Molecular characterization of DnaK from the halotolerant cyanobacterium Aphanothece halophytica for ATPase, protein folding, and copper binding undervarious salinity conditions

Previously, it was found that the dnaK1 gene of the halotolerant cyanobacte rium Aphanothece halophytica encodes a polypeptide of 721 amino acids which has a long C-terminal region rich in acidic amino acid residues. To unders tand whether the A. halophytica DnaK1 possesses chaperone activity at high salinity and to clarify the role of the extra C-terminal amino acids, a com parative study examined three kinds of DnaK molecules for ATPase activity a s well as the refolding activity of other urea-denatured proteins under var ious salinity conditions. DnaK1s from A. halophytica and Synechococcus sp. PCC 7942 and the C-terminal deleted A. halophytica DnaK1 were expressed in Escherichia coli and purified. The ATPase activity of A. halophytica DnaK1 was very high even at high salinity (1.0 M NaCl or KCl), whereas this activ ity in Synechococcus PCC 7942 DnaK1 decreased with increasing concentration s of NaCl or KCl. The salt dependence on the refolding activity of urea-den atured lactate dehydrogenase by DnaK1s was similar to that of ATPase activi ty of the respective DnaK1s. The deletion of the C-terminal amino acids of A. halophytica DnaK1 had no effect on the ATPase activity, but caused a sig nificant decrease in the refolding activity of other denatured proteins. Th ese facts indicate that the extra C-terminal region of A. halophytica DnaK1 plays an important role in the refolding of other urea-denatured proteins at high salinity. Furthermore, it was shown that DnaK1 could assist the cop per binding of precursor apo-plastocyanin as well as that of mature apo-pla stocyanin during the folding of these copper proteins.