On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor Gr oES cooperate in mediating the ATP-dependent folding of a wide range of pol ypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinde r, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, t hereby preventing protein aggregation during folding. The dynamic interacti on of GroEL and GroES is regulated by the GroEL ATPase and involves the for mation of asymmetrical GroEL:GroES(1) and symmetrical GroEL: GroES(2) compl exes. The proposed role of the symmetrical complex as a catalytic intermedi ate of the chaperonin mechanism has been controversial. It has also been su ggested that the formation of GroEL:GroES(2) complexes allows the folding o f two polypeptide molecules per GroEL reaction cycle, one in each ring of G roEL. By making use of a procedure to stabilize chaperonin complexes by rap id crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES(1) and GroEL:GroES(2) complexes in active ref olding reactions under a variety of conditions using mitochondrial malate d ehydrogenase (mMDH) as a substrate. Our results show that the symmetrical c omplexes are neither required for chaperonin function nor does their presen ce significantly increase the rate of mMDH refolding. In contrast, chaperon in-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES(1) complexes. These findings support the view that GroEL:GroES( 2) complexes have no essential role in the chaperonin mechanism.