IRREVERSIBLE THERMAL-DENATURATION OF GLUTATHIONE TRANSFERASE P1-1 - EVIDENCE FOR VARYING STRUCTURAL STABILITY OF DIFFERENT DOMAINS

Glutathione transferases (GSTs) belong to a dimeric multifunctional en zyme family in which each subunit is organized into two domains. Despi te numerous studies, the degree of the structural dependence of GST do mains, as well as the functional significance of interdomain interacti ons, are still unknown. In order to investigate these important aspect s of folding we decided to study the thermal denaturation of GSTP1-1. The protein transitions were followed by monitoring the loss of activi ty, far ultraviolet CD, intrinsic fluorescence and the aggregation sta te of the protein. The results show that thermal denaturation of the e nzyme is a multistep process. This substantially confirms our previous unfolding studies and indicates that the presence of intermediates du ring unfolding of the protein reflects an inherent property of the nat ive structure. Thermal denaturation of GSTP1-1 was essentially irrever sible because of the formation of inactive scrambled structures which were unable to refold spontaneously by lowering the temperature. The f act that the fluorescence changes occur before any other transitions i ndicates that the GST domain I region containing Trp28 and Tryp38 resi dues is relatively more flexible than the molecule as a whole, Moreove r, the present results, together with our previous unfolding and limit ed proteolysis studies on GSTP1-1, support the suggestion that the hig h mobility of the G-site polypeptide portion encompassing the Trp38 re sidue could have a role during catalysis. The present study also indic ates that domain II of GSTP1-1 has a higher intrinsic stability than t he other part of the molecule, including some regions of domain I and the active site. (C) 1998 Elsevier Science Ltd. All rights reserved.