Folding pathway for partially folded rabbit muscle creatine kinase

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrob enzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to pro duce a partially folded state with modified thiol groups. The partially fol ded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsi c fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calcu lated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to e valuate whether it can bind with partly folded CK during refolding, and 1-a nilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with Gro EL, although it had a larger area of hydrophobic surface relative to the na tive state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with pr eviously reported results that GroEL bound with substrate proteins mainly t hrough hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refol ding of CK. Schemes are given for explaining both the partial folding CK pa thway and the refolding pathway.