Irreversible thermal denaturation of glutathione transferase P1-1. Evidence for varying structural stability of different domains

Abstract

Glutathione transferases (GSTs) belong to a dimeric multifunctional enzyme family in which each subunit is organized into two domains. Despite numerous studies, the degree of the structural dependence of GST domains, as well as the functional significance of interdomain interactions, are still unknown. In order to investigate these important aspects of folding we decided to study the thermal denaturation of GSTP1-1. The protein transitions were followed by monitoring the loss of activity, far ultraviolet CD, intrinsic fluorescence and the aggregation state of the protein. The results show that thermal denaturation of the enzyme is a multistep process. This substantially confirms our previous unfolding studies and indicates that the presence of intermediates during unfolding of the protein reflects an inherent property of the native structure. Thermal denaturation of GSTP1-1 was essentially irreversible because of the formation of inactive scrambled structures which were unable to refold spontaneously by lowering the temperature. The fact that the fluorescence changes occur before any other transitions indicates that the GST domain I region containing Trp28 and Tryp38 residues is relatively more flexible than the molecule as a whole. Moreover, the present results, together with our previous unfolding and limited proteolysis studies on GSTP1-1, support the suggestion that the high mobility of the G-site polypeptide portion encompassing the Trp38 residue could have a role during catalysis. The present study also indicates that domain II of GSTP1-1 has a higher intrinsic stability than the other part of the molecule, including some regions of domain I and the active site.