For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional ¹³C/¹⁵N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton (¹H) detection schemes. We estimate that the entire preparative procedure until NMR experiments can be started takes 3–5 days.

长期以来，固态核磁共振 (ssNMR) 一直被用于在详细的化学、结构或动力学层面研究复杂的生物分子系统。高分辨率和高灵敏度 ssNMR 的最新进展，结合创新的样品制备和标记方案，为研究天然环境中的蛋白质提供了新的机会，而不管分子翻滚率如何。该协议描述了生化制备方案，以获取细菌中可溶性和不溶性或膜相关蛋白的细胞样本。为此，该协议适用于研究整个细胞和细胞包膜或分离膜制剂中感兴趣的蛋白质。在程序的第一阶段，适当的大肠杆菌菌株(DE3) 在诱导型 T7 启动子的控制下，用含有感兴趣蛋白质的感兴趣质粒进行转化。接下来，细胞适应于在最小 (M9) 培养基中生长。在生长进入稳定期之前，诱导蛋白质表达，此后不久，使用利福平抑制天然大肠杆菌RNA 聚合酶，以靶向标记感兴趣的蛋白质。表达后收获细胞并准备​​用于 ssNMR 转子填充。除了传统的¹³ C/ ¹⁵ N 检测 ssNMR 之外，我们还概述了如何使用动态核极化 (DNP) 或质子 ( ¹h) 检测方案。我们估计，直到 NMR 实验可以开始的整个准备过程需要 3-5 天。