The Purification And Biochemistry Study Of DnaK

Heat Shock Protein 70kDa, Hsp70s, are a class of highly conserved molecular chaperone which are essential for maintaining cellular protein homeostasis in organisms ranging from bacteria to mammal.DnaK, a major Hsp70 in E.coli, has been widely used as a paradigm for studying Hsp70s. The latest solved crystal structure of DnaK in complex with ATP shows there are two molecules of DnaK-ATP in the asymmetric unit. Interestingly, with several hydrogen bonds the interface between the two molecules of DnaK are large with good surface complementarity, suggesting functional importance of this crystallographic dimer.In this study, crosslinking experiment were done based on the DnaK-ATP structure and confirmed that DnaK forms specific dimer in an ATP-dependent manner. To understand the physiological function of the dimer, we mutated five residues on the dimer interface. The growth test in vivo showed that four mutations, R56A, T301 A, N537A and D540A, result in loss of chaperone activity, indicating the functional importance of the dimer. Then all above mutant DnaK protein were expressed in E.coli and purified with HisTrap column and HisTrap Q column. Furthermore we did fluorescence anisotropy peptide substrate binding assay. Dissociation constants(Kd) were determined by fitting anisotropy data using PRISM sofeware. The results didn’t show appreciable defect in substrate binding compared with WT DnaK. Another tryptophan fluorescence assay were did to test whether the disrupted dimer formation could influence ATP-induced allosteric coupling. Compared with 6 nm blue shift of WT DnaK, all the dimer mutants showed similar amount of blue shift, suggesting the ATP-induced allosteric coupling is largely intact.In summary, this study suggests that DnaK forms transient dimer upon ATP binding and this dimer structure is important to its molecular chaperone activity. Disrupting dimer formation reduced the chaperone activity. Surprisingly, neither the peptide binding ability nor the ATP-induced allosteric coupling is compromised in all the mutations.