Folding pathway for partially folded rabbit muscle creatine kinase.
Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated 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 evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.
Park
Y D
YD
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, China.
Ou
W B
WB
Yu
T W
TW
Zhou
H M
HM
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry / Q000378:metabolism*; D004136:Dinitrobenzenes; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60; D004136:Dinitrobenzenes; C027132:1-anilino-8-naphthalenesulfonate; D003402:Creatine Kinase; C010936:2,4-dinitrothiocyanatobenzene; D019791:Guanidine