Oxidized GroEL can function as a chaperonin

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Oxidative stress can inactivate enzymes like rhodanese.
• Molecular chaperones, such as GroEL, typically assist protein folding.
• The role of oxidized chaperones in enzyme reactivation is less understood.

Purpose of the Study:

• To investigate the potential of oxidized GroEL (ox-GroEL) in reactivating oxidatively inactivated rhodanese.
• To elucidate the mechanism by which ox-GroEL facilitates rhodanese reactivation.
• To characterize the structural and functional changes in GroEL upon oxidation.

Main Methods:

• Oxidation of native GroEL using hydrogen peroxide.
• Assessing rhodanese reactivation efficiency in the presence of ox-GroEL, reductant, and substrate.
• Employing differential centrifugation and fluorescence spectroscopy to detect protein complex formation.
• Using bis-ANS fluorescence to detect changes in protein surface hydrophobicity.
• Evaluating sensitivity to proteolysis by trypsin.

Main Results:

• Oxidized GroEL (ox-GroEL) achieved 85% reactivation of inactivated rhodanese.
• Reactivation required a reductant and the enzyme substrate, sodium thiosulfate.
• ox-GroEL formed a complex with inactivated rhodanese, mediated by hydrophobic interactions.
• Oxidized GroEL retained its structure but showed increased surface hydrophobicity and proteolysis sensitivity.
• Inactivated rhodanese also exhibited increased surface hydrophobicity.

Conclusions:

• Oxidized GroEL functions as a molecular chaperone to reactivate oxidatively damaged rhodanese.
• Hydrophobic interactions are key in the binding of ox-GroEL to inactivated rhodanese.
• GroEL maintains chaperone activity even after oxidation, suggesting a protective role during oxidative stress.