THE CHAPERONIN FROM THE ARCHAEON SULFOLOBUS-SOLFATARICUS PROMOTES CORRECT REFOLDING AND PREVENTS THERMAL-DENATURATION IN-VITRO

We have isolated a chaperonin from the hyperthermophilic archaeon Sulf olobus solfataricus based on its ability to inhibit the spontaneous re folding at 50 degrees C of dimeric S. solfataricus malic enzyme. The c haperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium -dependent ATPase activity with an optimum temperature at 80 degrees C . S. solfataricus chaperonin promotes correct refoldings of several gu anidine hydrochloride-denatured enzymes from thermophilic and mesophil ic sources. At a molar ratio of chaperonin oligomer to single polypept ide chain of 1:1, S. solfataricus chaperonin completely inhibits spont aneous refoldings and suppresses aggregation upon dilution of the dena turant; refoldings resume upon ATP hydrolysis, with yields of active m olecules and rates of folding notably higher than in spontaneous proce sses. S. solfataricus chaperonin prevents the irreversible inactivatio ns at 90 degrees C of several thermophilic enzymes by the binding of t he denaturation intermediate; the time-courses of inactivations are un affected and most activity is regained upon hydrolysis of ATP. S. solf ataricus chaperonin completely prevents the formation of aggregates du ring thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity. Tryptophan fluorescence measu rements provide evidence that S. solfataricus chaperonin undergoes a d ramatic conformational rearrangement in the presence of ATP/Mg, and th at the hydrolysis of ATP is not required for the conformational change . The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modific ation of the substrate binding sites. This is the first archaeal chape ronin whose involvement in protein folding has been demonstrated.