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Optimal isotope labelling for NMR protein structure determinations


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. Here we present stereo-array isotope labelling (SAIL), a technique that 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. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that 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.

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Figure 1: SAIL amino acids.
Figure 2: 1 H– 13 C CT-HSQC spectra of CaM.
Figure 3: 1 H– 13 C CT-HSQC spectra of MBP.
Figure 4: CaM and MBP solution and crystal structures.


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We thank M. Ikura and T. Yamazaki for providing the calmodulin and maltodextrin-binding protein genes, respectively, and M. Takeda for help with the preparation of figures. This work was supported by CREST/JST.

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Corresponding author

Correspondence to Masatsune Kainosho.

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Competing interests

Atomic coordinates of the SAIL-CaM and SAIL-MBP structures have been deposited in the Protein Data Bank with accession codes 1X02 and 2D21, respectively. Chemical shifts have been deposited in the BioMagResBank with accession numbers 6541 and 6807. Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Figure 1, which shows the isotope labelling patterns of the 20 SAIL amino acids. This file also contains Supplementary Table 1, which affords NMR structure statistics for SAIL–CaM and SAIL–MBP. (PDF 72 kb)

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Kainosho, M., Torizawa, T., Iwashita, Y. et al. Optimal isotope labelling for NMR protein structure determinations. Nature 440, 52–57 (2006).

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