All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat‐shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat‐induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O‐ClpX specific protein‐protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non‐hydrolysable analogue ATP‐gamma‐S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.

DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well. DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function. Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ. Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II). Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II). Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure. An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background.

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Using highly purified proteins, we have identified intermediate reactions that lead to the assembly of molecular chaperone complexes with wild-type or mutant p53R175H protein. Hsp90 possesses higher affinity for wild-type p53 than for the conformational mutant p53R175H. The presence of Hsp90 in a complex with wild-type p53 inhibits the binding of Hsp40 and Hsc70 to p53, consequently preventing the formation of wild-type p53-multiple chaperone complexes. The conformational mutant p53R175H can form a stable heterocomplex with Hsp90 only in the presence of Hsc70, Hsp40, Hop and ATP. The anti-apoptotic factor Bag-1 can dissociate Hsp90 from a pre- assembled complex wild-type p53 protein, but it cannot dissociate a pre-assembled p53R175H-Hsp40- Hsc70-Hop-Hsp90 heterocomplex. The results presented here provide possible molecular mechanisms that can help to explain the observed in vivo role of molecular chaperones in the stabilization and cellular localization of wild-type and mutant p53 protein.

Using the native proteins lambda P, lambda O, delta 32, and RepA, as well as permanently unfolded alpha-carboxymethylated lactalbumin, we show that DnaK and DnaJ molecular chaperones possess differential affinity toward these protein substrates. In this paper we present evidence that the DnaK protein binds not only to short hydrophobic peptides, which are in an extended conformation, but also efficiently recognizes large native proteins (RepA, lambda P). The best substrate for either the DnaK or DnaJ chaperone is the native P1 coded replication RepA protein. The native delta 32 transcription factor binds more efficiently to DnaJ than to DnaK, whereas unfolded alpha-carboxymethylated lactalbumin or native lambda P binds more efficiently to DnaK than to the DnaJ molecular chaperone. The presence of nucleotides does not change the DnaJ affinity to any of the tested protein substrates. In the case of DnaK, the presence of ATP inhibits, while a nonhydrolyzable ATP analogues markedly stimulates the binding of DnaK to all of these various protein substrates. ADP has no effect on these reactions. In contrast to substrate protein binding, DnaK binds to the DnaJ chaperone protein in a radically different manner, namely ATP stimulates whereas a nonhydrolyzable ATP analogue inhibits the DnaK-DnaJ complex formation. Moreover, the DnaKc94 mutant protein lacking 94 amino acids from its C-terminal domain, which still possesses at ATPase activity and forms a transient complex with protein substrates, does not interact with DnaJ protein. We conclude that the DnaK-ADP form, derived from ATP hydrolysis, possesses low affinity to the protein substrates but can efficiently interact with DnaJ molecular chaperone.