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Bioconjugation of Proteins with a Paramagnetic NMR and Fluorescent Tag

机译：具有顺磁性NMR和荧光标记的蛋白质生物缀合

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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. Lanthanidetagged 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 Tb3+ 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.
机译：利用镧系元素离子对蛋白质进行位点特异性标记，凭借镧系元素的独特性质，为研究蛋白质的结构，功能和动力学提供了巨大的机会。可以用NMR，X射线，荧光和EPR光谱研究标记有镧系元素的蛋白。但是，镧系元素标记在蛋白质标记中的刚性在确定蛋白质结构和相互作用中起着关键作用。伪接触位移（PCS）和顺磁弛豫增强（PRE）是结构生物学NMR光谱中有价值的远程结构限制。这些顺磁性约束物的产生通常依赖于靶蛋白具有顺磁性物质的位点特异性标记。为避免靶蛋白与顺磁性标签之间发生非特异性相互作用并获得可靠的顺磁性效应，镧系元素标签的刚性，稳定性和大小在蛋白质的顺磁性标记中非常重要。在此引入4'-巯基-2,2'：6'，2“-吡啶-6,6”-二羧酸（4MTDA）作为刚性顺磁性和荧光标记，可以通过形成将其位点特异性地附着到蛋白质上通过将蛋白质侧链与镧系元素离子配位，可以很容易地将4MTDA固定化;在顺磁性镧系元素离子的配合物中观察到4MTDA标记的蛋白质具有较大的PCS和RDC。由蛋白质4MTDA和Tb3 +形成的荧光在545 nm处具有高发射强度，这些有趣的特征使其成为非常有前途的标签，可用于NMR，荧光和EPR光谱研究蛋白质结构，相互作用和动力学。

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