Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies

Abstract

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and I-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (1 5 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.Keywords: creatine kinase; dimerization of subunits; folding intermediate; kinetics; molten globule; protein foldingIn spite of the accumulation of a large number of experimental studies, protein folding remains one of the most challenging subjects in structural biology. Characterization of folding intermediates is considered an important strategy for the elucidation of the mechanism of protein folding. A common equilibrium intermediate, the "molten globule" (MG) state, has been detected between the native (N) and the fully unfolded (U) states for many proteins (Fink, 1995; Ptitsyn, 1995). The MG state is characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface, and the absence of rigid side-chain packing

Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase

“…CK (EC 2.7.3.2) contains multiple cysteine residues and has been widely used as the model system to study enzymatic activity and protein folding. [13][14][15][16][17] Using biochemical and biophysical approaches, we found that the activity and stability of the Fc-III-tagged CK are better than those of the His-tagged CK, and the disulfide linkages in the Fc-III-tagged GFP and CK are different from those in the His-tagged GFP and CK. The results herein provided in-depth understanding of the fusion tag and suggested that the Fc-III tag is a useful tool for the expression and purification of reduced cysteinecontaining proteins.…”

Unfolding of Rabbit Muscle Creatine Kinase Induced by Acid

“…Chemical denaturants, such as guanidine hydrochloride and urea, have been widely used in the investigations of the unfolding of CK (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Because there is a direct interaction between a denaturant and a protein, some thermodynamic parameters may reflect protein-denaturant interaction rather than intrinsic parameters of the protein (15).…”

Unfolding kinetics of dimeric creatine kinase measured by stopped-flow small angle X-ray scattering

“…Synchrotron SAXS combined with the stopped-flow technique can be used to measure the kinetic course of protein unfolding from globular to random coil state, provided unfolding occurs on the second to minute time-scale or longer. In many cases, the backbone secondary structure and the rigid tertiary structure are lost simultaneously during protein unfolding [12]. The SAXS method used in combination with other physicochemical techniques [5] is able to throw light on the interrelation between secondary structure formation and globularization during protein folding.…”

“…Compared with the value derived from the integrated SAXS intensity, the rate constant obtained from I(0) is identical, while the value from R g is around 2.75-fold lower. This small difference is probably a reflection of the fact that CK unfolding is a complex process [12][13][14]. The different structural parameters derived from the SAXS data may vary in their sensitivity to the different intermediates populated during unfolding.…”