Bioconjugation of Proteins with a Paramagnetic NMR and Fluorescent Tag
Bioconjugation of Proteins with a Paramagnetic NMR and Fluorescent Tag
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蛋白质与顺磁 NMR 和荧光标签的生物共轭。
DOI：
10.1002/chem.201302273
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发表时间：
2013-12-09
影响因子：
4.3
通讯作者：
Su, Xun-Cheng
中科院分区：
文献类型：
--
作者：
Huang, Feng;Pei, Ying-Ying;Su, Xun-Cheng
利用镧系元素的独特性质，用镧系离子对蛋白质进行位点特异性标记，为研究蛋白质的结构、功能和动力学提供了巨大的机会。镧系元素标记的蛋白质可以通过核磁共振、x射线、荧光和EPR光谱来研究。然而，镧系标签在蛋白质标记中的刚性在蛋白质结构和相互作用的确定中起着关键作用。伪接触位移（PCS）和顺磁弛豫增强（PRE）是结构生物核磁共振光谱中有价值的远程结构约束。这些顺磁约束的产生通常依赖于用顺磁物质对靶蛋白进行位点特异性标记。为了避免靶蛋白与顺磁标签之间的非特异性相互作用，获得可靠的顺磁效果，镧系标签的刚性、稳定性和尺寸在蛋白质的顺磁标记中至关重要。4'-巯基-2,2':6',2 " -三吡啶-6,6 " -二羧酸（4MTDA）是一种刚性顺磁和荧光标记，可以通过形成二硫键特异性地附着在蛋白质上。mtda可以很容易地通过蛋白质侧链与镧系离子的配位来固定。在顺磁性镧系离子络合物中，观察到4mtda标记的蛋白具有较大的PCSs和rdc。在激发波长为340 nm时，蛋白- 4mtda与Tb（3+）形成的复合物产生高荧光，主要发射波长为545 nm。4MTDA的这些有趣的特征使其成为一个非常有前途的标签，可以用于核磁共振、荧光和EPR光谱研究蛋白质结构、相互作用和动力学。
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.