REAL-TIME KINETICS OF THE DNAK DNAJ GRPE MOLECULAR CHAPERONE MACHINE ACTION

Applying stopped-flow fluorescence spectroscopy for measuring conforma tional changes of the UnaK molecular chaperone (bacterial Hsp70 homolo gue) and its binding to target peptide, we found that after ATP hydrol ysis, DnaK is converted to the DnaK(ADP) conformation, which possesse s limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacter ial Hsp40 homologue), the DnaK(ADP) form is converted back to the Dna K conformation, and the resulting DnaJ . DnaK(ADP) complex binds to pe ptide substrates more tightly. Formation of the DnaJ(substrate-DnaK(AD P)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK(ADP) conformation. W e conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosph atase activity of DnaK.