| Range |
Table - link
|
| Organism |
Bacteria Escherichia coli |
| Reference |
Sharma SK, De los Rios P, Christen P, Lustig A, Goloubinoff P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat Chem Biol. 2010 Dec6(12):914-20. doi: 10.1038/nchembio.455. Supplementary Information p.17 Supplementary table 3PubMed ID20953191
|
| Primary Source |
See refs beneath table |
| Method |
P.914 right column 2nd paragraph: "Here [investigators] clarified the mechanistic subprocesses of the Escherichia coli version of the Hsp70-Hsp40-NEF chaperone system, the DnaK-DnaJ-GrpE system, by using a preformed and well-characterized misfolded substrate. [They] denatured a firefly luciferase mutant by a freeze-thaw treatment and isolated a near-homogenous population of stable, misfolded compact inactive monomers. The unique qualities of this chaperone substrate allowed for the first time the measuring of the rates of the various steps leading from a stable inactive misfolded state to a stable, native, enzymatically active refolded state. This permitted [them] to correlate rates of ATP consumption with rates of refolding to the native state. The data showed that bacterial Hsp70, DnaK, uses the energy of ATP to convert misfolded substrates into unfolded intermediates that, upon release from the chaperone, can spontaneously refold to their native state." |
| Comments |
P.918 right column 2nd paragraph: "Moreover, the special features of the new substrate allowed [investigators] to characterize the kinetics of chaperone-mediated renaturation in the presence of a large molar excess of substrate. Saturation kinetics were found that, under the present conditions, indicated a Vmax′ value of about 5 mol F-T–luciferase reactivated per mol DnaK per min and a Km′ value—the substrate concentration at which the rate of renaturation was half maximal—of about 2 μM. Intriguingly, ATP hydrolysis was half maximal already at about 0.2 μM substrate (Fig. 4b). The apparent lower efficiency of the chaperone machinery at low substrate concentration could be because of the simultaneous binding of more than one DnaK to a single substrate molecule, which would increase ATP consumption without improving the unfolding and thus produce an overall lower renaturation effect. Yet, at saturating substrate concentrations, every hydrolyzed ATP molecule was apparently accompanied by the successful locking and unfolding of the substrate. The fact that only five ATPs were needed to mediate the native refolding of one polypeptide [BNID 113282] is an unprecedentedly low value for any chaperone system thus far described (Supplementary Table 3). It agrees surprisingly well with the observation that at substoichiometric concentrations of the chaperone, the unfolding reaction is about five times faster than the subsequent refolding reaction, suggesting that only one out of five unfolded luciferase molecules take the folding path to the native state." |
| Entered by |
Uri M |
| ID |
113283 |