Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase

Area of Science:

• Molecular Biology
• Protein Folding Dynamics
• Chaperone-mediated Protein Regulation

Background:

• Chaperonins are essential molecular machines that assist protein folding.
• Dihydrofolate reductase (DHFR) is a model enzyme for studying protein refolding.
• The interaction between chaperonins and folding intermediates is crucial for cellular protein homeostasis.

Purpose of the Study:

• To investigate the mechanism by which chaperonin 60 (GroEL) inhibits DHFR refolding.
• To elucidate the role of ATP and chaperonin 10 (GroES) in the release of bound DHFR.
• To characterize the interaction between native DHFR and GroEL.

Main Methods:

• In vitro refolding assays of chemically denatured DHFR in the presence of GroEL.
• Proteolysis sensitivity assays to assess the structure of bound DHFR.
• ATP-dependent release assays using wild-type and non-hydrolyzable ATP analogs.
• Binding competition assays with radiolabeled DHFR and ribulose-1,5-bisphosphate carboxylase.

Main Results:

• GroEL completely arrests spontaneous DHFR refolding, forming stable complexes with folding intermediates.
• Bound DHFR exhibits increased sensitivity to proteolysis, indicating a nonnative conformation.
• ATP, potentiated by GroES, facilitates the release of DHFR from GroEL; ATP hydrolysis is not essential.
• Native DHFR also binds GroEL, but this interaction is slower and can be inhibited by DHFR substrates, suggesting an equilibrium of conformers.

Conclusions:

• GroEL actively inhibits DHFR refolding by sequestering folding intermediates.
• The GroEL-GroES-ATP system provides a mechanism for releasing misfolded or intermediate proteins.
• Native DHFR exists in dynamic equilibrium, with a substrate-binding conformer being less prone to GroEL interaction.