Skip to main content

Thank you for visiting 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.

NMR analysis of a 900K GroEL–GroES complex


Biomacromolecular structures with a relative molecular mass (Mr) of 50,000 to 100,000 (50K–100K) have been generally considered to be inaccessible to analysis by solution NMR spectroscopy. Here we report spectra recorded from bacterial chaperonin complexes ten times this size limit (up to Mr 900K) using the techniques of transverse relaxation-optimized spectroscopy and cross-correlated relaxation-enhanced polarization transfer1,2,3,4,5. These techniques prevent deterioration of the NMR spectra by the rapid transverse relaxation of the magnetization to which large, slowly tumbling molecules are otherwise subject. We tested the resolving power of these techniques by examining the isotope-labelled homoheptameric co-chaperonin GroES (Mr 72K), either free in solution or in complex with the homotetradecameric chaperonin GroEL (Mr 800K) or with the single-ring GroEL variant SR1 (Mr 400K). Most amino acids of GroES show the same resonances whether free in solution or in complex with chaperonin; however, residues 17–32 show large chemical shift changes on binding. These amino acids belong to a mobile loop region of GroES that forms contacts with GroEL6,7,8,9,10. This establishes the utility of these techniques for solution NMR studies that should permit the exploration of structure, dynamics and interactions in large macromolecular complexes.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


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

Figure 1: Two-dimensional [15N,1H]-correlation spectra at 25 °C of the uniformly 15N,2H-labelled co-chaperonin GroES free in solution and in a complex with the unlabelled GroEL variant SR1.
Figure 2: Chemical shift changes in [U-15N; > 97% 2H]GroES on binding to natural isotope abundance SR1 or GroEL.
Figure 3: Multiplet patterns in [15N,1H]-CRIPT-TROSY spectra of structures with an Mr of 72K and 472K.


  1. 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 

  2. Riek, R., Wider, G., Pervushin, K. & Wüthrich, K. Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules. Proc. Natl Acad. Sci. USA 96, 4918–4923 (1999)

    Article  ADS  CAS  Google Scholar 

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

    Book  Google Scholar 

  4. Wider, G. & Wüthrich, K. NMR spectroscopy of large molecules and multi-molecular assemblies in solution. Curr. Opin. Struct. Biol. 9, 594–601 (1999)

    Article  CAS  Google Scholar 

  5. Riek, R., Pervushin, K. & Wüthrich, K. TROSY and CRINEPT: NMR with large molecular and supramolecular structures in solution. Trends Biochem. Sci. 25, 462–468 (2000)

    Article  CAS  Google Scholar 

  6. Hunt, J. F., Weaver, A. J., Landry, S. J., Gierasch, L. & Deisenhofer, J. The crystal structure of the GroES co-chaperonin at 2.8 Å resolution. Nature 379, 37–45 (1996)

    Article  ADS  CAS  Google Scholar 

  7. Xu, Z., Horwich, A. L. & Sigler, P. B. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388, 741–750 (1997)

    Article  ADS  CAS  Google Scholar 

  8. Landry, S. J., Zeilstra-Ryalls, J., Fayet, O., Georgopoulos, C. & Gierasch, L. M. Characterization of a functionally important mobile domain of GroES. Nature 364, 255–258 (1993)

    Article  ADS  CAS  Google Scholar 

  9. Shewmaker, F., Maskos, K., Simmerling, C. & Landry, S. J. The disordered mobile loop of GroES folds into a defined β-hairpin upon binding GroEL. J. Biol. Chem. 276, 31257–31264 (2001)

    Article  CAS  Google Scholar 

  10. Landry, S. J., Taher, A., Georgopoulos, C. & van der Vies, S. M. Interplay of structure and disorder in cochaperonin mobile loops. Proc. Natl Acad. Sci. USA 93, 11622–11627 (1996)

    Article  ADS  CAS  Google Scholar 

  11. Horwich, A. L., Burston, S. G., Rye, H. S., Weissman, J. S. & Fenton, W. A. Construction of single-ring and two-ring hybrid versions of bacterial chaperonin GroEL. Methods Enzymol. 290, 141–146 (1998)

    Article  CAS  Google Scholar 

  12. Braig, K. et al. The crystal structure of the bacterial chaperonin GroEL at 2.8 Å. Nature 371, 578–586 (1994)

    Article  ADS  CAS  Google Scholar 

  13. Sigler, P. B. et al. Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67, 581–607 (1998)

    Article  CAS  Google Scholar 

  14. Roseman, A. M., Chen, S., White, H., Braig, K. & Saibil, H. R. The chaperonin ATPase cycle: Mechanism of allosteric switching and movements of substrate-binding domains in GroEL. Cell 87, 241–251 (1996)

    Article  CAS  Google Scholar 

  15. Weissman, J. S., Rye, H. S., Fenton, W. A., Beechem, J. M. & Horwich, A. L. Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction. Cell 84, 481–490 (1996)

    Article  CAS  Google Scholar 

  16. Mayhew, M. et al. Protein folding in the central cavity of the GroEL–GroES chaperonin complex. Nature 379, 420–426 (1996)

    Article  ADS  CAS  Google Scholar 

  17. Brinker, A. et al. Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223–233 (2001)

    Article  CAS  Google Scholar 

  18. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wüthrich, K. TROSY in triple resonance experiments: New perspectives for sequential assignment of large proteins. Proc. Natl Acad. Sci. USA 95, 13585–13590 (1998)

    Article  ADS  CAS  Google Scholar 

  19. Salzmann, M., Wider, G., Pervushin, K., Senn, H. & Wüthrich, K. TROSY-type triple-resonance experiments for sequential NMR assignments of large proteins. J. Am. Chem. Soc. 121, 844–848 (1999)

    Article  CAS  Google Scholar 

  20. Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wüthrich, K. NMR assignment and secondary structure determination of an octameric 110 kDa protein using TROSY in triple-resonance experiments. J. Am. Chem. Soc. 122, 7543–7548 (2000)

    Article  CAS  Google Scholar 

  21. Pellecchia, M., Sebbel, P., Hermanns, U., Wüthrich, K. & Glockshuber, R. Pilus chaperone FimC-adhesin FimH interactions mapped by TROSY-NMR. Nature Struct. Biol. 6, 336–339 (1999)

    Article  CAS  Google Scholar 

  22. Perham, R. N., Duckworth, H. W. & Roberts, G. C. K. Mobility of polypeptide chain in the pyruvate dehydrogenase complex revealed by proton NMR. Nature 292, 474–477 (1981)

    Article  ADS  CAS  Google Scholar 

  23. McEvoy, M. M., de la Cruz, A. F. & Dahlquist, F. W. Large modular proteins by NMR. Nature Struct. Biol. 4, 9 (1997)

    Article  CAS  Google Scholar 

  24. 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 

  25. Weissman, J. S. et al. Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES. Cell 83, 577–587 (1995)

    Article  CAS  Google Scholar 

  26. Morris, G. A. & Freeman, R. Enhancement of nuclear magnetic-resonance signals by polarization transfer. J. Am. Chem. Soc. 101, 760–762 (1979)

    Article  CAS  Google Scholar 

  27. Goldman, M. Interference effects in the relaxation of a pair of unlike spin-1/2 nuclei. J. Magn. Reson. 60, 437–452 (1984)

    ADS  CAS  Google Scholar 

  28. Güntert, P., Dötsch, V., Wider, G. & Wüthrich, K. Processing of multi-dimensional NMR data with the new software PROSA. J. Biomol. NMR 2, 619–629 (1992)

    Article  Google Scholar 

  29. Bartels, C., Xia, T., Billeter, M., Güntert, P. & Wüthrich, K. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biolmol. NMR 6, 1–10 (1995)

    Article  CAS  Google Scholar 

  30. DeMarco, A. & Wüthrich, K. Digital filtering with a sinusoidal window function: An alternative technique for resolution enhancement in FT NMR. J. Magn. Reson. 24, 201–204 (1976)

    ADS  CAS  Google Scholar 

Download references


This work was supported by the Schweizerischer Nationalfonds, by the Howard Hughes Medical Institute and by the NIH. We thank K. Furtak for help in constructing the plasmids used for expression of GroEL and SR1, and R. Riek for help with the NMR experiments at the outset of this project.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Arthur L. Horwich or Kurt Wüthrich.

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

Fiaux, J., Bertelsen, E., Horwich, A. et al. NMR analysis of a 900K GroEL–GroES complex. Nature 418, 207–211 (2002).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


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.


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