Kinetics of molecular chaperone action

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Folding

Background:

• Molecular chaperones, including heat shock proteins of the 70 kDa (Hsp70) family, are crucial for protein homeostasis.
• These proteins bind to unfolded polypeptide segments, preventing aggregation and promoting correct folding.
• The Hsp70 chaperone DnaK from Escherichia coli is a well-studied example involved in these processes.

Purpose of the Study:

• To investigate the real-time kinetics of peptide binding to the molecular chaperone DnaK.
• To characterize the binding and release cycle of DnaK in the presence and absence of adenosine triphosphate (ATP).
• To determine if the chaperone's binding-release cycle is coupled to its ATPase activity.

Main Methods:

• Utilized an environmentally sensitive fluorophore to label target peptides.
• Employed real-time fluorescence spectroscopy to monitor peptide binding to DnaK.
• Analyzed the binding kinetics using a two-step relaxation model and single-exponential processes.

Main Results:

• The binding of peptides to DnaK without ATP occurred via a two-step process with relaxation times of 27 and 200 seconds.
• In the presence of ATP, DnaK-peptide complex formation was significantly accelerated, showing a single-exponential process with a 0.4-second relaxation time.
• The binding-release cycle of DnaK operates on a timescale compatible with polypeptide chain elongation and folding.

Conclusions:

• The Hsp70 chaperone DnaK exhibits rapid binding and release kinetics, crucial for efficient protein folding.
• The chaperone's cycle is too fast to be stoichiometrically coupled to its ATPase activity.
• These findings provide insights into the dynamic mechanism of Hsp70-mediated protein folding.