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Journal Article

Nucleotide binding to the heat-shock protein DnaK as studied by ESR spectroscopy


Reinstein,  Jochen
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

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Neuhofen, S., Theyssen, H., Reinstein, J., Trommer, W. E., & Vogel, P. D. (1996). Nucleotide binding to the heat-shock protein DnaK as studied by ESR spectroscopy. European Journal of Biochemistry, 240(1), 78-82. doi:10.1111/j.1432-1033.1996.0078h.x.

Cite as: http://hdl.handle.net/11858/00-001M-0000-002C-262B-7
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 to the C8 position of the adenine moiety, 8-SL-AdoP3 and 8-SL-AdoP2 [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 Mg2+, only approximately 0.5 mol were bound. Subsequent addition of Mg2+, however, led to the previously observed maximum binding of 1 mol/mol. Both 8-SL-AdoP3 and 8-SL-AdoP2 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-AdoP2 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 nucleotide-substrates of the heat-shock protein. Small differences in the ESR spectra of 8-SL-AdoP3 and 8-SL-AdoP2 in complex with DnaK were observed.