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Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate
J A Hornby1, J K Luo, J M Stevens
1Protein Structure-Function Research Program, Department of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
Biochemistry
|October 4, 2000
Summary
Glutathione transferase stability was investigated using denaturation studies. Class mu enzymes exhibit a three-state unfolding process, differing from other glutathione transferase classes.
Area of Science:
- Biochemistry
- Protein Chemistry
- Enzymology
Background:
- Glutathione transferases (GSTs) are crucial enzymes involved in detoxification.
- Class mu GSTs, specifically GSTM1-1 and GSTM2-2, are homodimeric enzymes with high homology.
- Understanding their conformational stability is key to comprehending their function and regulation.
Purpose of the Study:
- To investigate and compare the conformational stabilities of GSTM1-1 and GSTM2-2.
- To elucidate the unfolding and refolding pathways of these class mu GSTs.
- To contrast the stability profiles of class mu GSTs with other GST classes.
Main Methods:
- Urea- and guanidinium chloride-induced denaturation.
- Far-ultraviolet circular dichroism spectroscopy.
- Tryptophan fluorescence spectroscopy.
- Enzyme activity assays.
- Chemical cross-linking.
- Size-exclusion chromatography.
Main Results:
- GSTM1-1 and GSTM2-2 exhibit reversible unfolding via a three-state process: N(2) <--> 2I <--> 2U.
- Dimer dissociation precedes secondary structure disruption.
- Tertiary structure changes occur in two distinct transitions, influenced by protein concentration.
- Catalytic activity loss occurs in one or two transitions, dependent on protein concentration.
- Monomeric intermediates are stable, catalytically inactive, and possess native-like secondary structure.
Conclusions:
- Class mu GSTs follow a three-state unfolding model, distinct from the two-state models of other GST classes.
- Despite high homology, GSTM1-1 and GSTM2-2 display differences in tertiary structure and dimer dissociation stability.
- Class mu GST subunits appear intrinsically more stable than those of other GST classes.