TY - JOUR
T1 - The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase
AU - Sharma, Sandeep K.
AU - De Los Rios, Paolo
AU - Christen, Philipp
AU - Lustig, Ariel
AU - Goloubinoff, Pierre
PY - 2010/12
Y1 - 2010/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=78649309029&partnerID=8YFLogxK
U2 - 10.1038/nchembio.455
DO - 10.1038/nchembio.455
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AN - SCOPUS:78649309029
SN - 1552-4450
VL - 6
SP - 914
EP - 920
JO - Nature Chemical Biology
JF - Nature Chemical Biology
IS - 12
ER -