Structural characterization of acid-induced intermediates of human glutathione transferase P1-1

The acid denaturation of human glutathione transferase P1-1 (hGSTP1-1) has been performed to investigate the unfolding intermediates of the protein an d their possible involvement in the refolding mechanism. The acid-induced s tructures of GSTP1-1 have been characterized by activity, gel filtration, i ntrinsic fluorescence and far-u.v, circular dichroism (CD) techniques. Beca use of the non-identity of the different transitions monitored, the acid de naturation of hGSTP1-1 appears to be a multistep process during which sever al intermediates coexist in equilibrium. The dependence of inactivation on the protein concentration, as well as gel-filtration experiments, indicate that the inactivation transition, centred at about pH 4.0, corresponds to t he monomerization of the protein. At pH 2.0, when the enzyme is completely inactive, the protein retains a small, but significant, amount of secondary structure. This means that the dimeric arrangement of the molecule is impo rtant for maintaining the native-like secondary structure of the monomer. T he results show that, at low pH, the compact state of the GST monomer, even upon the addition of salts, does not possess native-like secondary structu re as described for many monomeric proteins (molten globule). In the presen ce of physiological concentrations of salts, the protein solution at pH 2.0 leads to a dead-end aggregation process, suggesting that this compact stat e cannot represent a productive intermediate of the refolding pathway. (C) 2000 Elsevier Science Ltd. All rights reserved.