Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Nat Chem. 2012 Mar 18;4(5):410-7. doi: 10.1038/nchem.1299.
Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state NMR spectroscopy. However, the paucity of distance restraints >5 Å, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here, we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogues of the protein of interest containing covalently attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu(2+) mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone (15)N PREs corresponding to distances of ~10-20 Å were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å.
对于常规结构技术具有挑战性的生物大分子，可以通过固态 NMR 光谱学以原子分辨率进行研究。然而，传统上从蛋白质核之间的磁偶极子-偶极子耦合测量中得出的距离约束值<5 Å 的数量很少，这是阻碍此类结构阐明工作的主要瓶颈。在这里，我们描述了一种通用方法，通过测量含有共价连接的顺磁标记的感兴趣蛋白质的几个类似物中的核顺磁弛豫增强（PRE），可以在不使用传统核间距离约束的情况下，快速确定固相中的全局蛋白质折叠。该方法使用 56 残基蛋白 G 的 B1 免疫球蛋白结合域的六个半胱氨酸-EDTA-Cu(2+) 突变体进行了验证，为此获得了对应于约 10-20 Å 的距离的~230 个纵向骨架（15）N PRE。以这种方式确定的平均蛋白质折叠与 X 射线结构一致，骨架原子均方根偏差为 1.8 Å。
