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Optimal isotope labelling for NMR protein structure determinations

Nature volume 440, pages 52–57 (2006)Cite this article

Abstract

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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References

  1. Kennedy, M. A., Montelione, G. T., Arrowsmith, C. H. & Markley, J. L. Role for NMR in structural genomics. J. Struct. Funct. Genom. 2, 155–169 (2002)

    Article  CAS  Google Scholar 

  2. Clore, G. M. & Gronenborn, A. M. Structures of larger proteins, protein–ligand and protein–DNA complexes by multidimensional heteronuclear NMR. Prog. Biophys. Mol. Biol. 62, 153–184 (1994)

    Article  CAS  Google Scholar 

  3. Clore, G. M. & Gronenborn, A. M. NMR structure determination of proteins and protein complexes larger than 20 kDa. Curr. Opin. Chem. Biol. 2, 564–570 (1998)

    Article  CAS  Google Scholar 

  4. Gardner, K. H. & Kay, L. E. The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu. Rev. Biophys. Biomol. Struct. 27, 357–406 (1998)

    Article  CAS  Google Scholar 

  5. Goto, N. K. & Kay, L. E. New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr. Opin. Struct. Biol. 10, 585–592 (2000)

    Article  CAS  Google Scholar 

  6. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl Acad. Sci. USA 94, 12366–12371 (1997)

    Article  ADS  CAS  Google Scholar 

  7. Mueller, G. A. et al. Global folds of proteins with low densities of NOEs using residual dipolar couplings: Application to the 370-residue maltodextrin-binding protein. J. Mol. Biol. 300, 197–212 (2000)

    Article  CAS  Google Scholar 

  8. Markley, J. L., Putter, I. & Jardetzky, O. High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease. Science 161, 1249–1251 (1968)

    Article  ADS  CAS  Google Scholar 

  9. LeMaster, D. M. Chiral β and random fractional deuteration for the determination of protein side-chain conformation by NMR. FEBS Lett. 223, 191–196 (1987)

    Article  CAS  Google Scholar 

  10. Farmer, B. T. II & Venters, R. A. NMR of perdeuterated large proteins. Mod. Techniques Protein NMR 16, 75–120 (1999)

    Article  Google Scholar 

  11. Arata, Y., Kato, K., Takahashi, H. & Shimada, I. Nuclear-magnetic-resonance study of antibodies—a multinuclear approach. Methods Enzymol. 239, 440–464 (1994)

    Article  CAS  Google Scholar 

  12. Yamazaki, T. et al. Segmental isotope labeling for protein NMR using peptide splicing. J. Am. Chem. Soc. 120, 5591–5592 (1998)

    Article  CAS  Google Scholar 

  13. Oba, M., Kobayashi, M., Oikawa, F., Nishiyama, K. & Kainosho, M. Synthesis of 13C/D doubly labelled l-leucines: Probes for conformational analysis of the leucine side-chain. J. Org. Chem. 66, 5919–5922 (2001)

    Article  CAS  Google Scholar 

  14. Oba, M., Terauchi, T., Miyakawa, A., Kamo, H. & Nishiyama, K. Stereoselective deuterium-labelling of diastereotopic methyl and methylene protons of l-leucine. Tetrahedron Lett. 39, 1595–1598 (1998)

    Article  CAS  Google Scholar 

  15. Oba, M., Terauchi, T., Miyakawa, A. & Nishiyama, K. Asymmetric synthesis of l-proline regio- and stereoselectively labelled with deuterium. Tetrahedron Asymmetry 10, 937–945 (1999)

    Article  CAS  Google Scholar 

  16. Torizawa, T., Shimizu, M., Taoka, M., Miyano, H. & Kainosho, M. Efficient production of isotopically labelled proteins by cell-free synthesis: A practical protocol. J. Biomol. NMR 30, 311–325 (2004)

    Article  CAS  Google Scholar 

  17. Kigawa, T., Muto, Y. & Yokoyama, S. Cell-free synthesis and amino acid-selective stable-isotope labeling of proteins for NMR analysis. J. Biomol. NMR 6, 129–134 (1995)

    Article  CAS  Google Scholar 

  18. Zubay, G. In-vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7, 267–287 (1973)

    Article  CAS  Google Scholar 

  19. McIntosh, L. P. & Dahlquist, F. W. Biosynthetic incorporation of 15N and 13C for assignment and interpretation of nuclear-magnetic-resonance spectra of proteins. Q. Rev. Biophys. 23, 1–38 (1990)

    Article  CAS  Google Scholar 

  20. Kay, L. E., Nicholson, L. K., Delaglio, F., Bax, A. & Torchia, D. A. Pulse sequences for removal of the effects of cross-correlation between dipolar and chemical-shift anisotropy relaxation mechanism on the measurement of heteronuclear T1 and T2 values in proteins. J. Magn. Reson. 97, 359–375 (1992)

    ADS  CAS  Google Scholar 

  21. Vuister, G. W. & Bax, A. Resolution enhancement and spectral editing of uniformly 13C-enriched proteins by homonuclear broadband 13C decoupling. J. Magn. Reson. 98, 428–435 (1992)

    ADS  CAS  Google Scholar 

  22. Güntert, P., Braun, W., Billeter, M. & Wüthrich, K. Automated stereospecific 1H NMR assignments and their impact on the precision of protein structure determinations in solution. J. Am. Chem. Soc. 111, 3997–4004 (1989)

    Article  Google Scholar 

  23. Nilges, M., Clore, G. M. & Gronenborn, A. M. 1H-NMR stereospecific assignments by conformational data-base searches. Biopolymers 29, 813–822 (1990)

    Article  CAS  Google Scholar 

  24. Neri, D., Szyperski, T., Otting, G., Senn, H. & Wüthrich, K. Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28, 7510–7516 (1989)

    Article  CAS  Google Scholar 

  25. Cavanagh, J., Palmer, A. G., Fairbrother, W. & Skelton, N. Protein NMR Spectroscopy: Principles and Practice (Academic, San Diego, 1996)

    Google Scholar 

  26. Torizawa, T., Ono, M. A., Terauchi, T. & Kainosho, M. NMR assignment methods for the aromatic ring resonances of phenylalanine and tyrosine residues in proteins. J. Am. Chem. Soc. 127, 12620–12626 (2005)

    Article  CAS  Google Scholar 

  27. Sharff, A. J., Rodseth, L. E. & Quiocho, F. A. Refined 1.8-Å structure reveals the mode of binding of β-cyclodextrin to the maltodextrin binding protein. Biochemistry 8, 10553–10559 (1993)

    Article  Google Scholar 

  28. Marion, D., Kay, L. E., Sparks, S. W., Torchia, D. A. & Bax, A. 3-dimensional heteronuclear NMR of 15N-labelled proteins. J. Am. Chem. Soc. 111, 1515–1517 (1989)

    Article  CAS  Google Scholar 

  29. Güntert, P. Automated NMR protein structure calculation. Prog. NMR Spectrosc. 43, 105–125 (2003)

    Article  Google Scholar 

  30. Herrmann, T., Güntert, P. & Wüthrich, K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319, 209–227 (2002)

    Article  CAS  Google Scholar 

  31. Chattopadhyaya, R., Meador, W. E., Means, A. R. & Quiocho, F. A. Calmodulin structure refined at 1.7 Å resolution. J. Mol. Biol. 228, 1177–1192 (1992)

    Article  CAS  Google Scholar 

  32. Chou, J. J., Li, S., Klee, C. B. & Bax, A. Solution structure of Ca2+-calmodulin reveals flexible hand-like properties of its domains. Nature Struct. Biol. 8, 990–997 (2001)

    Article  CAS  Google Scholar 

  33. Güntert, P., Mumenthaler, C. & Wüthrich, K. Torsion angle dynamics for NMR structure calculation with the new program Dyana. J. Mol. Biol. 273, 283–298 (1997)

    Article  Google Scholar 

  34. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999)

    Article  CAS  Google Scholar 

  35. Cornell, W. D. et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179–5197 (1995)

    Article  CAS  Google Scholar 

  36. Koradi, R., Billeter, M. & Güntert, P. Point-centered domain decomposition for parallel molecular dynamics simulation. Comput. Phys. Commun. 124, 139–147 (2000)

    Article  ADS  CAS  Google Scholar 

  37. Ikura, M. et al. Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy. Biochemistry 30, 9216–9228 (1991)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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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Authors and Affiliations

  1. CREST/JST and Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-ohsawa, 192-0397, Hachioji, Japan

    Masatsune Kainosho, Takuya Torizawa, Yuki Iwashita, Tsutomu Terauchi & Akira Mei Ono

  2. Tatsuo Miyazawa Memorial Program, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, 230-0045, Yokohama, Tsurumi, Japan

    Peter Güntert

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  1. Masatsune Kainosho
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  2. Takuya Torizawa
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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 npg.nature.com/reprintsandpermissions. 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). https://doi.org/10.1038/nature04525

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