DnaK protein vitro

Hence, several in vitro activities have demonstrated a participation of stress-70 proteins in the disassembly of macromolecular complexes. The participation of dnaK in initiation of replication for X and PI phages appears to be dependent on ancillary proteins, specifically dnaJ and grpE. This raises the possibility that substrate specificity, in these cases, may be intrinsic to the ancillary proteins rather than residing solely in the dnaK protein. In the case of in vitro dissassembly of clathrin cages, the HSC70 protein can accomplish the reaction without accessory proteins. In all the above cases, ATP hydrolysis is essential for the activities. 

Under certain conditions, the stress-70 proteins can participate in the renaturation of denatured or inactivated proteins. The renaturation capabilities of E. coli dnaK protein have been most extensively documented. It has been shown that in vitro, dnaK can protect E. coli RNA polymerase from aggregation when the polymerase is incubated at elevated temperatures that would normally result in loss of activity, and, further, that dnaK can disaggregate and reactivate polymerase, once it has been inactivated by heat denaturation (Skowyra et al., 1990). These activities are absolutely dependent on ATP hydrolysis. The mutant dnaK756 protein is effective in protecting active RNA polymerase against heat inactivation, but is incapable of disaggregating and reactivating polymerase, once it has been heat inactivated. 

Clarke, C.F., Cheng, K., Frey, A.B., Stein, R., Hinds, P.W.. Levine, A.J. (1988). Purification of complexes of nuclear oncogene p53 with rat and E. coli heat shock protein In vitro dissociation of hsc70 and dnaK from murine p53 by ATP. Mol. Cell. Biol. 8, 1206-1215. 

A second demonstration of participation in renaturation of heat-inactivated proteins by dnaK is provided by experiments with a temperature-sensitive repressor protein of bacteriophage X, XcI857 protein (Gaitanaris et ai, 1990). Activities of the Xcl wild-type and mutant proteins were measured in an in vitro operator DNA binding assay and by in vivo expression from a Xcl-regulated operon fusion of XPrOr and the... 

In vitro, with CaATP as a substrate, E. coli dnaK autophosphorylates exclusively at Thr-199 (McCarty and Walker, 1991). It does not auto-phosphorylate when MgATP is used as a substrate. In vivo, dnaK is found to be phosphorylated on serine as well as on threonine residues (Rieul et al., 1987). Under normal growth conditions, phosphorylation is primarily on serine when E. coli is infected with bacteriophage M13, the phosphorylation shifts predominantly to threonine, with minor phosphorylation of serine. The specific target residues of in vivo phosphorylation of dnaK have not yet been determined. The disparity between the in vitro results (autophosphorylation exclusively at Thr-199 an absolute requirement for CaATP) and the in vivo results (phosphorylation on both serine and threonine residues, under conditions in which MgATP would be presumed to be the available intracellular substrate nucleotide) raises the questions of (1) whether the specific autophosphorylation of Thr-199 observed in vitro also occurs in vivo, or whether it may be an artifactual side reaction when the larger Ca ion is substituted for Mg" at the active site of the protein, and (2) whether there is a serine/threonine protein kinase that specifically phosphorylates dnaK in vivo. 

It has also been shown that mitochondiral stress-70 proteins autophos-phorylate in vitro in the presence of CaATP (but not MgATP), which is consistent with the observations on dnaK and BiP (Mizzen et al., 1991). 

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