State Key Laboratory of Elemento-organic Chemistry and College of Chemistry, Nankai University, Weijin Road 94, Tianjin 300071 (P. R. China), Fax: (+86) 22-23500623.
Chemistry. 2013 Dec 9;19(50):17141-9. doi: 10.1002/chem.201302273. Epub 2013 Nov 7.
Site-specific labeling of proteins with lanthanide ions offers great opportunities for investigating the structure, function, and dynamics of proteins by virtue of the unique properties of lanthanides. Lanthanide-tagged proteins can be studied by NMR, X-ray, fluorescence, and EPR spectroscopy. However, the rigidity of a lanthanide tag in labeling of proteins plays a key role in the determination of protein structures and interactions. Pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) are valuable long-range structure restraints in structural-biology NMR spectroscopy. Generation of these paramagnetic restraints generally relies on site-specific tagging of the target proteins with paramagnetic species. To avoid nonspecific interaction between the target protein and paramagnetic tag and achieve reliable paramagnetic effects, the rigidity, stability, and size of lanthanide tag is highly important in paramagnetic labeling of proteins. Here 4'-mercapto-2,2':6',2''-terpyridine-6,6''-dicarboxylic acid (4MTDA) is introduced as a a rigid paramagnetic and fluorescent tag which can be site-specifically attached to a protein by formation of a disulfide bond. 4MTDA can be readily immobilized by coordination of the protein side chain to the lanthanide ion. Large PCSs and RDCs were observed for 4MTDA-tagged proteins in complexes with paramagnetic lanthanide ions. At an excitation wavelength of 340 nm, the complex formed by protein-4MTDA and Tb(3+) produces high fluorescence with the main emission at 545 nm. These interesting features of 4MTDA make it a very promising tag that can be exploited in NMR, fluorescence, and EPR spectroscopic studies on protein structure, interaction, and dynamics.
镧系离子的蛋白质定点标记为研究蛋白质的结构、功能和动力学提供了很好的机会，这是由于镧系元素的独特性质。通过 NMR、X 射线、荧光和 EPR 光谱可以研究镧系标记的蛋白质。然而，在蛋白质标记中，镧系标记的刚性在确定蛋白质结构和相互作用中起着关键作用。赝接触位移（PCS）和顺磁弛豫增强（PRE）是结构生物学 NMR 光谱学中非常有价值的长程结构约束。这些顺磁约束的产生通常依赖于目标蛋白质与顺磁物种的定点标记。为了避免目标蛋白与顺磁标签之间的非特异性相互作用并实现可靠的顺磁效应，在蛋白质的顺磁标记中，镧系标记的刚性、稳定性和尺寸非常重要。此处介绍了 4'-巯基-2,2':6',2''-三联吡啶-6,6''-二羧酸（4MTDA）作为刚性的顺磁和荧光标记物，其可以通过形成二硫键而被定点连接到蛋白质上。4MTDA 可以通过蛋白质侧链与镧系离子的配位而被轻易固定。在与顺磁镧系离子形成的复合物中，观察到 4MTDA 标记的蛋白质具有较大的 PCS 和 RDC。在 340nm 的激发波长下，蛋白质-4MTDA 和 Tb(3+) 形成的复合物产生高荧光，其主要发射峰在 545nm。4MTDA 的这些有趣特性使其成为一种非常有前途的标记物，可以用于蛋白质结构、相互作用和动力学的 NMR、荧光和 EPR 光谱学研究。
