Chemical denaturation and elevated folding temperatures are required for wild‐type activity and stability of recombinant Methanococcus jannaschii 20S proteasome

Abstract

The 20S proteasome from the extreme thermophile Methanococcus jannaschii ( Mj ) was purified and sequenced to facilitate production of the recombinant proteasome in E. coli . The recombinant proteasome remained in solution at a purity level of 80–85% (according to SDS PAGE) following incubation of cell lysates at 70°C. Temperature–activity profiles indicated that the temperature optima of the wild‐type and recombinant enzymes differed substantially, with optimal activities occurring at 119°C and 95°C, respectively. To ameliorate this discrepancy, two recombinant enzyme preparations were produced, each of which included denaturation of the proteasome by 4 M urea followed by high‐temperature (85°C) dialysis. The wild‐type temperature optimum was restored, but only if proteasome subunits were denatured and refolded prior to assembly (a preparation designated as α & β). In contrast, when proteasome assembly preceded denaturation (designated α + β) the optimum temperature was raised to a lesser degree. Moreover, the α & β and α + β preparations had apparent thermal half‐lives at 114°C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild‐type activity and stability thus required the proper folding of both the α‐ and β‐subunits prior to proteasome assembly. Consistent with this behavior, dual‐scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high‐temperature activity.