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NMR analysis of a 900K GroEL–GroES complex

Nature volume 418, pages 207–211 (2002)Cite this article

Abstract

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.

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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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, CH-8093, Zürich, Switzerland

    Jocelyne Fiaux & Kurt Wüthrich

  2. Howard Hughes Medical Institute and Department of Genetics, Yale School of Medicine, New Haven, Connecticut, 06510, USA

    Eric B. Bertelsen & Arthur L. Horwich

Authors
  1. Jocelyne Fiaux
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  2. Eric B. Bertelsen
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  3. Arthur L. Horwich
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  4. Kurt Wüthrich
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Correspondence to Arthur L. Horwich or Kurt Wüthrich.

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Fiaux, J., Bertelsen, E., Horwich, A. et al. NMR analysis of a 900K GroEL–GroES complex. Nature 418, 207–211 (2002). https://doi.org/10.1038/nature00860

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