Recombinant homo- and hetero-oligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro

The archaeon Pyrodictium occultum is one of the most thermophilic organisms presently known. Previous experiments provided support for the significant contribution of a high-molecular-mass protein complex to the extreme therm otolerance of P. occultum. This protein complex, the 'thermosome', is compo sed of two subunits, a and P, which form a hexadecameric double ring comple x. In order to obtain the thermosome in amounts sufficient for structural a nd functional investigations, we produced the two subunits jointly and sepa rately in Escherichia coli BL21(DE3). In all three cases, we isolated solub le, high-molecular-mass double-ring complexes from E. coli BL21(DE3). On el ectron micrographs, the recombinant complexes were indistinguishable from e ach other and from the natural thermosome. To characterize the quaternary s tructure of the recombinant particles, we used native gel electrophoresis, analytical gel filtration, and analytical ultracentrifugation. Spectral ana lysis, using absorption, fluorescence emission and far-UV circular dichrois m spectroscopy were applied to compare the three recombinant protein comple xes with the natural thermosome from P. occultum. All three recombinant com plex species exhibit ATPase activity. Furthermore, we could demonstrate tha t the recombinant complexes slow down the aggregation of citrate synthase, alcohol dehydrogenase, and insulin. Thus, we conclude that the recombinant protein complexes exhibit a chaperone-like activity, interacting with non-n ative proteins; they do so at temperatures far below the lower physiologica l limit of growth.