Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination
Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination
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DOI：
10.1007/s10858-009-9360-9
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发表时间：
2010-01-01
影响因子：
2.7
通讯作者：
Kainosho, Masatsune
中科院分区：
文献类型：
--
作者：
Takeda, Mitsuhiro;Ono, Akira M.;Kainosho, Masatsune
涉及芳环信号的 NOE 约束数据的广泛收集对于准确确定蛋白质结构至关重要，尽管在实践中它经常受到普遍的信号重叠和芳环的紧密自旋耦合的阻碍。我们制备了各种类型的立体阵列同位素标记的苯丙氨酸（epsilon-和 zeta-SAIL Phe）和酪氨酸（epsilon-SAIL Tyr）来克服这些问题（Torizawa 等人，2005），并证明这些 SAIL 氨基酸为芳环 NMR 信号提供了显着的光谱简化和灵敏度增强。除了这些 SAIL 芳香族氨基酸之外，我们最近还合成了 delta-SAIL Phe 和 delta-SAIL Tyr，这使我们能够非常有效地观察和分配 delta-C-13/H-1 信号。各种类型的 SAIL Phe 和 SAIL Tyr 分别对 delta-、epsilon- 或 zeta-C-13/H-1 信号产生良好解析的共振，这些共振可以通过简单而稳健的脉冲序列轻松分配。由于 Phe/Tyr 残基的 delta-、ε-和 zeta-质子信号会产生互补的 NOE 限制，因此与使用传统的统一 C-13、N-15 双标记蛋白获得的结构相比，同时使用各种类型的 SAIL-Phe 和 SAIL-Tyr 将产生更准确的蛋白结构。我们以 18.2 kDa 蛋白质大肠杆菌肽基脯氨酰顺反异构酶 b (EPPIb) 为例说明了这一点，并得出结论，zeta-SAIL Phe 和 epsilon-SAIL Tyr 的联合使用实际上是蛋白质结构测定的最佳选择。
The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-C-13/H-1 signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-C-13/H-1 signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly C-13, N-15-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.