Seebald Leah M, DeMott Christopher M, Ranganathan Srivathsan, Asare Okai Papa Nii, Glazunova Anastasia, Chen Alan, Shekhtman Alexander, Royzen Maksim
Department of Chemistry, University at Albany, SUNY , 1400 Washington Avenue, Albany, New York 12222, United States.
Inorg Chem. 2017 Apr 3;56(7):3773-3780. doi: 10.1021/acs.inorgchem.6b02286. Epub 2017 Mar 22.
Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA-protein interactions. The extent of the attenuation was related to the probe's proximity allowing us to construct the protein's contact surface map.
顺磁核磁共振技术可用于研究RNA-蛋白质复合物的三维结构。特别是，顺磁弛豫增强（PRE）数据能够提供有关不同结构组分之间长程距离的宝贵信息。对于PRE核磁共振实验，寡核苷酸通常使用氮氧化物试剂进行自旋标记。当前的工作描述了一种替代方法，该方法涉及一种基于铜（II）环烯的探针，它可以使用“点击”化学共价连接到蛋白质结合位点附近的RNA链上。该方法已被应用于研究HIV-1核衣壳蛋白7（NCp7）与模型RNA五核苷酸5'-ACGCU-3'的结合。顺磁金属与NCp7的谷氨酸残基配位降低了探针的灵活性，从而简化了PRE数据的解释。核磁共振实验表明，由于RNA-蛋白质相互作用，位于顺磁探针附近的蛋白质残基的信号强度减弱。衰减程度与探针的接近程度有关，这使我们能够构建蛋白质的接触表面图谱。
