Equilibrium and kinetic unfolding properties of dimeric human glutathione transferase A1-1.

The equilibrium and kinetic unfolding properties of homodimeric class alpha glutathione transferase (hGST A1-1) were characterized. Urea-induced equilibrium unfolding data were consistent with a folded dimer/unfolded monomer transition. Unfolding kinetics were investigated, using stopped-flow fluorescence, as a function of denaturant concentration (3.5-8.9 M urea) and temperature (10-40 degrees C). The unfolding pathway, monitored by tryptophan fluorescence, was biphasic with a fast unfolding event (millisecond time range with enhanced fluorescence properties) and a slow unfolding event (seconds to minutes time range with quenched fluorescence properties). Both events occurred simultaneously from 3.5 M urea. Each phase displayed single-exponential behavior, consistent with two unimolecular reactions. Urea-dependence studies and thermodynamic activation parameters (transition-state theory) suggest that the transition state for each phase is well-structured and is closely related to native protein in terms of solvent exposure. The apparent activation Gibbs free energy change in the absence of denaturant, DeltaG (H2O), indicates that the slow unfolding event represents the transition state for the overall unfolding pathway. The rate and urea independence of each phase on the initial condition exclude the possibility of a preexisting equilibrium between various native forms in the pretransition baseline. The unfolding pathways monitored by energy transfer to or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding event (described above). A sequential unfolding kinetic mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed.