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.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004734:Energy Metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C063842:dnaK protein, E coli