Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase.
The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.
Zhu
L
L
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 100101, Beijing, People's Republic of China.
Fan
Y X
YX
Zhou
J M
JM
D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014508:Urea / Q000737:chemistry*
D004220:Disulfides; D014508:Urea; D003402:Creatine Kinase; D005990:Glycerol