Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
J Mol Biol. 2010 Jun 4;399(2):268-82. doi: 10.1016/j.jmb.2010.04.012. Epub 2010 Apr 13.
We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.
我们提出了通过魔角旋转固态 NMR 对大蛋白的化学位移进行分配的策略，以 21kDa 的二硫键形成酶 DsbA 作为原型。先前的研究表明，对于大约 17 kDa 的蛋白质，完全从头分配是可能的，并且已经对几个较大的蛋白质进行了部分分配。在这里，我们表明，同位素标记策略、高场相关光谱学以及三维（3D）和四维（4D）骨架相关实验的组合可产生超过 90%的 DsbA 骨架共振的高度置信分配。通过透析程序制备纳米晶沉淀物作为样品，导致不均匀线宽低于 0.2 ppm。因此，高磁场、选择性解耦脉冲序列和稀疏的同位素标记都提高了光谱分辨率。通过氨基酸类型的分配通过脉冲序列和同位素标记的特定组合得到促进；例如，转移回波双共振实验增强了 Pro 和 Gly 残基的灵敏度；[2-(13)C]甘油标记澄清了 Val、Ile 和 Leu 的分配；同相反相相关谱使解释 otherwise crowded Glx/Asx 侧链区域成为可能；并且 [2-(13)C]甘油样品上的 3D NCACX 实验提供了独特的芳香族（Phe、Tyr 和 Trp）相关物。与高灵敏度 CANCOCA 4D 实验和 CANCOCX 3D 实验一起，可以在序列的大部分区域进行明确的骨架遍历。在 189 个残基中，DsbA 是迄今为止基本上实现了完整固态 NMR 分配的最大单体单元。这些结果将促进对纳米晶 DsbA 结构和动力学的研究，并能够分析其与膜蛋白 DsbB 的 41 kDa 共价复合物，我们为此证明了高分辨率二维（13)C-（13)C 谱。
