The Impact Of ATPase Activity To The Function Of Chaperonin From Sulfolobus Solfataricus P2

Members of group II chaperonins occur widely in the cytosol of eukaryotes and archaea. They have important physiological functions in promoting protein folding and preventing the aggregation of denatured protein and play important roles in vitro and vivo .As a strain of extreme thermophilic archrean, Sulfolobus solfataricus P2 is belonging to Sulfolobus , Crenarcheaota. Its chaperonins consist of different kinds of subunits, which form two stacked rings with eightfold symmetry. The chaperonins have ATPase activity and don't need co-chaperonin. The gene sequences of this chaperonins have been known. However, the relations between the function of the group II chaperonins and ATPase activity and how ATPase acting on the group II chaperonins have not been reported at home and abroad up to now.In this study ,we based on conservative sequence alignment of Sulfolobus solfataricus P2 chaperonin and other members of group II chaperonins and spatial structure to find sites related to ATPase activity and successfully constructed two P2 chaperonins site-directed mutation by the use of overlap extension PCR technique. The two gene-mutations were respectively constructed in pET-21a(+) vectors, and effectively expressed in BL21(DE3) strain of E. coli. Moreover, the characteristics and functions of the mutative chaperonins were further studied.The reconstructed engineering bacteria harboring chaperonin gene mutations of Sulfolobus solfataricus P2 were induced by IPTG for protein expression. The monomers of chaperon were obtained through dealing with the collected cells by means of sonication, heat treatment at 75°C for 30 minute, ammonium sulfate of 80% saturation salting out, and column chromatography purification of ion on Resource Q . The purified chaperonin monomers were able to automatically assemble and form a typical double-ring structure of octamer chaperonins in the presence of ATP and Mg²⁺. The ATPase activity of two chaperonin mutants were measured through malachite green method at different temperatures and the results show that one mutants lost ATPase activity and the other improved ATPase activity and that the genetic mutation sites we have designed is indeed related to ATPase activity. The GFP (The green fluorescent protein) and CS were used to study the function of promoting acid denatured protein re-folding of the P2 chaperonin wildtype and mutant-type. The results showed that the chaperonin played a dramatic role in both promoting the refolding of denatured GFP, and increasing the thermal stability of CS in an ATPase-dependent manner.This study shows that the aspartate 104 and phenylalanine 418 of the chaperonin gene from Sulfolobus solfataricus P2 are closely related with the site of ATPase activity and their mutants can cause the ATPase activity changes. The mutant of higher ATPase activity can better promote the renaturation of denatured proteins and prevent protein aggregation of the heat. The mutant of ATPase activity loss had no affect on protein aggregation and thermal renaturation . ATPase is necessary to chaperonin function of correctly folding and it laid a good foundation for the further study the mechanism of the process of groud II chaperonin .