ATP lowers the activation enthalpy barriers to DnaK-peptide complex formation and dissociation.

The role of nucleotide in controlling the pre-steady-state kinetics of peptide binding to the Escherichia coli 70-kDa molecular chaperone DnaK was investigated using stopped-flow fluorescence. The peptide used in this study, fVSV13 (representing amino acids 490-502 of the vesicular stomatitis virus glycoprotein), was dansylated specifically at its N-terminus. We found that (i) between 17 and 35 degrees C in the presence of ATP the second-order rate constant (k(on)) for fVSV13 binding to DnaK exhibited almost no dependence on temperature and did not deviate significantly from 3.8 x 10(5) M(-1) s(-1). In contrast, over the same temperature range in the presence of ADP, k(on) increased by a factor of 32 (7.3 x 10(4) to 2.3 x 10(6) M(-1) s(-1)); and (ii) ATP increased the apparent first-order rate constant for the release of fVSV13 from preformed DnaK-fVSV13 complexes by several orders of magnitude relative to ADP. The activation energy parameters for fVSV13 binding to and dissociation from DnaK are compared to the activation parameters for the binding of an unrelated peptide to DnaK and are also discussed in terms of an open-to-closed equilibrium in the polypeptide-binding domain. On the basis of this comparison, it is suggested that the activation entropy term deltaS++, which is related to the structure of the DnaK-bound peptide or the degree of solvation of the peptide, is a controlling factor in the kinetics of peptide binding to DnaK.

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.

Visualization of a slow, ATP-induced structural transition in the bacterial molecular chaperone DnaK.

Recent reports have shown that the binding of ATP to a 70-kDa molecular chaperone induces a rapid global conformational transition from a "high affinity" state to a "low affinity" state, where these states are defined by tight and weak binding to (poly)peptides, respectively. To complete the activity cycle, a chaperone molecule must ultimately return to the high affinity state. In this report, this return to the high affinity state was studied using a chemical cross-linking assay in conjunction with SDS-polyacrylamide gel electrophoresis. The basis for this assay is that in the absence of nucleotide or in the presence of ADP, conditions that stabilize the high affinity state, cross-linking of the Escherichia coli molecular chaperone DnaK yielded two monomeric forms, with apparent molecular masses of 70 kDa (77%) and 90 kDa (23%), whereas cross-linking yielded only the 70-kDa monomeric form in the presence of ATP. This ATP-dependent difference in cross-linking was used to follow the kinetics of the low affinity to high affinity transition under single turnover conditions. The rate of this transition (kobs = 3.4 (+/-0.6) x 10(-4) s-1 at 25 degrees C) is almost identical to the reported rate of ATP hydrolysis (khy = 2.7 (+/-0.7) x 10(-4) s-1 at 22 degrees C). These results are consistent with a two-step sequential reaction where rate-limiting ATP hydrolysis precedes the conformational change. Models for the formation of two cross-linked DnaK monomers in the absence of ATP are discussed.

Large activation energy barriers to chaperone-peptide complex formation and dissociation.

To probe the mechanism of chaperone substrate selection, we have investigated the kinetics of complex formation and dissociation between the molecular chaperone DnaK and a short peptide (Cro, representing amino acids 1-12 of the cro repressor protein). The Cro protein was N-terminally labeled with the environmentally sensitive fluorophore dansyl chloride (Cro*), and steady-state and stopped-flow fluorescence spectroscopies and fluorescence-detected high-performance size exclusion chromatography (HPSEC) were used to monitor complex formation and dissociation over a range of temperatures in the absence of ATP. The results are summarized as follows: (i) Cro* binds to DnaK with a second-order rate constant, k(on), which varies from 8 to 200 M-1 s-1 between 15 and 37 degrees C. The slow on-rate is a consequence of a large activation energy barrier. The activation enthalpy (delta H*) and the prefactor [omega exp delta S*/R)] are 26 kcal mol-1 and 7 x 10(20) M-1 s-1, respectively. (ii) Once formed, DnaK-Cro* complexes are long-lived, especially at low temperatures (T < 15 degrees C). The off-rate is unusually temperature-sensitive, for example, there is a 478-fold increase in k(off) from 2.3 x 10(6) to 1.1 x 10(-3) s-1 over a range of only 30 degrees C (5-35 degrees C). The steep temperature-dependence of the off-rate is a consequence of a very large activation energy barrier to DnaK-Cro* complex dissociation [delta H* = 34.6 kcal mol-1 and omega exp (delta S*/R) = 2 x 10(21) s-1]. The relatively low affinity of the Cro* peptide for DnaK is due to a large kinetic barrier to binding. We discuss possible causes for these large kinetic barriers.