Quenched Ligand-Directed Tosylate Reagents for One-Step Construction of Turn-On Fluorescent Biosensors
Quenched Ligand-Directed Tosylate Reagents for One-Step Construction of Turn-On Fluorescent Biosensors
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DOI：
10.1021/ja902486c
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发表时间：
2009-07-01
影响因子：
15
通讯作者：
Hamachi, Itaru
中科院分区：
文献类型：
--
作者：
Tsukiji, Shinya;Wang, Hangxiang;Hamachi, Itaru
由蛋白质框架和合成荧光团组成的半合成荧光生物传感器是特异性检测生物相关分子的强大分析工具。我们在此报告了一种新的方法，允许在一个步骤的方式构建打开荧光半合成生物传感器。该策略是基于配体导向的甲苯磺酰基（LDT）化学，一种新型的亲和导向的蛋白质标记方案，它可以位点特异性地将合成探针引入蛋白质表面，同时释放亲和配体。通过将有机染料连接到蛋白质配体和荧光猝灭剂的缀合物上，设计了新型猝灭配体导向的甲苯磺酸酯（Q-LDT）试剂。Q-LDT介导的标记直接将天然蛋白质转化为荧光标记的蛋白质，其保持与切割的配体束缚的猝灭剂非共价复合。该标记蛋白的荧光最初被淬灭，并且仅在特定分析物存在下，由于配体-淬灭剂片段的排出，荧光增强（打开）。使用一个单一的标记步骤，这种方法被成功地应用到碳酸酐酶II（CAII）和Src同源2（SH 2）域，分别产生对CAII抑制剂和磷酸酪氨酸肽的开启荧光生物传感器。详细的研究表明，所获得的生物传感器表现出其天然的配体选择性。LDT化学的高靶特异性还允许我们不仅以纯化的形式而且以细菌细胞裂解物的形式制备基于SH 2结构域的生物传感器。这些结果表明，实用的Q-LDT为基础的方法，以扩大半合成生物传感器的应用。
Semisynthetic fluorescent biosensors consisting of a protein framework and a synthetic fluorophore are powerful analytical tools for specific detection of biologically relevant molecules. We report herein a novel method that allows for the construction of turn-on fluorescent semisynthetic biosensors in a one-step manner. The strategy is based on the ligand-directed tosyl (LDT) chemistry, a new type of affinity-guided protein labeling scheme which can site-specifically introduce synthetic probes to the surface of proteins with concomitant release of the affinity ligands. Novel quenched ligand-directed tosylate (Q-LDT) reagents were designed by connecting an organic dye to a conjugate of a protein ligand and a fluorescence quencher through a tosyl linker. The Q-LDT-mediated labeling directly converts a natural protein to a fluorescently labeled protein that remains noncovalently complexed with the cleaved ligand-tethered quencher. The fluorescence of this labeled protein is initially quenched and only in the presence of specific analytes is the fluorescence enhanced (turned on) due to the expulsion of the ligand-quencher fragment. Using a single labeling step, this approach was successfully applied to carbonic anhydrase II (CAII) and a Src homology 2 (SH2) domain to generate turn-on fluorescent biosensors toward CAII inhibitors and phosphotyrosine peptides, respectively. Detailed investigations revealed that the obtained biosensors exhibit their natural ligand selectivity. The high target-specificity of the LDT chemistry also allowed us to prepare the SH2 domain-based biosensor not only in a purified form but also in a bacterial cell lysate. These results demonstrate the utility of the Q-LDT-based approach to expand the applications of semisynthetic biosensors.