Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D ¹³C–¹³C spectra. Sparse labeling of ¹³C spins is an effective approach to simplify the ¹³C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein E. coli aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate ¹³C–¹²C labeling, and demonstrate an enhanced dilution effect in ¹³C–¹³C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate ¹³C–¹²C labeling). The signal intensities of some other types of residues in 2D ¹³C–¹³C spectra are observed to be 20–50% weaker because of the unwanted reverse labeling. The extensively sparse ¹³C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D ¹³C–¹³C spectra of membrane proteins.

固态核磁共振 (ssNMR) 是一种新兴技术，用于研究人工或天然膜环境中膜蛋白的结构和动力学。然而，由于 2D ¹³ C– ¹³ C 光谱中的信号严重重叠，ssNMR 对蛋白质的结构研究通常会受到信号分配的延长或阻碍，特别是用于收集距离限制的信号分配。¹³ C 自旋的稀疏标记是简化¹³C 光谱并促进距离限制的提取。在这里，我们提出了一种新的六种氨基酸残基（Ile、Leu、Phe、Trp、Tyr 和 Lys）的反向标记组合，并对模型膜蛋白大肠杆菌水通道蛋白 Z (AqpZ)显示了干净的反向标记效果. 我们进一步结合了这种反向标记组合和交替的¹³ C– ¹² C 标记，并在¹³ C– ¹³ C 光谱中证明了增强的稀释效果。此外，在两种策略中定量分析了反向标记对其他类型残基标记的影响（1，反向标记和 2，反向标记结合交替¹³ C– ¹²C 标记）。由于不需要的反向标记，观察到二维¹³ C– ¹³ C 光谱中一些其他类型的残基的信号强度要弱 20–50%。本研究中提出的广泛稀疏的¹³ C 标记有望用于使用膜蛋白的2D ¹³ C– ¹³ C 光谱收集距离限制。