Unfolding and refolding of dimeric creatine kinase equilibrium and kineticstudies

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfo lding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilin onaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding c urve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. Thi s intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state mode l indicate that this intermediate is similar in energy to the fully unfolde d state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-fl ow apparatus. Further refolding to the native state after the burst phase f ollows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these i ntermediates are similar in some respects, but different in others. Both ar e characterized by pronounced secondary structure, compact globularity, exp osed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermedia te shows more secondary structure, more exposed hydrophobic surface area, a nd more flexible side-chain packing than the equilibrium intermediate. Foll owing the burst phase, there is a fast phase corresponding to folding of th e monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protei n concentration dependent. The refolding kinetics are also not concentratio n dependent. This suggests that association of the subunits is not rate lim iting for refolding, and that under equilibrium conditions, dissociation oc curs in the region between the two unfolding transitions. Based upon the ab ove results, schemes of unfolding and refolding of creatine kinase are prop osed.