Microcalorimetric investigation of the ATPase activity and the refolding activity of GroEL system

GroEL, a member of the Hsp60 class molecular chaperones, has been extensively studied by general molecular biological method not only on its ATPase activities but also on refolding mechanism. Here, a C80 isothermal calorimeter was applied to understand the molecular mechanism of GroEL-assisted refolding. The power–time curves were obtained from different reaction systems, and the integral heat was calculated and analyzed. The ATPase activity of HSP60 was proven to be very stable at high temperature and had a high temperature dependence. In ATP hydrolysis experiments, we found that ATP hydrolysis catalyzed by GroEL was an exothermic reaction, in which divalent cations especially magnesium cation played an important role. And the ATP concentration also influences the ATP hydrolysis rate. Moreover, GroEL can assist in refolding thermodenatured xylanase with the presence of ATP and co-chaperone GroES, and the refolding process was demonstrated to be endothermic reaction, indicating that non-native proteins interacted with hydrophobic apical domains of GroEL, mainly hydrophobic interaction. The thermodynamic parameters presented in this study illustrated the work mechanism of GroEL by microcalorimetry and have important implications for further study of other interaction mechanism.