Significant Expansion of the Fluorescent Protein Chromophore through the Genetic Incorporation of a Metal-Chelating Unnatural Amino Acid
Significant Expansion of the Fluorescent Protein Chromophore through the Genetic Incorporation of a Metal-Chelating Unnatural Amino Acid
复制标题
通过金属螯合非天然氨基酸的基因掺入显着扩展荧光蛋白发色团
DOI：
10.1002/anie.201301307
复制
发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Wang, Jiangyun
中科院分区：
文献类型：
--
作者：
Liu, Xiaohong;Li, Jiasong;Wang, Jiangyun
结合金属的非天然氨基酸的基因整合[1]（UAA）是蛋白质传感器设计，[2]金属酶工程[3]和蛋白质NMR光谱学的有力方法。[4]然而，这种方法目前未被化学生物学家充分利用，因为合成UAA的路线复杂。在这里，我们报告了一个一步，高产率的酶促路线的合成一种新的UAA轴承8-羟基喹啉基团（HqAla，方案1），它形成了高度稳定的配合物与大多数过渡金属离子。[5]通过用HqAla取代不同荧光蛋白（FP）中荧光团的Tyr残基，我们首次表明UAA掺入可以导致显着红移的激发和发射光谱。我们解析了在其发色团中具有HqAla的超折叠GFP [6]（sfGFP）的晶体结构，揭示了新的8-羟基喹啉-咪唑啉酮（HQI）发色团的形成（图2），与在维多利亚水母（Aequorea victoria）绿色荧光蛋白（GFP）中发现的亲本4-（对羟基苯亚甲基）-5-咪唑啉酮（HBI）发色团相比，其具有显著更大的共轭π系统。我们的研究结果表明，HqAla纳入FP荧光团，使其具有独特的金属螯合和金属离子传感能力。在所有生物学相关的金属离子中，只有与HQI结合的ZnII离子导致荧光显著增加（7.2倍）。我们首先尝试利用野生型弗氏柠檬酸杆菌（ATCC 8090）酪氨酸酚裂解酶（wt TPL）将双齿螯合剂8-羟基喹啉（8-hydroxyquinoline）转化为2-氨基-3-（8-hydroxyquinolin-5-yl）丙酸（HqAla），因为之前已经报道wt TPL具有相对宽的底物范围。[3d然而，使用茚三酮薄层色谱法（TLC;图1B，泳道1），我们不能检测到任何HqAla的形成。检查
Genetic incorporation of metal-binding unnatural amino acids [1](UAA) is a powerful method for protein sensor design,[2] metalloenzyme engineering,[3] and protein NMR spectroscopy.[4] However, this method is currently underused by chemical biologists, because of the complex synthetic routes to UAAs. Herein, we report a one-step, high-yield enzymatic route for the synthesis of a novel UAA bearing an 8-hydroxyquinoline group (HqAla, Scheme 1), which forms highly stable complexes with most transition metal ions.[5] By substituting the Tyr residue of the fluorophore in diverse fluorescent proteins (FPs) with HqAla, we show for the first time that UAA incorporation can result in significantly redshifted excitation and emission spectra. We solved the crystal structure of superfolder GFP [6](sfGFP) with HqAla in its chromophore, revealing the formation of a novel 8-hydroxyquinolin-imidazolinone (HQI) chromophore (Figure 2), which has a significantly larger conjugated π-system in comparison to the parental 4-(p-hydroxybenzylidene)-5-imidazolinone (HBI) chromophore found in Aequorea victoria green fluorescent protein (GFP). Our results indicate that HqAla incorporation into the FP fluorophore gives it unique metal-chelating and metal-ion-sensing abilities. Among all biologically relevant metal ions, only ZnII ion binding to HQI causes a significant increase (7.2-fold) in fluorescence. This selective turn-on FP sensor was then applied for ZnII ion sensing in vivo.We first attempted to transform 8-hydroxyquinoline, a bidentate chelating agent, to 2-amino-3-(8-hydroxyquinolin-5-yl) propanoic acid (HqAla) by using the wild-type Citrobacter freundii (ATCC8090) tyrosine phenol lyase (wt TPL), because it has previously been reported that wt TPL has a relatively broad substrate scope.[3d, 7] However, we could not detect any formation of HqAla, using ninhydrin thin-layer chromatography (TLC; Figure 1B, lane 1). Inspection of the