Effect of Zn2+ during reactivation and refolding of urea-denatured creatine kinase

The courses of refolding and reactivation of urea-denatured creatine kinase (CK) (ATP:creatine N-phosphotrans-ferase, EC 2.7.3.2) have been studied in the absence and presence of zinc ions. The presence of Zn²⁺ at low concentrations blocks the reactivation and refolding of urea-denatured CK and keeps it in a partially folded state. The partially folded state proved to be a monomeric state which resembles the molten globule state in the CK folding pathway. During refolding in the presence of Zn²⁺ , creatine kinase forms aggregates with the aggregation dependent on zinc concentration and temperature. In the presence of EDTA, the partially folded creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results also suggest that low concentrations of zinc ions might be toxic to some proteins such as creatine kinase by disrupting their proper folding.     

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.     

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I₁ (existing in 1.8–1.4 M urea) and I₂ (existing in 0.8–0.4 M urea). I₁ was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I₂ was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates (I_B). Comparison of the properties of these intermediates suggested that I_B in the kinetic process corresponded to I₁ in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.     

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.     

Effect of Mg2+ during reactivation and refolding of guanidine hydrochloride-denatured creatine kinase

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin. 25B, 1296–1302; Yao et al. (1984), Biochemistry 23, 2740–2744; Yao et al. (1982), Sci. Sin. 25B, 1186–1193]. Under suitable conditions, about 60–70% of the activity can be recovered in the presence of different Mg²⁺ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg²⁺ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg²⁺; however, the rates of reactivation are independent of the Mg²⁺ concentration. In addition, Mg²⁺ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg²⁺ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg²⁺ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations.     

Stable intermediates can be trapped during the reversible refolding of urea-denatured rhodanese

The enzyme rhodanese (EC 2.8.1.1) could be reversibly refolded from urea in the presence of lauryl maltoside, beta-mercaptoethanol, and sodium thiosulfate. The unfolding/folding transition monitored using intrinsic fluorescence was resolved into two two-state transitions with midpoints at 3.6 and 5.0 M urea. The analysis assumed an intermediate with an emission maximum at 345 nm. Monitoring anisotropy of intrinsic fluorescence also gave an asymmetric transition. Activity followed one two-state transition centered at 3.6 M urea with no major change of secondary structure. Without thiosulfate or mercaptoethanol, there was one two-state transition at 5.0 M urea giving a species, in dilute urea, with a fluorescence maximum at 345 nm. This intermediate slowly relaxed toward 335 nm (t1/2 = 85 min) if only thiosulfate was absent but without regaining activity. Subsequent addition of thiosulfate led to a first-order recovery of activity (t1/2 = 75 min). Thus, a possible folding intermediate can be trapped which displays increased access of water and solutes to its fluorescent tryptophans. This intermediate conformer, which is flexible, has considerable secondary structure, is inactive, has exposed hydrophobic surfaces, and requires specific reducing conditions to regain full activity. Refolding probably involves an initial, rapid, hydrophobic collapse with acquisition of secondary structure to form the intermediate, followed by slower adjustment to the native global conformation. Final reactivation requires reduction localized at the active site.     

Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation

The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups. In concentrated urea solutions, the denaturation of the enzyme results in negative peaks at 285 nm with shoulders at 280 and 290 nm and positive peaks at 244 and 302 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission maximum of the enzyme red shifts with increasing intensity in urea solutions of increasing concentrations. At least part of these changes can be attributed to direct effects of urea on the exposed Tyr and Trp residues as shown by experiments with model compounds. The inactivation of this enzyme has been followed and compared with the conformational changes observed during urea denaturation. A marked decrease in enzyme activity is already evident at low urea concentrations before significant conformational changes can be detected by the exposure of hidden SH groups or by ultraviolet absorbance and fluorescence changes. At higher urea concentrations, the enzyme is inactivated at rates 3 orders of magnitude faster than the rates of conformational changes. The above results are in accord with those reported previously for guanidine denaturation of this enzyme [Yao, Q., Hou, L., Zhou, H., & Tsou, C.-L. (1982) Sci. Sin. (Engl. Ed.) 25, 1186-1193] and can best be explained by assuming that the active site of this enzyme is situated near the surface of the enzyme molecule and is sensitive to very slight conformational changes.     

Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: evidence for two equilibrium intermediates in the refolding pathway

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.     

Monomeric creatine kinase aggregation and sodium dodecyl sulfate-cyclodextrin assisted refolding

The monomeric state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a denaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as alpha-, beta-, and gamma-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS-binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.     

Reactivation and refolding of reassociated dimers of rabbit muscle creatine kinase

Creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a good model for studying dissociation and reassociation during unfolding and refolding. This study compares self-reassociated CK dimers and CK dimers that contain hybrid dimers under proper conditions. Creatine kinase forms a monomer when denatured in 6 M urea for 1 h which will very quickly form a dimer when the denaturant is diluted under suitable conditions. After modification by DTNB, CK was denatured in 6 M urea to form a modified CK monomer. Dimerization of this modified subunit of CK occurred upon dilution into a suitable buffer containing DTT. Therefore, three different types of reassociated CK dimers including a hybrid dimer can be made from two different CK monomers in the proper conditions. The CK monomers are from a urea-denatured monomer of DTNB-modified CK and from an unmodified urea dissociated monomer. Equal enzyme concentration ratios of these two monomers were mixed in the presence of urea, then diluted into the proper buffer to form the three types of reassociated CK dimers including the hybrid dimer. Reassociated CK dimers including all three different types recover about 75% activity following a two-phase course (k ₁ = 4.88 × 10^–3 s^–1, k ₂ = 0.68 × 10^–3 s^–1). Intrinsic fluorescence spectra of the three different CK monomers which were dissociated in 6 M urea, dissociated in 6 M urea after DTNB modification, and a mixture of the first two dissociated enzymes were studied in the presence of the denaturant urea. The three monomers had different fluorescence intensities and emission maxima. The intrinsic fluorescence maximum intensity changes of the reassociated CK dimers were also studied. The refolding processes also follow biphasic kinetics (k ₁ = 3.28 × 10^–3 s^–1, k ₂ = 0.11 × 10^–3 s ^–1) after dilution in the proper solutions. Tsou's method [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381–436] was also used to measure the kinetic reactivation rate constants for the different three types of reassociated CK dimers, with different kinetic reactivation rate constants observed for each type. CK dissociation and reassociation schemes are suggested based on the results.     

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.     

Blocking creatine kinase refolding by trace amounts of copper ions

Creatine kinase (CK) is a key enzyme to maintain the energy homeostasis in vertebrate excitable tissues. Due to its importance in cellular energetics, the activity and level of CK are crucial to cellular and body functions. CK is sensitive to oxidative stresses and is thought to be one of the main targets of oxidative modification in neurodegenerative diseases. In this research, we investigated the effect of copper, an essential trace element for all organisms and an inducer of the reactive oxygen species, on CK refolding. It was found that trace amounts of Cu(2+) (3mol eq of Cu(2+)) could efficiently block the refolding of CK. The Cu(2+)-trapped CK could not be reactivated by the addition of EDTA, but could be reactivated by DTT. Spectroscopic experiments suggested that copper ions blocked CK refolding by specifically binding with the monomeric refolding intermediate, which further retarded CK refolding and promoted the formation of off-pathway aggregates. The results herein suggested that Cu(2+)-induced CK dysfunction might be caused not only by the post-translational oxidation, but also by the direct binding of copper ions with the newly-synthesized polypeptides.     

Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies.

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 1-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 (15 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.     

Effects of zinc on creatine kinase: activity changes, conformational changes, and aggregation

The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in a-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37°C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.To whom correspondence should be addressed.     

Effect of osmolytes as folding aids on creatine kinase refolding pathway.

The influence of osmolytes, including dimethysulfoxide, glycine, proline and sucrose, on the refolding and reactivation courses of guanidine-denatured creatine kinase was studied by fluorescence emission spectra, circular dichroism spectra, recovery of enzymatic activity and aggregation. The results showed that low concentrations of dimethysulfoxide (<20%), glycine (<0.5 M), proline (<1 M) and sucrose (<0.75 M) improved the refolding yields of creatine kinase, but high osmolyte concentrations decreased its recovery. Sucrose favored the secondary structural formation of creatine kinase. Proline and sucrose facilitated refolding of the protein to its original conformation, while dimethysulfoxide and proline accelerated the hydrophobic collapse of creatine kinase to a packed protein. During the aggregation of creatine kinase, dimethysulfoxide and sucrose inhibited aggregation of creatine kinase, as did proline, but glycine was unable to inhibit aggregation. These systematic observations further support the suggestion that osmolytes, including low concentrations of dimethysulfoxide, proline or sucrose, possibly play a chaperone role in the refolding of creatine kinase. The results also indicate that sucrose and free amino acids are not only energy substrates and organic components in vivo, but also help correct protein folding.     

Effects of macromolecular crowding on refolding of recombinant human brain-type creatine kinase

In this study, we quantitatively measured the effects of the macromolecular crowding agents, polyethylene glycol 2000 (PEG 2000), dextran 70, and calf thymus DNA (CT DNA), on the refolding and aggregation of recombinant human brain-type creatine kinase (rHBCK) denatured by guanidine hydrochloride (GdnHCl). The results showed that there is more aggregation in the presence of either a single crowding agent or in a mixture of crowding agents than in the absence of crowding agents, especially in the presence of a mixture containing CT DNA and PEG 2000 (or dextran 70). In the presence of high concentrations of PEG 2000 (100 g/L), dextran 70 (100 g/L), and CT DNA (15 g/L), the refolding yield remarkably decreased from 70% to 20%, 52% and 57%, respectively. A remarkable decrease in the refolding yield and rate with mixed crowding agent containing CT DNA and PEG 2000 (or dextran 70) was also observed. In comparison to refolding in the presence of 100 g/L PEG 2000, the refolding yields and rates improved in the presence of a mixture of PEG 2000 and dextran 70. We speculate that the crowding agents can favor both correct folding and misfolding/aggregation of denatured-rHBCK. Though it is not known what combination of crowding agents most accurately reflects the physiological environment within a cell, we believe our study could contribute to the understanding of protein folding and the factors that contribute to proper conformation and function in the intracellular environment.     

Reactivation and refolding of rabbit muscle creatine kinase denatured in 2,2,2-trifluoroethanol solutions

The unfolding and refolding of creatine kinase (ATP:creatine N-phosphotransferase (CK), EC 2.7.3.2) during denaturation and reactivation by trifluoroethanol (TFE) have been studied. Significant aggregation was observed when CK was denatured at TFE concentrations between 10% and 40% (v/v). 50% TFE (v/v) was used to study the denaturation and unfolding of CK. The activity loss of CK was a very quick process, as was the marked conformational changes during denaturation followed by fluorescence emission spectra and far-ultraviolet CD spectra. DTNB modification and size exclusion chromatography were used to find that CK dissociated and was in its monomer state after denaturation with 50% TFE. Reactivation and refolding were observed after 80-fold dilution of the denatured CK into 0.05 M Tris-HCl buffer, pH 8.0. The denatured CK recovered about 38% activity following a two phase course (k(1)=4.82+/-0.41x10(-3) s(-1), k(2)=0.60+/-0.01x10(-3) s(-1)). Intrinsic fluorescence maximum intensity changes showed that the refolding process also followed biphasic kinetics (k(1)=4.34+/-0.27x10(-3) s(-1), k(2)=0.76+/-0.02x10(-3) s(-1)) after dilution into the proper solutions. The far-ultraviolet CD spectra ellipticity changes at 222 nm during the refolding process also showed a two phase course (k(1)=4.50+/-0.07x10(-3) s(-1), k(2)=1.13+/-0.05x10(-3) s(-1)). Our results suggest that TFE can be used as a reversible denaturant like urea and GuHCl. The 50% TFE induced CK denaturation state, which was referred to as the 'TFE state', and the partially refolded CK are compared with the molten globule state. The aggregation caused by TFE during denaturation is also discussed in this paper.