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The SAIL Wiki
The SAIL Wiki provides information on stereo-array isotope labeling (SAIL). It is managed by the groups of Prof. Masatsune Kainosho and Prof. Peter Güntert at Tokyo Metropolitan University, Nagoya University, and the University of Frankfurt.
Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Stereo-array isotope labelling (SAIL) can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.
Stereo-array isotope labeling
The basic strategy of the SAIL approach is to prepare amino acids with the following features:
- Stereo-selective replacement of one 1H in methylene groups by 2H.
- Replacement of two 1H in each methyl group by 2H.
- Stereo-selective modification of the prochiral methyl groups of Leu and Val such that one methyl is -12C(2H)3 and the other is -13C1H(2H)2.
- Labelling of six-membered aromatic rings by alternating 12C-2H and 13C-1H moieties.
This labeling pattern preserves through-bond connectivity information needed for backbone and side chain assignments, eliminates the need for stereospecific assignments, simplifies measurements of couplings, and removes the most serious sources of spin diffusion so as to improve the accuracy of inter-proton distance measurements. Lines are sharpened both by decreasing long-range couplings and by eliminating dipolar relaxation pathways. The methyl and methylene labeling patterns simplify the analysis of the motional properties of the side chains from relaxation measurements. The aromatic ring labeling strategy removes one-bond 13C-13C couplings, which often complicate spectra or require the use of constant time data collection methods so as to reduce spectral complexity.
The 20 protein-component SAIL amino acids are prepared based on these design concepts by chemical and enzymatic syntheses.
The SAIL approach reduces the number of non-exchangeable side-chain protons, which are prone to overlap but essential for defining the side-chain conformations, to less than half, and decreases the number of expected NOESY cross-peaks in proteins by 40–45%. Most of the additional NOEs from uniformly labeled proteins either involve fixed (geminal) distances or become redundant in the absence of stereospecific assignments and thus contribute to spectral overlap without furnishing independent information.
In practice SAIL is expected to increase rather than decrease the number of identifiable NOE cross-peaks. The expected increase is moderate in regions without overlap but is significant in regions with strong overlap and therefore for larger proteins, for which SAIL is expected to yield two or more times the number of relevant conformational restraints than uniform labeling.
