Automated structure determination of proteins with the SAIL-FLYA NMR method.
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.
Takeda
Mitsuhiro
M
Graduate School of Science, Center for Structural Biology, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
Ikeya
Teppei
T
Güntert
Peter
P
Kainosho
Masatsune
M
D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D012984:Software
D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins