Institute of Pharmacology and Toxicology, Rudolf Virchow Center University of Wurzburg, Versbacher Strasse 9, 97078 Wurzburg, Germany.
Bioconjug Chem. 2010 May 19;21(5):853-9. doi: 10.1021/bc900394j.
The fusion of fluorescent proteins to proteins of interest has greatly advanced fluorescence microscopy, but is often limited by their large size. Here, we report site-specific, orthogonal labeling of two cellular proteins in intact cells with two small fluorescent dyes: fluorescein arsenical hairpin binder, FlAsH, and its red analogue, ReAsH, which bind to tetracysteine motifs. Development of a sequential labeling method to two different motifs, CCPGCC and FLNCCPGCCMEP, allowed site-specific labeling with FlAsH and ReAsH, respectively. Using the cell surface receptor for parathyroid hormone and its cytosolic binding protein, beta-arrestin2, we show their selective visualization in intact cells and analyze their interaction by colocalization and fluorescence resonance energy transfer (FRET). We propose that this method may be widely applied to label intracellular proteins and to study their interactions in intact cells with minimal disturbance of their function.
荧光蛋白与目的蛋白的融合极大地推动了荧光显微镜的发展，但通常受到其较大尺寸的限制。在这里，我们报告了在完整细胞中使用两种小分子荧光染料——荧光素砷发夹结合物（FlAsH）及其红色类似物 ReAsH——对两种细胞内蛋白质进行特异性、正交标记。开发出一种对两个不同基序（CCPGCC 和 FLNCCPGCCMEP）进行顺序标记的方法，分别用 FlAsH 和 ReAsH 进行特异性标记。我们利用甲状旁腺激素的细胞表面受体及其胞质结合蛋白β-arrestin2，分别对其进行了选择性可视化，并通过共定位和荧光共振能量转移（FRET）分析了它们的相互作用。我们提出，该方法可广泛应用于标记细胞内蛋白，并在不干扰其功能的情况下研究其在完整细胞中的相互作用。
