Extensively sparse 13C labeling to simplify solid-state NMR 13C spectra of membrane proteins
Extensively sparse 13C labeling to simplify solid-state NMR 13C spectra of membrane proteins
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广泛稀疏的 13C 标记可简化膜蛋白的固态 NMR 13C 谱
DOI：
10.1007/s10858-021-00372-y
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发表时间：
2021-06-20
影响因子：
2.7
通讯作者：
Yang，Jun
中科院分区：
文献类型：
--
作者：
Tong，Qiong;Tan，Huan;Yang，Jun
固态核磁共振（ssNMR）是一种新兴的研究人工或天然膜环境中膜蛋白结构和动力学的技术。然而，由于2D13C-13C光谱中信号的严重重叠，ssNMR对蛋白质结构的研究通常会受到信号赋值的延长或阻碍，特别是距离约束采集信号的赋值。13c自旋的稀疏标记是简化13c谱和方便提取距离约束的有效方法。在这里，我们提出了一种新的6种氨基酸残基（Ile, Leu, Phe, Trp， Tyr和Lys）的反向标记组合，并在模型膜蛋白上显示了干净的反向标记效果。大肠水通道蛋白Z （AqpZ）。我们进一步将这种反向标记组合与交替标记13c - 12c结合起来，并在13c - 13c光谱中证明了增强的稀释效应。此外，定量分析了两种策略（1、反标记和2、反标记结合交替13c - 12c标记）中反标记对其他类型残基标记的影响。由于不必要的反向标记，在2D13C-13C光谱中观察到其他类型残基的信号强度弱20-50%。本研究中提出的广泛稀疏13c标记有望用于利用膜蛋白的2D13C-13C光谱收集距离限制。
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 2D13C–13C spectra. Sparse labeling of13C spins is an effective approach to simplify the13C 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 proteinE. coliaquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate13C–12C labeling, and demonstrate an enhanced dilution effect in13C–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 alternate13C–12C labeling). The signal intensities of some other types of residues in 2D13C–13C spectra are observed to be 20–50% weaker because of the unwanted reverse labeling. The extensively sparse13C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D13C–13C spectra of membrane proteins.