Research Center for State-of-the-Art Functional Protein Analysis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan; Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
Biochim Biophys Acta Gen Subj. 2020 Feb;1864(2):129439. doi: 10.1016/j.bbagen.2019.129439. Epub 2019 Oct 5.
The structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals.
The 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra.
The MSG proteins, selectively labeled with either [δ,ε,ε,η]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously.
We demonstrated that the aromatic ring CH pairs without directly bonded C and adjacent H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available.
The method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.
如果迄今为止尚未探索到的芳环 NMR 信号除了当前使用的骨架酰胺和侧链甲基信号之外能够被利用，那么大蛋白质复合物的原子水平的结构-功能关系将得到全面理解。
选择标记有 Trp 和 Phe 的 82 kDa 苹果酸合酶 G（MSG）蛋白，以研究获得芳族 TROSY 光谱的最佳条件。
用[δ，ε，ε，η]-SAIL Trp 或 ζ-SAIL Phe 选择性标记的 MSG 蛋白为 MSG 中的 12 个 Trp 和 19 个 Phe 残基提供了分离良好、狭窄的 TROSY 信号。使用一组单氨基酸取代突变体，这些信号被序列特异性地分配。每个 Phe 被 Tyr 或 Leu 的定点取代会引起其他芳环信号的显著化学位移，使我们能够识别 MSG 中的芳族簇，这与先前提出的结构域相当。
我们证明，如果环被完全氘化的氨基酸包围，则没有与直接键合的 C 和相邻 H 自旋的芳环 CH 对提供令人惊讶的狭窄 TROSY 信号。如果有甲基分配，则可以通过单氨基酸取代或芳族和甲基质子之间的 NOE 轻松分配观察到的信号。
这里描述的方法应该普遍适用于困难的靶标，例如双层脂膜中的蛋白质或可能在活细胞中的蛋白质，从而为在结构生物学中使用这些新探针提供了前所未有的机会。
