The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase

Sandeep K. Sharma, Paolo De Los Rios, Philipp Christen, Ariel Lustig, Pierre Goloubinoff

Research output: Contribution to journalArticlepeer-review

181 Scopus citations

Abstract

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 V( max)′ and K(m)′ 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.

Original languageEnglish
Pages (from-to)914-920
Number of pages7
JournalNature Chemical Biology
Volume6
Issue number12
DOIs
StatePublished - Dec 2010
Externally publishedYes

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