PMID:21821046
| Citation |
Nager, AR, Baker, TA and Sauer, RT (2011) Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease. J. Mol. Biol. 413:4-16 |
|---|---|
| Abstract |
In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis. |
| Links |
PubMed PMC3184388 Online version:10.1016/j.jmb.2011.07.041 |
| Keywords |
Adenosine Triphosphate/metabolism; Endopeptidase Clp/metabolism; Escherichia coli/enzymology; Escherichia coli/metabolism; Escherichia coli Proteins/metabolism; Green Fluorescent Proteins/metabolism; Hydrolysis; Models, Chemical; Models, Molecular; Protein Folding |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
See also
References
See Help:References for how to manage references in EcoliWiki.