Nucleotide Binding to the Heat‐Shock Protein DnaK as Studied by ESR Spectroscopy

We employed ESR spectroscopy using spin‐labeled adenine nucleotides to investigate nucleotide binding to the 70‐kDa heat shock protein, DnaK, from Escherichia coli. Binding stoichiometries of 1 mol/mol for both ATP and ADP to previously nucleotide‐depleted protein in the presence of Mg 2+ were determined directly and under equilibrium binding conditions. Of the spin‐labeled adenine nucleotides available to us, only the derivatives with the spin label attached to the C8 position of the adenine moiety, 8‐SL‐Ado P 3 and 8‐SL‐Ado P 2 [8‐(2,2,6,6‐tetramethyl‐piperidin‐4‐yl‐1‐oxyl‐)amino‐adenosine‐5′‐triphosphate or diphosphate], were bound sufficiently tightly by the heat‐shock protein, resulting in ESR spectra typical for immobilized radicals. In the absence of Mg 2+ , only approximately 0.5 mol were bound. Subsequent addition of Mg 2+ , however, led to the previously observed maximum binding of 1 mol/mol. Both 8‐SL‐Ado P 3 , and 8‐SL‐Ado P 2 were fully exchangeable upon addition of excess ATP or ADP suggesting that the analogs bound directly to the nucleotide binding sites within the protein. 8‐SL‐Ado P 2 release was also observed in the presence of the co‐chaperone GrpE, indicating that the spin‐labeled analogs of adenine nucleotides function like the natural nucleotidesubstrates of the heat‐shock protein. Small differences in the ESR spectra of 8‐SL‐Ado P 3 and 8‐SL‐Ado P 2 in complex with DnaK were observed.