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Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery

Nature Communications volume 5, Article number: 4192 (2014) | Download Citation

Abstract

In living organisms, most proteins work in complexes to form multicomponent protein machines. The function of such multicomponent machines is usually addressed by dividing them into a collection of two state systems at equilibrium. Many molecular machines, like Hsp90, work far from equilibrium by utilizing the energy of ATP hydrolysis. In these cases, important information is gained from the observation of the succession of more than two states in a row. We developed a four-colour single-molecule FRET system to observe the succession of states in the heat shock protein 90 (Hsp90) system, consisting of an Hsp90 dimer, the cochaperone p23 and nucleotides. We show that this multicomponent system is a directional ATP-dependent machinery. This reveals a previously undescribed mechanism on how cochaperones can modify Hsp90, namely by strengthening of the coupling between ATP hydrolysis and a kinetic step involved in the Hsp90 system resulting in a stronger directionality.

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Acknowledgements

This work was supported by the German Science Foundation (Hu997/9-2, SFB 863) and the Nanosystems Initiative Munich. We thank Markus Götz for help with the data analysis; Edward Lemke and Peter Schultz for help with unnatural amino acid incorporation; Johannes Buchner, Carlos Bustamante, Jeff Gore, Martin Hessling, Markus Jahn, Alexandra Rehn, Matthias Rief and Sonja Schmid for helpful discussions.

Author information

Author notes

    • C. Ratzke

    Present address: Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

Affiliations

  1. Physik-Department E22 and IMETUM, Technische Universität München, München 85748, Germany

    • C. Ratzke
    • , B. Hellenkamp
    •  & T. Hugel

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Contributions

C.R., B.H. and T.H. designed experiments; C.R. and B.H. carried out experiments; all authors analysed data; B.H. and T.H. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to T. Hugel.

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DOI

https://doi.org/10.1038/ncomms5192

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