Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, USA.
Magn Reson Chem. 2010 Dec;48(12):918-24. doi: 10.1002/mrc.2687.
Saturation transfer difference (STD)-NMR has been widely used to screen ligand compound libraries for their binding activities to proteins and to determine the binding epitopes of the ligands. We report herein, a Clean STD-NMR method developed to overcome false positives (artifacts) observed in the STD-NMR spectrum due to the power spillover of RF irradiation. The method achieved higher degree of resonance saturation through digital editing of two STD-NMR spectra to generate a concatenated difference spectrum and three times of sensitivity enhancement for a loose binding complex involving DNA oligonucleotide and an RNA-binding protein, CUGBP-1ab (25.2 kDa). The interesting binding characteristics of the complex dCTGTCT-CUGBP1ab were obtained. The method was applied to a mixture of small ligand and bovine serum albumin protein (BSA, 66.3 kDa), and detected the intermolecular contacts at a BSA concentration as low as 0.1 µM, a working concentration useful for the detection of proteins of low solubility at biologically relevant conditions.
饱和转移差异（STD）-NMR 已被广泛用于筛选配体化合物文库与蛋白质的结合活性，并确定配体的结合表位。我们在此报告了一种清洁 STD-NMR 方法的开发，以克服由于 RF 照射的功率溢出而在 STD-NMR 光谱中观察到的假阳性（假象）。该方法通过对两个 STD-NMR 光谱进行数字编辑，生成串联差分光谱，从而实现了更高程度的共振饱和，对于涉及 DNA 寡核苷酸和 RNA 结合蛋白 CUGBP-1ab（25.2 kDa）的松散结合复合物，灵敏度提高了三倍。获得了该复合物 dCTGTCT-CUGBP1ab 的有趣结合特性。该方法应用于小分子配体和牛血清白蛋白（BSA，66.3 kDa）的混合物中，并在 BSA 浓度低至 0.1 µM 的情况下检测到分子间接触，这是在生物学相关条件下检测低溶解度蛋白质的有用工作浓度。
