Mechanisms of chemical protein 19F-labeling and NMR-based biosensor construction in vitro and in cells using self-assembling ligand-directed tosylate compounds
Mechanisms of chemical protein 19F-labeling and NMR-based biosensor construction in vitro and in cells using self-assembling ligand-directed tosylate compounds
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DOI：
10.1039/c0sc00513d
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发表时间：
2011-01-01
期刊：
影响因子：
8.4
通讯作者：
Hamachi, Itaru
中科院分区：
文献类型：
--
作者：
Takaoka, Yousuke;Sun, Yedi;Hamachi, Itaru
化学标记方法将特定的内源性蛋白质转化为半合成生物传感器，为生物研究和药物发现提供了许多新的机会。我们最近开发了一种新的蛋白质标记方案，称为配体定向tosyl （LDT）化学，它可以定位特异性地将合成探针引入蛋白质，同时释放亲和配体。在之前的工作中，我们证明了LDT试剂1可以用F-19探针修饰碳酸酐酶I (CAI)，将其转化为基于F-19核磁共振的生物传感器，用于体外或红细胞（红细胞）中的CAI抑制剂。本文报道了1的化学性质，以及控制生物传感器构建的机制。结果表明，LDT试剂在缺乏靶蛋白的情况下形成自组装聚集体。在聚合状态下，试剂的非生产性水解被显著抑制，这表明自组装在设计稳定性更高的标记试剂方面具有潜在的实用性。在目标蛋白存在的情况下，聚集物被破坏形成非共价蛋白-试剂复合物，蛋白f -19标记继续生成f -19标记的CAI。标记的CAI的配体结合袋在体外保留了被切割的配体片段，而在红细胞中则是空的。进一步的生化研究表明，阴离子转运体可能在消除被切割的配体从细胞内部到外部的过程中起作用。这些发现为合理设计适用于生物环境中选择性蛋白质标记和生物传感器构建的试剂提供了基础依据。
Chemical labeling methods that convert a specific endogenous protein into a semisynthetic biosensor offer numerous new opportunities for biological research and drug discovery. We recently developed a novel protein labeling scheme, termed ligand-directed tosyl (LDT) chemistry, which can site-specifically introduce a synthetic probe to a protein with the concomitant release of the affinity ligand. In previous work, we demonstrated that LDT reagent 1 can be used to modify carbonic anhydrase I (CAI) with a F-19 probe, converting it into a F-19 NMR-based biosensor for CAI inhibitors either in vitro or in red blood cells (RBCs). We herein report the chemical properties of 1, and the mechanisms controlling biosensor construction. It was revealed that the LDT reagent forms self-assembled aggregates in the absence of the target protein. In the aggregated state, nonproductive hydrolysis of the reagent was significantly suppressed, which suggests the potential utility of self-assembly in the design of labeling reagents that have increased stability. In the presence of the target protein, the aggregates were disrupted to form a noncovalent protein-reagent complex, and protein F-19-labeling proceeded to generate F-19-labeled CAI. The ligand-binding pocket of the labeled CAI retained the cleaved ligand fragment in vitro, whereas the pocket was vacant in RBC. Further biochemical studies suggested that an anion transporter might play a role in eliminating the cleaved ligand from the interior to the exterior of the cells. The findings provide a fundamental basis for the rational design of reagents applicable to selective protein labeling and biosensor construction in biological contexts.