Unfolding and refolding of Escherichia coli chaperonin GroES is expressed by a three-state model

The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding chara cteristics of the co-chaperonin GroES from Escherichia coli, a homo-heptame r of subunit molecular mass 10,000 Da, were studied by using intrinsic fluo rescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusi on HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at arou nd 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HP LC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescen t probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fl uorescence showed a single transition curve with a transition midpoint at 0 .5 M Gdn-HCl. This unfolding transition curve as well as the refolding kine tics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the ext ent of inhibition of GroEL ATPase activity and the enhancement of lactate d ehydrogenase refolding yields in the presence of GroEL and ADP. These resul ts clearly demonstrated that the GroES heptamer first dissociated to monome rs and then unfolded completely upon increasing the concentration of Gdn-HC l, and that both transitions were reversible. From the thermodynamic analys is of the dissociation reaction, it was found that the partially folded mon omer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits. (C) 1999 Academic Press .