Global unfolding of a substrate protein by the Hsp100 chaperone ClpA.
The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.
Weber-Ban
E U
EU
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Reid
B G
BG
Miranker
A D
AD
Horwich
A L
AL
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D018833:Chaperonins / Q000378:metabolism*; D003903:Deuterium; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D003903:Deuterium; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D018833:Chaperonins; C069775:ClpB protein, E coli