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 Mg2+ 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 tu the C8 position of the adenine moiety, 8-SL-AdoP3 and 8-SL-AdoP2 [8-(2,2,6,6-tetramethyl-piperidin-4-yl-l-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 Mg2+, only approximately 0.5 mol were bound. Subsequent addition of Mg", however, led to the previously observed maximum binding of 1 molhol. Both 8-SL-AdoP3 and g-SL-AdoP, were fully exchangeable upon addition of excess ATP or ADP suggesting and CrpE [8]. DnaJ accelerates the rate of hydrolysis of DnaKbound ATP, while GrpE stimulates nucleotide release from the heat-shock protein and is therefore referred to as the nucleotide exchange factor [8, 91. Recent experiments as described in [lo] indicate a 2: 1 stoichiometry of the GrpE binding to DnaK. The same binding stoichiometry was also found for the ATP-binding domain of the protein, indicating that GrpE-binding sites are located in the N-terminal portion of DnaK. A conformational change was reported previously to take place within DnaK upon hydrolysis of ATP [ll].ESR spectroscopy has been widely applied in investigations of structure/function relationships in biochemical systems (for reviews see 112-141). In addition to binding studies to determine the stoichiometry of accessible nucleotide-binding sites, ESR spectroscopy is also a conformation-sensitive technique and allows investigation of conformational changes in the vicinity of the reporter-group, here the spin-labeled (SL) nucleotides.Burgard and coworkers [ 151 demonstrated such conformational changes taking place within the nucleotide binding sites of F,-ATPases in the presence or absence of divalent cations.Even though the stoichiometry of adenine nucleotide binding to DnaK is widely discussed as 1 mol/niol due to similarities to other proteins, these data have always been assumed but have never been shown in direct experiments. Here we present the application of ESR spectroscopy to investigate the heat-shock protein DnaK from Escherichia coli. We used spin-labeled adenine nucleotides to determine the binding stoichiometry for both ATP and ADP and probe for conformational differences within the nucleotide-binding site when either the ATP-analog or ADPanalog was bound to DnaK. The effects of Mg2+ and the nucleotide-exchange factor, GrpE, on nucleotide binding were also tested.
