Evidence for Kinetic Intermediate States during the Refolding of GdnHCl-Denatured MM-Creatine Kinase. Characterization of a Trapped Monomeric Species

Abstract

The kinetics of refolding of guanidinium chloride-denatured rabbit MM-creatine kinase was investigated. Recovery of enzymatic activity is biphasic, depending on the temperature but not on the protein or DTT concentration. Only 45% of the original, active dimeric form is recovered even after several hours of refolding. The reactivation yield is limited by the accumulation of a highly stable but nonproductive monomeric species. The ratio of "correct" to "incorrect" forms depends on the duration of exposure to the denaturant, which may be consistent with the existence of a heterogeneous population of unfolded states with regard to proline isomerization. The first fast reaction observed during renaturation results in the appearance of collapsed monomeric states, displaying features of a pre-molten globule state. These burst species are rapidly transformed into more structured monomers resembling a molten globule state possessing a partially folded C-terminal domain. A proportion of these latter transient intermediates (45%) associates into an active dimer, while the remainder (55%) is trapped by reshuffling in a monomeric dead-end product. Our results strongly indicate that (i) the dimeric state is a prerequisite for the expression of catalytic activity, (ii) the kinetic intermediates of refolding are very similar to those observed during equilibrium unfolding, and (iii) refolding of creatine kinase in these conditions is limited by the accumulation of inactive misfolded nondimerizable monomer.

Catalysis of Creatine Kinase Refolding by Protein Disulfide Isomerase Involves Disulfide Cross-link and Dimer to Tetramer Switch

“…Similar to the results reported previously (19), GdnHCldenatured CK could refold to about 45% of the activity of native CK after being diluted into 30 mM Tris-HCl buffer to a final concentration of 2 M, and nonnative aggregation was observed along with the refolding process (Fig. 1).…”

Towards creatine kinase aggregation due to the cysteine modification at the flexible active site and refolding pathway

“…The muscle type of CK is a good model to use for studying its folding pathways because of several characteristics: (i) it is a dimer that consists of two identical subunits, each with an N-terminal domain with about 100 residues and a C-terminal domain with about 250 residues connected by a long linker [7]; (ii) extensively denatured CK can be renatured spontaneously with restoration of its enzymatic activity in the absence of any external assistance [8]; (iii) its folding pathway is complicated and involves several intermediates [9][10][11][12][13][14]; (iv) conformational changes of the secondary and tertiary structures can be easily measured with monitoring activity changes [12,15]; (v) protein-protein interactions, including molecular chaperones are observed during refolding [16][17][18].…”

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).…”