Reversible site-selective labeling of membrane proteins in live cells
Reversible site-selective labeling of membrane proteins in live cells
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DOI：
10.1038/nbt954
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发表时间：
2004-04-01
影响因子：
46.9
通讯作者：
Vogel, H
中科院分区：
文献类型：
--
作者：
Guignet, EG;Hovius, R;Vogel, H
化学和生物标记是阐明生物化学细胞网络中蛋白质功能的基础。特别地，荧光探针允许以高时间和空间分辨率检测活细胞中蛋白质的分子相互作用、迁移率和构象变化（1-3）。我们提出了一种通用的方法来标记蛋白质在体内选择性，快速（秒）和可逆的，小分子探针，可以有各种各样的属性。这些探针包含发色团和金属离子螯合次氮基三乙酸酯（NTA）部分，其可逆且特异性地结合到感兴趣的蛋白质中的工程化寡聚组氨酸序列（4）。我们通过将NTA-发色团缀合物（5）结合到代表性的配体门控离子通道和G蛋白偶联受体（各自含有多组氨酸序列）来证明该方法的可行性。我们通过荧光测量研究了离子型5-羟色胺受体，以表征体内探针-受体相互作用，获得受体结构和质膜分布的信息。
Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution(1-3). We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest(4). We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates(5) to a representative ligand-gated ion channel and G protein-coupled receptor, each containing a polyhistidine sequence. We investigated the ionotropic 5HT(3) serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.