Clean STD‐NMR spectrum for improved detection of ligand‐protein interactions at low concentration of protein
Clean STD‐NMR spectrum for improved detection of ligand‐protein interactions at low concentration of protein
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DOI：
10.1002/mrc.2687
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发表时间：
2010-12
影响因子：
2
通讯作者：
Youlin Xia;Qi Zhu;Kyu‐Yeon Jun;Jingchun Wang;Xiaolian Gao
中科院分区：
文献类型：
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作者：
Youlin Xia;Qi Zhu;Kyu‐Yeon Jun;Jingchun Wang;Xiaolian Gao
饱和转移差（STD）-NMR已广泛用于筛选配体化合物库的蛋白质结合活性并确定配体的结合表位。我们在此报告，开发了一种清洁 STD-NMR 方法，以克服由于 RF 辐射的功率溢出而在 STD-NMR 谱中观察到的假阳性（伪影）。该方法通过对两个 STD-NMR 谱进行数字编辑以生成串联差异谱，实现了更高程度的共振饱和度，并且对于涉及 DNA 寡核苷酸和 RNA 结合蛋白 CUGBP-1ab (25.2 kDa) 的松散结合复合物，灵敏度增强了三倍。获得了复合物 dCTGTCT-CUGBP1ab 有趣的结合特征。该方法适用于小配体和牛血清白蛋白（BSA，66.3 kDa）的混合物，并在 BSA 浓度低至 0.1 µM 时检测分子间接触，该工作浓度可用于在生物学相关条件下检测低溶解度蛋白质。版权所有 © 2010 约翰威利父子有限公司
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. Copyright © 2010 John Wiley & Sons, Ltd.