Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species.

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.

High salt concentrations induce dissociation of dimeric rabbit muscle creatine kinase. Physico-chemical characterization of the monomeric species.

Incubation of dimeric MM-creatine kinase (MM-CK) with high NaCl or LiCl concentrations results in dissociation of the subunits and complete enzyme inactivation. In NaCl, this process, which depends on protein concentration, may be described according to a two-state model where the dimer can be reversibly converted into compact folded monomers (D <--> 2M). At LiCl concentrations higher than 2-2.5 M, MM-CK is recovered in two monomeric states: an inactive compact species (M) and a more expanded form (EF), which represents 15-20% of the population. Thus, in LiCl, a three-state model (D <--> 2M --> 2EF) more adequately accounts for our experimental results. The monomeric species (M) obtained in NaCl and LiCl exhibits some properties of the molten globule state described in guanidine hydrochloride. Indeed, this form is compact and devoid of any enzymatic activity; it maintains a high degree of secondary structure and binds 8-anilino-1-naphthalenesulfonate. The formation of this intermediate induces the exposure of a second tryptophan (among the four present) which is located at the monomer-monomer interface in the native structure. In LiCl, the monomeric species (M) is irreversibly converted into a less compact form (EF) which seems to have lost a large part of its secondary structure.

Involvement of a tyrosine residue in the ADP binding site of creatine kinase. A second-derivative UV-spectroscopy study.

Second-derivative spectroscopy was used to determine the percentage of tyrosine residues that are exposed to solvent in rabbit MM-creatine kinase. Six residues, among the ten present per monomer, are solvent-exposed. The presence of creatine in the incubation medium does not modify this value. However, this number is decreased by one when the enzyme is incubated with saturating concentrations of MgADP. A dissociation constant for MgADP can be estimated and the obtained value (0.085 mM) is comparable to the Km for this substrate. Thus, a tyrosine residue is located near the MgADP binding site or is masked during protein conformational change induced by adenyl nucleotide binding.

Denaturation by guanidinium chloride of dimeric MM-creatine kinase and its proteinase K-nicked form: evidence for a multiple-step process.

Cytosolic MM-creatine kinase is a homodimeric protein. Each monomer can be cleaved by proteinase K at an exposed surface loop, into two fragments K1 and K2, which remain associated. The nicked protein is thus a heterotetrameric protein, named (K1K2)2, made up of two heterodimers K1K2 linked together by their K1 subunit. In non-denaturing conditions, the cleaved protein does not present any measurable difference compared with uncleaved MM-creatine kinase, except for the loss of enzymatic activity. Comparative equilibrium denaturation of the two oligomeric proteins by guanidinium chloride indicates a multistep process with formation of either compact monomer or compact K1K2 dimer, a molten globule and a pre-molten globule state. In the case of the nicked-enzyme, the molten globule is composed of the two peptides K1 and K2, whereas in the pre-molten globule the interactions between K1 and K2 are too weak to maintain their cohesion. At low guanidinium chloride concentration, the proteinase K-nicked protein exhibits a higher accessibility of one of its tryptophan accompanied by a small decrease in its molar ellipticity suggesting a secondary structure loosening of the K1 peptide. Our results suggest that K1 and K2 are not strictly autonomous unfolding units and thus cannot be considered as independent domains.

Proteinase K processing of rabbit muscle creatine kinase.

Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328-K380). However, the C-terminal end of the K1 fragment is not A327 as expected, but D325. Thus, the amino acids residues T326 and A327 have been eliminated by the protease.

Refolding of SDS- and thermally denatured MM-creatine kinase using cyclodextrins.

We have tried to refold thermally-denatured MM-CK using detergent and cyclodextrins as protein folding assistants. This procedure, named artificial chaperone-assisted refolding, has been extensively tested to refold carbonic anhydrase B. Here, we describe a study which shows that this procedure can be applied to refold a dimeric multidomain protein : MM-creatine kinase. The pair SDS/hydroxy-propyl beta-cyclodextrin was used in this sequential refolding method. In the first step, the protein was denatured by SDS which is able to strongly inhibit aggregation. In the second step, hydroxy-propyl beta-cyclodextrin, an efficient SDS-stripping agent, is added and the denatured enzyme can regain its native structure as shown by the 75% reactivation. In conclusion, this study suggests that this procedure can be widely used to refold monomeric, as well as oligomeric, multidomain proteins.

Denaturation of MM-creatine kinase by sodium dodecyl sulfate.

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm x 11 nm). These results are compatible with recent models of SDS-protein complexes: the "protein decorated micelle structure" or the "necklace structure".

Variations of muscle mitochondrial creatine kinase activity in mitochondrial diseases.

Mitochondrial creatine kinase (mtCK) activity has been measured in the mitochondria isolated from the muscle of 69 patients suspected of mitochondrial diseases. The isolated mitochondria did not contain significant amounts of the muscle isoform of creatine kinase, as checked by an immunoassay performed after electrophoretic separation of the various isoforms. Hence, the enzyme assay reliably represented the mtCK activity. Therefore, a simple measurement of CK activity in isolated mitochondria permitted the measurement of mtCK activity. An absence of mtCK activity in muscle was never observed. The lowest activities were not associated to defined mitochondrial diseases linked to defects of respiratory chain complexes or to defects of citric cycle enzymes. On the contrary, mtCK activity was significantly increased in the muscle of patients exhibiting ragged red fibers. This increase was generally associated to an increase of citrate synthase activity. Since ragged-red fibers and elevated mtCK activities were generally not found in children younger than 3 years, even in cases of characteristic oxidative phosphorylation deficiency, it is suggested that the increase in mtCK activity as well as the appearance of ragged-red fibers are not the first events which occur during the evolution of mitochondrial diseases but would rather be long-term secondary processes which slowly develop in deficient mitochondria.

Effects of pH and KCl on the conformations of creatine kinase from rabbit muscle. Infrared, circular dichroic and fluorescence studies.

The activity loss of creatine kinase (CK), observed at low pH (midpoint was 4.8) corresponded to the monomerization of the dimeric protein and was correlated with structural changes. The acid-induced unfolding was not complete at this pH, as probed by circular dichroic (CD) and fluorescence methods. Further decrease of pH, led to a second transition (midpoint was pH 3.5). The loss of activity was irreversible at pH 4.8 (< 20% native activity was recovered) while it was almost fully reversible (> 90% of native activity was recovered) for the enzyme incubated at pH 0.9-2.5. The amount of intermolecular beta-sheets (monitored with the 1620 cm-1 infrared component band) was maximal when the enzyme was incubated at pH 4.8, as a consequence of protein aggregation, while it was minimal at extremes of pH and at low ionic strength. Acid-induced and alkaline-induced denaturations promoted different structural changes, leading to distinct partially unfolded conformational states. The addition of KCl (from 0.05 M to 0.5 M) to an acidic solution of monomeric creatine kinase (pH 1.6) resulted in a highly cooperative transition from the partially unfolded conformation (UA) to the more compact conformation (A) with the properties of a molten globule, as probed by CD spectra and by fluorescence. The formation of intermolecular beta-sheets in the compact conformation was observed by infrared spectroscopy, indicating formation of unstable aggregates.

Reversible dissociation and unfolding of dimeric creatine kinase isoenzyme MM in guanidine hydrochloride and urea.

The unfolding of dimeric cytoplasmic creatine kinase (MM) by guanidine hydrochloride and by urea has been investigated using activity measurements, far-ultraviolet circular dichroism, sedimentation velocity and fluorescence energy transfer experiments to monitor global structural changes. Intrinsic (cysteine and tryptophan residues) and extrinsic probes (1-anilinonaphthalene-8-sulfonate) were also used. The reversibility of the unfolding was checked by monitoring activity and tryptophan fluorescence. The unfolding of creatine kinase in guanidine hydrochloride is a reversible multistep process, as suggested by the non-coincidence of denaturation curves at equilibrium. Inactivation of the dimer precedes its dissociation into two monomers and an intermediate state was identified during the unfolding of the monomer. This intermediate state is characterized by a relatively high degree of secondary structure (as shown by far-ultraviolet circular dichroism), of compactness (as shown by fluorescence energy transfer measurements and sedimentation experiments), a fluctuating tertiary structure (as shown by near-ultraviolet circular dichroism) and a strong affinity for anilinonaphthalene sulfonate (as demonstrated by fluorescence). These results clearly indicate that the intermediate state detected possesses some of the properties of a molten globule. In urea, the unfolding pathway is reversible but differs from that observed in guanidine hydrochloride. Indeed inactivation, dissociation and loss of tertiary structure are coincident but the ellipticity curve is slightly shifted to a higher urea concentration. The dimer is dissociated into two expanded monomers possessing some secondary structure which is progressively lost at a higher urea concentration (6.4M). These results show that guanidine hydrochloride is approximately six times more effective than urea for inactivation and dissociation, underlining the fact that electrostatic interactions are very important in the stabilization of the active site and of the dimeric state.

Creatine kinase compactness and thiol accessibility during sodium dodecyl sulfate denaturation estimated by resonance energy transfer and 2-nitro-5-thiocyanobenzoic acid cleavage.

We have investigated the effect of increasing sodium dodecyl sulfate (SDS) concentrations on rabbit muscle cytosolic creatine kinase structure by two methods. We have first determined the variation of accessibility of the thiol groups of the enzyme during SDS denaturation by a technique which involves an irreversible chemical modification of CK accessible thiol groups, followed by NTCB cleavage before the unmodified cysteines in 8 M urea (pH 9) and analysis of the peptides obtained by resolutive gel electrophoresis, without sequencing. We have determined that the order of accessibility of CK MM cysteine residues during SDS denaturation is Cys-282, Cys-145 and then Cys-253. The fourth cysteine residue, Cys-73, is never titrated even at high SDS/CK molar ratio. In contrast, the three last residues are simultaneously titrated when CK is denatured in guanidinium chloride. Thus, SDS-denatured CK seems to retain some residual organized structure. In order to confirm this hypothesis, compactness of the molecule was estimated by fluorescence energy transfer between CK tryptophans and AEDANS, an extrinsic fluorophore. The location of this fluorophore on the accessible thiol of Cys-282 was verified by the previous technique. The results of these experiments do indicate that SDS-denatured CK is more compact than CK completely unfolded in guanidinium chloride.