Global unfolding of a substrate protein by the Hsp100 chaperone ClpA

The bacterial protein ClpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP (refs 1, 2). ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences3,4,5, much as the 19S ATPase ‘cap’ of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins6,7,8. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins9,10; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates11. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder12,13,14. 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 degradation5,15. Fluorescence studies both with and without a ‘trap’ version of the chaperonin GroEL, which binds non-native forms of GFP16, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.