Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences , Wuhan, 430071, PR China.
J Am Chem Soc. 2013 Jun 5;135(22):8294-303. doi: 10.1021/ja4021149. Epub 2013 May 24.
Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromolecules which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is shown that high-resolution structure of proteins in solid phase can be determined without the use of traditional dipolar-dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomethyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunoglobulin-binding domain of protein G (GB1). The long-range structural restraints with metal-nucleus distance of up to ∼20 Å are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances back-calculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic susceptibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 Å root-mean-square deviation relative to X-ray structure.
魔角旋转固态 NMR 是一种独特的技术，可用于研究抗结晶或太大而无法通过溶液 NMR 技术研究的生物大分子的原子分辨率结构。然而，基于偶极耦合的光谱获得足够数量的长程距离约束的困难阻碍了固态 NMR 中蛋白质结构确定的过程。在这项研究中，通过将赝接触位移（PCS）测量与 Rosetta 计算相结合，表明可以在不使用传统基于偶极-偶极耦合的距离约束的情况下确定固态蛋白质的高分辨率结构。PCS 是通过螯合外源性顺磁金属离子到标签 4-巯基甲基-二吡啶酸来产生的，该标签共价连接到蛋白质 G（GB1）的 56 残基免疫球蛋白结合域中的不同残基位点。从实验观察到的 PCS 中定量提取了长达约 20 Å 的长程结构约束，并且这些约束与使用 X 射线结构模型反向计算的距离很好地吻合。此外，我们证明，使用几种具有不同顺磁磁化率的顺磁离子以及在不同位置引入顺磁标记，可以显著增加长程约束的数量并覆盖蛋白质的不同区域。从固态 NMR PCS 约束和 Rosetta 计算生成的结构与 X 射线结构相比，均方根偏差为 0.7 Å。
