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Peptide-induced conformational changes in the molecular chaperone DnaK
1Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
Biochemistry
|December 8, 1998
Summary
Molecular chaperone DnaK (Deoxyribonucleic acid K) transitions between high- and low-affinity states for peptide binding, regulated by ATP. The study reveals peptide binding can reverse this switch, influencing chaperone function.
Area of Science:
- Molecular Biology
- Biochemistry
- Protein Dynamics
Background:
- DnaK, a 70 kDa molecular chaperone from Escherichia coli, exhibits distinct high-affinity (ADP-bound) and low-affinity (ATP-bound) states for target peptide binding.
- A significant decrease in DnaK's tryptophan fluorescence accompanies the transition from the high- to low-affinity state.
- Understanding the dynamics of these conformational changes is crucial for elucidating chaperone-substrate interactions.
Purpose of the Study:
- To investigate the reversibility of the structural transition in DnaK, specifically how peptide binding affects its affinity state.
- To characterize the kinetic parameters governing the interaction between DnaK and its target peptide in the presence of ATP.
Main Methods:
- Utilized rapid mixing and equilibrium fluorescence spectroscopy to monitor real-time changes in DnaK's tryptophan fluorescence.
- Employed the Cro peptide (MQERITLKDYAM) as a model unfolded substrate to probe DnaK's binding and release kinetics.
- Analyzed the data to determine rate constants and binding affinities under specific experimental conditions.
Main Results:
- Rapid addition of the Cro peptide to preformed low-affinity DnaK-ATP complexes induced a swift increase in tryptophan fluorescence (kobs = 3–30 s−1).
- This fluorescence increase suggests the peptide promotes the transition of DnaK from its low-affinity to a high-affinity state, independent of ATP hydrolysis.
- Kinetic parameters were determined: ATP binding (K1) = 22 μM, peptide release (koff) = 3.3 s−1, and peptide binding (kon) = 2.4 x 10^4 M−1 s−1 at 25°C.
Conclusions:
- The findings support a minimal mechanism where ATP binding induces conformational change and peptide release, while peptide binding to the low-affinity state drives a transition back to the high-affinity state.
- DnaK exists in an equilibrium between closed and open conformations even in the absence of ATP and peptide, indicating inherent structural flexibility.
- Peptide binding acts as a trigger to shift DnaK towards its high-affinity state, facilitating chaperone-substrate interaction dynamics.