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Equilibrium Folding of Dimeric Class μ Glutathione Transferases Involves a Stable Monomeric Intermediate†

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Protein Structure−Function Research Program, Department of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa, and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Cite this: Biochemistry 2000, 39, 40, 12336–12344
Publication Date (Web):September 13, 2000
https://doi.org/10.1021/bi000176d
Copyright © 2000 American Chemical Society
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    Abstract

    The conformational stabilities of two homodimeric class μ glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class μ glutathione transferases M1-1 and M2-2 proceed via a three-state process:  N2 ↔ 2I ↔ 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N2 tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class μ enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes α/π/Sj26 GSTs. Class μ subunits appear to be intrinsically more stable than those of the other GST classes.

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    †

     This work was supported by the University of the Witwatersrand, the South African National Research Foundation, the Fogarty International Collaboration Award TW00779, Grant GM30910 from the National Institutes of Health, and the Alexander von Humboldt Foundation.

    ‡

     University of the Witwatersrand.

    §

     Vanderbilt University School of Medicine.

    *

     To whom correspondence should be addressed:  e-mail [email protected]; fax +27 11 403 1733; phone +27 11 716 2265.

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