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 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.
Neuhofen
S
S
Fachbereich Chemie/Biochemie, Universität Kaiserslautern, Germany.
Theyssen
H
H
Reinstein
J
J
Trommer
W E
WE
Vogel
P D
PD
D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels
D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D003497:Cyclic N-Oxides; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011994:Recombinant Proteins; D013113:Spin Labels; C063842:dnaK protein, E coli; C001803:tempol