Kinetic evidence for peptide-induced oligomerization of the molecular chaperone DnaK at heat shock temperatures.
The pre-steady-state kinetics of the binding of a fluorescent peptide (dansyl-KLIGVLSSLFRPK, fVSV13) to the Escherichia coli molecular chaperone DnaK were investigated over a range of temperatures (25-42 degrees C). At 42 degrees C, over a wide range of peptide concentrations, the fVSV13 peptide bound to DnaK with biphasic kinetics: a rapid burst in the DnaK-fVSV13 signal in the first 5 s was followed by a gradual reduction in the signal over the next 100 s. The descending portion of each biphasic trace followed the equation F(t) = DeltaF exp(-kdt) + Finfinity, where DeltaF, kd, and Finfinity are the amplitude, the apparent first-order rate constant, and the fluorescence end point, respectively. Both DeltaF and kd increased with increasing concentrations of DnaK, which suggests that the loss of the DnaK-fVSV13 signal is caused by a bimolecular reaction. We propose that (i) the fVSV13 peptide binds to and induces a conformational change in the DnaK monomer [E + P right harpoon over left harpoon (EP)*]; and (ii) the conformational change promotes the formation of oligomeric DnaK-peptide complexes [En + (EP)* right harpoon over left harpoon En-EP]. The term (EP)* denotes a monomeric DnaK-peptide complex in which the bound peptide is fluorescent; En-EP denotes an oligomeric DnaK-peptide complex in which the fluorescence of the bound peptide is quenched. Numerical fitting of the stopped-flow data to reactions (i) and (ii) yielded values for the four rate constants. When the proposed kinetic model was tested by conducting experiments in the presence of excess peptide or excess ATP&sbd;conditions which inhibit oligomerization&sbd;DnaK-fVSV13 complex formation proceeded to stable asymptotes, with no reduction in the DnaK-fVSV13 signal at long times.
Farr
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, 71130-3932, USA.
Witt
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D003619:Dansyl Compounds; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D016018:Least-Squares Analysis; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*
D001426:Bacterial Proteins; D003619:Dansyl Compounds; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D010455:Peptides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli