Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy

Nature volume 420, pages 99–102 (2002)Cite this article

Abstract

The determination of a representative set of protein structures is a chief aim in structural genomics. Solid-state NMR may have a crucial role in structural investigations of those proteins that do not easily form crystals or are not accessible to solution NMR, such as amyloid systems1 or membrane proteins2,3,4. Here we present a protein structure determined by solid-state magic-angle-spinning (MAS) NMR. Almost complete 13C and 15N resonance assignments for a micro-crystalline preparation of the α-spectrin Src-homology 3 (SH3) domain5 formed the basis for the extraction of a set of distance restraints. These restraints were derived from proton-driven spin diffusion (PDSD) spectra of biosynthetically site-directed, labelled samples obtained from bacteria grown using [1,3-13C]glycerol or [2-13C]glycerol as carbon sources. This allowed the observation of long-range distance correlations up to ∼7 Å. The calculated global fold of the α-spectrin SH3 domain is based on 286 inter-residue 13C–13C and six 15N–15N restraints, all self-consistently obtained by solid-state MAS NMR. This MAS NMR procedure should be widely applicable to small membrane proteins that can be expressed in bacteria.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Labelling patterns and NMR spectra for the different α-spectrin SH3 domain preparations.
Figure 2: Assignment strategy.
Figure 3: Solid-state structure of the α-spectrin SH3 domain.

Similar content being viewed by others

References

  1. Tycko, R. Biomolecular solid state NMR: Advances in structural methodology and applications to peptide and protein fibrils. Annu. Rev. Phys. Chem. 52, 575–606 (2001)

    Article  ADS  CAS  Google Scholar 

  2. Opella, S. J. NMR and membrane proteins. Nature Struct. Biol. 4 suppl., 845–848 (1997)

    CAS  PubMed  Google Scholar 

  3. Griffin, R. G. Dipolar recoupling in MAS spectra of biological solids. Nature Struct. Biol. 5 NMR suppl., 508–512 (1998)

    Article  CAS  Google Scholar 

  4. De Groot, H. J. M. Solid-state NMR spectroscopy applied to membrane proteins. Curr. Opin. Struct Biol. 10, 593–600 (2000)

    Article  CAS  Google Scholar 

  5. Pauli, J., Baldus, M., van Rossum, B., de Groot, H. & Oschkinat, H. Backbone and side-chain 13C and 15N signal assignments of the α-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 tesla. ChemBioChem 2, 272–281 (2001)

    Article  CAS  Google Scholar 

  6. Straus, S. K., Bremi, T. & Ernst, R. R. Experiments and strategies for the assignment of fully 13C/15N-labelled polypeptides by solid-state NMR. J. Biomol. NMR 12, 39–50 (1998)

    Article  CAS  Google Scholar 

  7. Hong, M. Resonance assignment of 13C/15N labelled solid proteins by two-and three-dimensional magic-angle-spinning NMR. J. Biomol. NMR. 15, 1–14 (1999)

    Article  CAS  Google Scholar 

  8. Hong, M. Determination of multiple φ-torsion angles in proteins by selective and extensive 13C labeling and two-dimensional solid-state NMR. J. Magn. Reson. 139, 389–401 (1999)

    Article  ADS  CAS  Google Scholar 

  9. Jaroniec, C. P., Tounge, B. A., Herzfeld, J. & Griffin, R. G. Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate 13C-15N distance measurements in uniformly 13C,15N-labeled peptides. J. Am. Chem. Soc. 123, 3507–3519 (2001)

    Article  CAS  Google Scholar 

  10. Brown, S. P. & Spiess, H. W. Advanced solid-state NMR methods for the elucidation of structure and dynamics of molecular, macromolecular, and supramolecular systems. Chem. Rev. 101, 4125–4156 (2001)

    Article  CAS  Google Scholar 

  11. Ketchem, R. R., Lee, K.-C., Huo, S. & Cross, T. A. Macromolecular structural elucidation with solid-state NMR-derived orientational constraints. J. Biomol. NMR 8, 1–14 (1996)

    Article  CAS  Google Scholar 

  12. Shochat, S. et al. 13C MAS NMR evidence for a homogeneously ordered environment of tyrosine M210 in reaction centres of Rhodobacter sphaeroides. Spectrochim. Acta 51A, 135–144 (1995)

    Article  ADS  CAS  Google Scholar 

  13. Verhoeven, M. A. et al. Ultra-high-field MAS NMR assay of a multispin labeled ligand bound to its G-protein receptor target in the natural membrane environment: electronic structure of the retilylidene chromophore in rhodopsin. Biochemistry 40, 3282–3288 (2001)

    Article  CAS  Google Scholar 

  14. Egorova-Zachernyuk, T. A. et al. Characterization of pheophytin ground states in Rhodobacter sphaeroides R26 photosynthetic reaction centers from multispin pheophytin enrichment and 2-D 13C MAS NMR dipolar correlation spectroscopy. Biochemistry 36, 7513–7519 (1997)

    Article  CAS  Google Scholar 

  15. Griffiths, J. M. et al. Dipolar correlation NMR spectroscopy of a membrane protein. J. Am. Chem. Soc. 116, 10178–10181 (1994)

    Article  CAS  Google Scholar 

  16. Hodgkinson, P. & Emsley, L. The accuracy of distance measurements in solid-state NMR. J. Magn. Reson. 139, 46–59 (1999)

    Article  ADS  CAS  Google Scholar 

  17. Kiihne, S. et al. Distance measurements by dipolar recoupling two-dimensional solid-state NMR. J. Phys. Chem. A 102, 2274–2282 (1998)

    Article  CAS  Google Scholar 

  18. Nielsen, N. C., Bildsoe, H., Jakobsen, H. J. & Levitt, M. H. Double-quantum homonuclear rotary resonance: efficient dipolar recovery in magic-angle spinning nuclear magnetic resonance. J. Chem. Phys. 101, 1805–1812 (1994)

    Article  ADS  CAS  Google Scholar 

  19. Costa, P. R., Sun, B. Q. & Griffin, R. G. Rotational resonance tickling: accurate internuclear distance measurement in solids. J. Am. Chem. Soc. 119, 10821–10830 (1997)

    Article  CAS  Google Scholar 

  20. Pauli, J., van Rossum, B., Förster, H., De Groot, H. J. M. & Oschkinat, H. Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the α-spectrin SH3 domain. J. Magn. Reson. 143, 411–416 (2000)

    Article  ADS  CAS  Google Scholar 

  21. LeMaster, D. M. & Kushlan, D. M. Dynamical mapping of E. coli thioredoxin via 13C NMR relaxation analysis. J. Am. Chem. Soc. 118, 9255–9264 (1996)

    Article  Google Scholar 

  22. Szeverenyi, N. M., Sullivan, M. J. & Maciel, G. E. 13C Spin exchange by 2D FT 13C CP/MAS. J. Magn. Reson. 47, 462–475 (1982)

    ADS  CAS  Google Scholar 

  23. Wüthrich, K. NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)

    Book  Google Scholar 

  24. van Rossum, B. J., Castellani, F., Rehbein, K., Pauli, J. & Oschkinat, H. Assignment of the non exchanging protons of the α-spectrin SH3 domain by two- and three-dimensional 1H-13C solid-state magic-angle-spinning NMR and comparison of solution and solid-state proton chemical shifts. ChemBioChem 2, 906–914 (2001)

    Article  CAS  Google Scholar 

  25. Brünger, A. T. et al. Crystallography and NMR system (CNS): a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  26. Musacchio, A., Noble, M., Pauptit, R., Wierenga, R. & Saraste, M. Crystal structure of a Src-homology 3 (SH3) domain. Nature 359, 851–855 (1992)

    Article  ADS  CAS  Google Scholar 

  27. Reif, B., Jaroniec, C. P., Rienstra, C. M., Hohwy, M. & Griffin, R. G. 1H-1H MAS correlation spectroscopy and distance measurements in a deuterated peptide. J. Magn. Reson. 151, 320–327 (2001)

    Article  ADS  CAS  Google Scholar 

  28. Bennett, A. E., Rienstra, C. M., Auger, M., Lakshmi, K. V. & Griffin, R. G. Heteronuclear decoupling in rotating solids. J. Chem. Phys. 103, 6951–6958 (1995)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

H. de Groot is acknowledged for access to the high-field NMR facility in Leiden. The authors thank R. Kühne, P. Schmieder, G. Krause and C. Glaubitz for discussions, and L. Ball, K. Heuer and K. Zierler for carefully reading the manuscript.

Author information

Authors and Affiliations

  1. Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125, Berlin

    Federica Castellani, Barth van Rossum, Annette Diehl, Mario Schubert, Kristina Rehbein & Hartmut Oschkinat

  2. Freie Universität Berlin, Takustrasse 3, 14195, Berlin, Germany

    Federica Castellani, Barth van Rossum, Annette Diehl, Mario Schubert, Kristina Rehbein & Hartmut Oschkinat

Authors
  1. Federica Castellani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barth van Rossum
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Annette Diehl
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mario Schubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kristina Rehbein
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hartmut Oschkinat
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hartmut Oschkinat.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Castellani, F., van Rossum, B., Diehl, A. et al. Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420, 99–102 (2002). https://doi.org/10.1038/nature01070

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature01070

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing