Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate

The conformational stabilities of two homodimeric class mu glutathione tran sferases (GSTM 1-1 and GSTM2-2) were studied by urea- and guanidinium chlor ide-induced denaturation. Unfolding is reversible and structural changes we re followed with far-ultraviolet circular dichroism, tryptophan fluorescenc e, enzyme activity, chemical cross-linking, and size-exclusion chromatograp hy. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occ ur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2), The seco nd transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transit ion for GSTM2-2, These transitions are dependent upon protein concentration . The first deactivation transition coincides with the first tertiary struc ture transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of th e class Fl glutathione transferases M1-1 and M2-2 proceed via a three-state process: N-2 <-> 21 <-> 2U. Although GSTM1-1 and GSTM2-2 are homologous (7 8% identity/94% homology), their N2 tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lowe r denaturant concentrations than for GSTM2-2, The monomeric intermediate fo r GSTM1-1 is, however, more stable than the intermediate for GSTM2-2, The i ntermediates are catalytically inactive and display nativelike secondary st ructure. Guanidinium chloride-induced denaturation yields monomeric interme diates, which have a more loosely packed tertiary structure displaying enha nced solvent exposure of its tryptophans and enhanced ANS binding. The thre e-state model for the class mu enzymes is in contrast to the equilibrium tw o-state models previously proposed for representatives of classes alpha/pi/ Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than th ose of the other GST classes.