Monomeric creatine kinase aggregation and sodium dodecyl sulfate-cyclodextrin assisted refolding

Area of Science:

• Biochemistry
• Protein Folding
• Enzymology

Background:

• Monomeric creatine kinase (CK) is prone to aggregation when denatured.
• Hydrophobic interactions between CK subunits drive aggregation of partially folded monomers.
• Developing methods to prevent aggregation and refold proteins is crucial for understanding their function.

Purpose of the Study:

• To stabilize monomeric creatine kinase (CK) in a non-aggregating state.
• To investigate the aggregation kinetics of partially folded monomeric CK (PF-CK).
• To develop and apply an artificial chaperone system for refolding PF-CK.

Main Methods:

• Employing cysteine residue modifications to stabilize monomeric CK.
• Utilizing SDS-cyclodextrin as an artificial chaperone to prevent aggregation.
• Applying size exclusion chromatography and intrinsic fluorescence to monitor refolding.
• Investigating the kinetics of aggregation and refolding pathways.

Main Results:

• Stable monomeric CK was captured in partially folded states.
• Partially folded monomeric CK (PF-CK) aggregation followed first-order kinetics due to hydrophobic interactions.
• SDS treatment effectively blocked PF-CK aggregation.
• Refolding of PF-CK was achieved using cyclodextrins and DTT, with different cyclodextrins influencing the process.
• Intrinsic fluorescence confirmed protein reactivation and conformational changes during refolding.
• Size exclusion chromatography identified various intermediate monomeric CK species.

Conclusions:

• Monomer CK aggregation was demonstrated for the first time.
• The SDS-cyclodextrin system effectively prevents aggregation and facilitates CK reassociation.
• Refolding pathways of CK with the artificial chaperone follow first-order kinetics.