Automated structure determination of proteins with the SAIL-FLYA NMR method

被引:36
作者
Takeda, Mitsuhiro [1 ,2 ]
Ikeya, Teppei [1 ,2 ]
Guentert, Peter [2 ,3 ,4 ]
Kainosho, Masatsune [1 ,2 ]
机构
[1] Nagoya Univ, Ctr Struct Biol, Grad Sch Sci, Chikusa Ku, Nagoya, Aichi 4648602, Japan
[2] Tokyo Metropolitan Univ, Dept Chem, Grad Sch Sci, Hachioji, Tokyo 1920397, Japan
[3] Goethe Univ Frankfurt, Inst Biophys Chem, D-60438 Frankfurt, Germany
[4] Goethe Univ Frankfurt, Ctr Biol Magnet Resonance, Dept Biochem Chem & Pharm, D-60438 Frankfurt, Germany
基金
日本科学技术振兴机构; 日本学术振兴会;
关键词
D O I
10.1038/nprot.2007.423
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The labeling of proteins with stable isotopes enhances the NMR method for the determination of 3D protein structures in solution. Stereo-array isotope labeling ( SAIL) provides an optimal stereospecific and regiospecific pattern of stable isotopes that yields sharpened lines, spectral simplification without loss of information, and the ability to collect rapidly and evaluate fully automatically the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as those that can be analyzed using conventional methods. Here, we describe a protocol for the preparation of SAIL proteins by cell-free methods, including the preparation of S30 extract and their automated structure analysis using the FLYA algorithm and the program CYANA. Once efficient cell-free expression of the unlabeled or uniformly labeled target protein has been achieved, the NMR sample preparation of a SAIL protein can be accomplished in 3 d. A fully automated FLYA structure calculation can be completed in 1 d on a powerful computer system.
引用
收藏
页码:2896 / 2902
页数:7
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