates a product with a twisted chromophore, corroborating the observed "turn-off" fluorescence response. Thus, this study extends the color palette of genetically encoded peroxynitrite biosensors, provides insight into the response mechanism of the new biosensor, and demonstrates the versatility of using protein scaffolds to modulate chemoreactivity.
含硼酸的荧光分子已被广泛用于检测过氧化氢和过氧亚硝酸盐，它们是生物系统中重要的活性氧和氮物种。然而，获得特异性一直具有挑战性。我们之前的研究通过在环状排列的绿色荧光蛋白（cpGFP）的发色团中基因整合含有硼酸的非天然氨基酸（ncAA）-硼苯丙氨酸（BoF），开发了遗传编码的绿色荧光过氧亚硝酸盐生物传感器。在这项工作中，我们将 BoF 引入到与增强型环状排列红色荧光蛋白（ecpApple）的发色团空间接近的氨基酸残基中。我们的努力得到了两个响应性的 ecpApple 突变体：一个赋予对过氧亚硝酸盐和过氧化氢的反应性，而另一个，即 pnRFP，是一种选择性的红色荧光过氧亚硝酸盐生物传感器。我们对 pnRFP 进行了表征，并在活哺乳动物细胞中进行了研究。我们进一步使用 X 射线晶体学、B-NMR 和计算方法研究了 pnRFP 的结构和传感机制。pnRFP 中的硼原子在疏水环境中采用 sp 杂化几何形状，pnRFP 与过氧亚硝酸盐的反应生成具有扭曲发色团的产物，证实了观察到的“关闭”荧光响应。因此，这项研究扩展了遗传编码过氧亚硝酸盐生物传感器的颜色范围，深入了解了新生物传感器的响应机制，并展示了使用蛋白质支架来调节化学选择性的多功能性。
