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Folding pathway for partially folded rabbit muscle creatine kinase
1Department of Biological Science and Biotechnology, Tsinghua University, Beijing, China.
Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
|August 31, 2001
Summary
Rabbit muscle creatine kinase (CK) was partially unfolded and modified, yielding a monomeric intermediate. This intermediate regained significant activity upon dithiothreitol (DTT) treatment, synchronized with refolding, and did not bind to GroEL.
Area of Science:
- Biochemistry
- Protein Folding
- Enzymology
Background:
- Creatine kinase (CK) is a crucial enzyme in cellular energy metabolism.
- Understanding protein folding pathways, including intermediate states, is vital for comprehending enzyme function and dysfunction.
- Molecular chaperones like GroEL play a role in protein folding, but their substrate recognition mechanisms are not fully elucidated.
Purpose of the Study:
- To characterize a partially folded monomeric intermediate of rabbit muscle creatine kinase (CK).
- To investigate the refolding pathway and activity recovery of modified CK.
- To examine the interaction of the partially folded CK intermediate with the molecular chaperone GroEL.
Main Methods:
- Chemical modification of rabbit muscle CK using 5,5'-dithio-bis(2-nitrobenzoic acid) and guanidine hydrochloride denaturation.
- Analysis of protein state using size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and fluorescence spectroscopy.
- Enzyme activity assays following dithiothreitol (DTT) treatment, GroEL binding assays, and hydrophobic surface detection using 1-anilinonaphthalene-8-sulfonate.
Main Results:
- A monomeric, partially folded state of CK with modified thiol groups was generated.
- Dithiothreitol (DTT) treatment restored approximately 70% of CK activity in a two-phase kinetic course, synchronized with refolding.
- The monomeric CK intermediate exhibited increased hydrophobic surface area but did not bind to GroEL, challenging existing models of GroEL substrate recognition.
Conclusions:
- The study provides evidence for a monomeric intermediate trapped during CK refolding due to SH group modification.
- Refolding and activity recovery in the modified CK are synchronized processes.
- The lack of GroEL binding to the partially folded CK, despite exposed hydrophobic surfaces, suggests specific structural requirements for GroEL recognition beyond hydrophobicity.
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