Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent Vmax′ and Km′ values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.
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We thank A.S. Spirin (Institute of Protein Research, Russian Academy of Sciences) for the luciferase plasmid, H.J. Schönfeld (F. Hoffmann-La Roche) for DnaK, DnaJ and GrpE, A. Azem (Tel Aviv University) for discussions and AUC analyses, M. Muriset and R.U.H. Mattoo for discussions and technical assistance, G. Lorimer and J. Buchner for suggesting, respectively, the experiments in Figures 5b and 2b, and A. Finka and S. Priya for discussions and manuscript correction. This research was financed by grant 3100A0-109290 from the Swiss National Science Foundation and in part by the Zwahlen Grant from the Faculty of Biology and Medicine, University of Lausanne.
The authors declare no competing financial interests.
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Sharma, S., De Los Rios, P., Christen, P. et al. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat Chem Biol 6, 914–920 (2010). https://doi.org/10.1038/nchembio.455
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