PREVENTION OF IN-VITRO PROTEIN THERMAL AGGREGATION BY THE SULFOLOBUS-SOLFATARICUS CHAPERONIN - EVIDENCE FOR NONEQUIVALENT BINDING SURFACES ON THE CHAPERONIN MOLECULE

We have studied the effects of the Sulfolobus solfataricus chaperonin on the aggregation and inactivation upon heating of four model enzymes : chicken egg white lysozyme (one 14.4-kDa chain), yeast alpha-glucosi dase (one 68.5-kDa chain), chicken liver malic enzyme (four 65-kDa sub units), and yeast alcohol dehydrogenase (four 37.5-kDa subunits). When the proteins were heated in the presence of an equimolar amount of ch aperonin, 1) the aggregation was prevented in all solutions; 2) the in activation profiles of the single-chain enzymes were comparable with t hose detected in the absence of the chaperonin, and enzyme activities were regained in the solutions heated in the presence of the chaperoni n upon ATP hydrolysis (78 and 55% activity regains for lysozyme and al pha-glucosidase, respectively); 3) the inactivation of the tetrameric enzymes was completely prevented, whereas the activities decreased in the absence of the chaperonin. We demonstrate by gel filtration chroma tography that the chaperonin interacted with the structures occurring during thermal denaturation of the model proteins and that the interac tion with the single-chain proteins (but not that with the tetrameric proteins) was reversed upon ATP hydrolysis, The chaperonin had nonequi valent surfaces for the binding of the model proteins upon heating: th e thermal denaturation intermediates of the single-chain proteins shar e Surfaces I, while the thermal denaturation intermediates of the tetr americ proteins share Surfaces II, ATP binding to the chaperonin induc ed a conformation that lacked Surfaces I and carried Surfaces II, Thes e data support the concept that chaperonins protect native proteins ag ainst thermal aggregation by two mechanistically distinct strategies ( an ATP-dependent strategy and an ATP-independent strategy), and provid e the first evidence that a chaperonin molecule bears functionally spe cialized surfaces for the binding of the protein substrates.