Class sigma glutathione transferase unfolds via a dimeric and a monomeric intermediate: impact of subunit interface on conformational stability in the superfamily.

Solvent-induced equilibrium unfolding of a homodimeric class sigma glutathione transferase (GSTS1-1, EC 2.5.1.18) was characterized by tryptophan fluorescence, anisotropy, enzyme activity, 8-anilino-1-naphthalenesulfonate (ANS) binding, and circular dichroism. Urea induces a triphasic unfolding transition with evidence for two well-populated thermodynamically stable intermediate states of GSTS1-1. The first unfolding transition is protein concentration independent and involves a change in the subunit tertiary structure yielding a partially active dimeric intermediate (i.e., N2 left and right arrow I2). This is followed by a protein concentration dependent step in which I2 dissociates into compact inactive monomers (M) displaying enhanced hydrophobicity. The third unfolding transition, which is protein concentration independent, involves the complete unfolding of the monomeric state. Increasing NaCl concentrations destabilize N2 and appear to shift the equilibrium toward I2 whereas the stability of the monomeric intermediate M is enhanced. The binding of substrate or product analogue (i.e., glutathione or S-hexylglutathione) to the protein's active site stabilizes the native dimeric state (N2), causing the first two unfolding transitions to shift toward higher urea concentrations. The stability of M was not affected. The data implicate a region at/near the active site in domain I (most likely alpha-helix 2) as being highly unstable/flexible which undergoes local unfolding, resulting initially in I2 formation followed by a disruption in quaternary structure to a monomeric intermediate. The unfolding/refolding pathway is compared with those observed for other cytosolic GSTs and discussed in light of the different structural features at the subunit interfaces, as well as the evolutionary selection of this GST as a lens crystallin.