A topologically conserved aliphatic residue in α-helix 6 stabilizes the hydrophobic core in domain II of glutathione transferases and is a structural determinant for the unfolding pathway

A topologically conserved residue in a-helix 6 of domain II of\ud human glutathione transferase (hGST) A1-1 was mutated to\ud investigate its contribution to protein stability and the unfolding\ud pathway. The replacement of Leu-164 with alanine (L164A) did\ud not impact on the functional and gross structural properties of\ud native hGST A1-1. The wild-type protein unfolds via a threestate\ud pathway in which only folded dimer and unfolded monomer\ud were highly populated at equilibrium; a native-like dimeric\ud intermediate with partially dissociated domains I and II was\ud detected using stopped-¯ow ¯uorescence studies [Wallace, Sluis-\ud Cremer and Dirr (1998) Biochemistry 37, 5320±5328]. In the\ud present study, urea-induced equilibrium unfolding of L164A\ud hGST A1-1 indicated a destabilization of the native state and suggested the presence of a stable dimeric intermediate. The\ud unfolding kinetic pathway for L164A hGST A1-1, like that for\ud the wild type, is biphasic, with a fast and a slow unfolding\ud event; the cavity-forming mutation has a substantially greater\ud effect on the rate of unfolding of the fast event. The equilibrium\ud and kinetic unfolding data for L164A hGST A1-1 suggest that a\ud rapid pre-equilibrium is established between the native dimer\ud and a dimeric intermediate before complete domain and subunit\ud dissociation and unfolding. It is proposed that the topologically\ud conserved bulky residue in a-helix 6 plays a role in specifying and\ud stabilizing the core of domain II and the interface of domains I\ud and II