Article: article from journal or magazin.
The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase.
Nature Chemical Biology
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.
Adenosine Triphosphatases/metabolism, Adenosine Triphosphate/metabolism, Energy Metabolism/physiology, Escherichia coli/metabolism, Fluorescent Dyes, Freezing, Genes, Reporter, HSP70 Heat-Shock Proteins/metabolism, HSP70 Heat-Shock Proteins/physiology, Kinetics, Luciferases/metabolism, Molecular Chaperones/metabolism, Molecular Chaperones/physiology, Polynucleotide 5'-Hydroxyl-Kinase/metabolism, Protein Folding, Substrate Specificity, Thiazoles, Urea/chemistry
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