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 protea se ClpP (refs 1, 2). ClpA directs the ATP-dependent degradation of substrat e proteins bearing specific sequences(3-5), much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated prote ins(6-8). In isolation, ClpA and its relative ClpX can mediate the disassem bly of oligomeric proteinsg(9,10); another similar eukaryotic protein, Hsp1 04, can dissociate low-order aggregates(11). ClpA has been proposed to dest abilize protein structure, allowing passage of proteolysis substrates throu gh a central channel into the ClpP proteolytic cylinder(12-14). Here we tes t the action of ClpA on a stable monomeric protein, the green fluorescent p rotein GFP, onto which has been added an Il-amino-acid carboxy-terminal rec ognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation(5,15). Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP (16), and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.