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Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry

Nature Methods volume 2, pages 371376 (2005) | Download Citation

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Abstract

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.

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Acknowledgements

We thank E. Kroezinga and J. Hendriks, who contributed to some of the work reported in this article. The research was supported by Fundamenteel Onderzoek der Materie, project number 01FB12.

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Affiliations

  1. Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

    • Esther van Duijn
    • , Ron M A Heeren
    • , Robert H H van den Heuvel
    •  & Albert J R Heck
  2. Section Biochemistry and Molecular Biology, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

    • Esther van Duijn
    • , Patrick J Bakkes
    • , Harm van Heerikhuizen
    •  & Saskia M van der Vies
  3. Fundamenteel Onderzoek der Materie–Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands.

    • Ron M A Heeren

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The authors declare no competing financial interests.

Corresponding authors

Correspondence to Saskia M van der Vies or Albert J R Heck.

Supplementary information

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  1. 1.

    Supplementary Fig. 1

    Various ratios of GroEL-gp23 complexes analyzed by gel filtration

  2. 2.

    Supplementary Methods

    Protein purification

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DOI

https://doi.org/10.1038/nmeth753

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