The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase.
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.
Département de Biologie Moléculaire Végétale, Université de Lausanne, Lausanne, Switzerland.
Sandeep K
SK
Sharma
Paolo
P
De los Rios
Philipp
P
Christen
Ariel
A
Lustig
Pierre
P
Goloubinoff
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D052160:Benzothiazoles; D004734:Energy Metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes; D005615:Freezing; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism / Q000502:physiology*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D017510:Protein Folding; D013379:Substrate Specificity; D013844:Thiazoles; D014508:Urea / Q000737:chemistry
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D013844:Thiazoles; C009462:thioflavin T; D000255:Adenosine Triphosphate; D014508:Urea; D008156:Luciferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D000251:Adenosine Triphosphatases