Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations
Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations
复制标题
DOI：
10.1023/a:1008334930603
复制
发表时间：
1999-05-01
影响因子：
2.7
通讯作者：
Jakes, K
中科院分区：
文献类型：
--
作者：
Hong, M;Jakes, K
报道了25 kDa膜蛋白结肠素Ia通道结构域中主要疏水氨基酸残基的选择性和广泛的C-13标记。新的C-13标记方法利用了细菌中氨基酸的生物合成途径，抑制了柠檬酸循环中氨基酸产物的合成。标记的选择性和广泛性显著简化了固体核磁共振谱，减少了谱线加宽，并且应该允许同时测量多个结构约束。我们根据蛋白质的特征化学位移、C-13标记模式和氨基酸组成，将大多数C-13共振指定为特定的氨基酸类型。魔角旋转下的2D同核双量子滤光片实验部分证实了这一点。这种C-13标记方法具有很高的灵敏度和光谱分辨率，被称为十个氨基酸的选择性和广泛标记。
The selective and extensive C-13 labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel C-13 labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most C-13 resonances to specific amino acid types based on the characteristic chemical shifts, the C-13 labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this C-13-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated.