ATP HYDROLYSIS IS REQUIRED FOR THE DNAJ-DEPENDENT ACTIVATION OF DNAK CHAPERONE FOR BINDING TO BOTH NATIVE AND DENATURED PROTEIN SUBSTRATES

Using two independent experimental approaches to monitor protein-prote in interactions (enzyme-linked immunosorbent assay and size exclusion high performance liquid chromatography) we describe a general mechanis m by which DnaJ modulates the binding of the DnaK chaperone to various native protein substrates, e.g. lambda P, lambda O, sigma(32), P1, Re pA, as well as permanently denatured alpha-carboxymethylated lactalbum in, The presence of DnaJ promotes the DnaK for efficient DnaK-substrat e complex formation, ATP hydrolysis is absolutely required for such Dn aJ-dependent activation of DnaK for binding to both native and denatur ed protein substrates. Although ADP can stabilize such an activated Dn aK-protein complex, it cannot substitute for ATP in the activation rea ction, Ln the presence of DnaJ and ATP, DnaK possesses the affinity to different substrates which correlates well with the affinity of DnaJ alone for these protein substrates, Only when the affinity of the DnaJ chaperone for its protein substrate is relatively high (e.g. sigma(32 ), RepA) can a tertiary complex DnaK-substrate-DnaJ be detected. In th e case that DnaJ binds weakly to its substrate (lambda P, alpha-carbox ymethylated lactalbumin), DnaJ is only transiently associated with the DnaK-substrate complex, but the DnaK activation reaction still occurs , albeit less efficiently,