Novartis Institutes for Biomedical Research, Novartis Pharma AG, 4002 Basel, Switzerland.
J Am Chem Soc. 2011 Jan 19;133(2):210-3. doi: 10.1021/ja108383x. Epub 2010 Dec 16.
The detection and structural characterization of protein-ligand interactions by solution NMR is central to functional biology research as well as to drug discovery. Here we present a robust and highly automated procedure for obtaining the resonance assignments necessary for studies of such interactions. The procedure relies on a combination of three automated projection spectroscopy (APSY) experiments, including the new 4D APSY-HNCACB, and the use of fractionally deuterated protein samples. This labeling pattern increases the experimental sensitivity on the one hand, but it leads to peak multiplets on the other hand. The latter complications are however overcome by the geometric APSY analysis of the projection spectra. The three APSY experiments thus provide high precision chemical shift correlations of the backbone and side chain methyl groups, allowing a reliable and robust assignment of the protein by suitable algorithms. The present approach doubles the molecular size limit of APSY-based assignments to 25 kDa, thus providing the basis for efficient characterization of protein-ligand interactions at atomic resolution by NMR, such as structure-based drug design. We show the application to two human proteins with molecular weights of 15 and 22 kDa, respectively, at concentrations of 0.4 mM and discuss the general applicability to studies of protein-protein and protein-nucleic acid complexes.
通过溶液 NMR 检测和结构表征蛋白质-配体相互作用是功能生物学研究以及药物发现的核心。这里我们提出了一种稳健且高度自动化的方法，用于获得研究此类相互作用所需的共振分配。该程序依赖于三种自动化投影光谱（APSY）实验的组合，包括新的 4D APSY-HNCACB，以及使用部分氘代蛋白质样品。这种标记模式一方面增加了实验灵敏度，但另一方面会导致峰多重峰。然而，投影光谱的几何 APSY 分析克服了后一种复杂性。因此，这三个 APSY 实验提供了骨架和侧链甲基的高精度化学位移相关性，允许通过合适的算法对蛋白质进行可靠和稳健的分配。目前的方法将基于 APSY 的分配的分子大小限制提高了一倍，达到 25 kDa，从而为通过 NMR 以原子分辨率高效表征蛋白质-配体相互作用提供了基础，例如基于结构的药物设计。我们展示了该方法在浓度为 0.4 mM 时分别对两个分子量为 15 和 22 kDa 的人蛋白的应用，并讨论了其在研究蛋白质-蛋白质和蛋白质-核酸复合物中的一般适用性。
