Preparation of a protein sample for solid-state NMR is in many aspects similar to solution-state NMR approaches, mainly with respect to the need for stable isotope labeling. But the possibility of using solid-state NMR to investigate membrane proteins in (native) lipids adds the important requirement of adapted membrane-reconstitution schemes. Also, dynamic nuclear polarization and paramagnetic NMR in solids need specific schemes using metal ions and radicals. Sample sedimentation has enabled structural investigations of objects inaccessible to other structural techniques, but rotor filling using sedimentation has become increasingly complex with smaller and smaller rotors, as needed for higher and higher magic-angle spinning (MAS) frequencies. Furthermore, solid-state NMR can investigate very large proteins and their complexes without the concomitant increase in line widths, motivating the use of selective labeling and unlabeling strategies, as well as segmental labeling, to decongest spectra. The possibility of investigating sub-milligram amounts of protein today using advanced fast MAS techniques enables alternative protein synthesis schemes such as cell-free expression. Here we review these specific aspects of solid-state NMR sample preparation.

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用于固态 NMR 研究的蛋白质样品制备

用于固态 NMR 的蛋白质样品的制备在许多方面类似于溶液态 NMR 方法，主要是在需要稳定同位素标记方面。但是，使用固态 NMR 研究（天然）脂质中的膜蛋白的可能性增加了对适应膜重建方案的重要要求。此外，固体中的动态核极化和顺磁 NMR 需要使用金属离子和自由基的特定方案。样品沉淀使其他结构技术无法实现的物体的结构研究成为可能，但随着越来越高的魔角旋转 (MAS) 频率的需要，使用沉淀的转子填充变得越来越复杂，转子越来越小。此外，固态 NMR 可以研究非常大的蛋白质及其复合物，而不会同时增加线宽，从而推动使用选择性标记和未标记策略以及分段标记来解充谱。如今，使用先进的快速 MAS 技术研究亚毫克量的蛋白质的可能性使替代蛋白质合成方案成为可能，例如无细胞表达。在这里，我们回顾了固态 NMR 样品制备的这些特定方面。