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Protein Science (2006), 15:171-181. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Capture of monomeric refolding intermediate of human muscle creatine kinase

Sen Li1,3, Ji-Hong Bai2,3,4, Yong-Doo Park2 and Hai-Meng Zhou2

1 Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing 100875, P.R. China
2 Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, P.R. China

Reprint requests to: Hai-Meng Zhou, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, P.R. China; e-mail: zhm-dbs{at}tsinghua.edu.cn; fax: +86-10-6277-2245; or Sen Li, Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing 100875, P.R. China; e-mail: lisen{at}bnu.edu.cn; fax: +86-10-5880-7721.

(RECEIVED July 29, 2005; FINAL REVISION September 27, 2005; ACCEPTED October 5, 2005)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
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.

Keywords: human muscle creatine kinase; monomeric refolding intermediate; aggregation; GroEL; {alpha}s-casein

Abbreviations: ANS, 8-anilino-1-naphthalene sulfonate • CK, creatine kinase • GuHCl, guanidine hydrochloride

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051738406.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
One of the defining characteristics of a living system is the ability of even the most intricate of the component molecular structures to self-assemble with precision and fidelity. Uncovering the mechanisms through which such processes take place is one of the grand challenges of modern science (Vendruscolo et al. 2003). The folding of proteins into their compact three-dimensional structures is the most fundamental and universal example of biological self-assembly; understanding this complex process will therefore provide an unique insight into the way in which evolutionary selection has influenced the properties of a molecular system for functional advantages (Radford and Dobson 1999).

Numerous investigations over the past 40 years have focused on the folding and structural determinants that govern how a polypeptide adopts its native structure. Most of our knowledge on protein folding derives from studies on small monomeric proteins (Mathews 1993). However, the majority of native proteins are more complicated in structure, composed of several subunits that in turn consist of domains. The extrapolation of results obtained in the study of small proteins to larger ones is not always appropriate, and it is therefore important to investigate proteins composed of more than one subunit. Folding of many large proteins with several folding domains and/or subunits often shows multiphasic kinetics with fast and slow folding phases, suggesting the presence of either multiple paths or intermediate states in the process (Dobson et al. 1998). For proteins with >100 residues, experiments generally reveal that one (or more) intermediate is significantly populated during the folding process. There has, however, been considerable discussion about the significance of such species; whether they assist the protein to find its correct structure or whether they are traps that inhibit the folding process (Roder and Colon 1997; Khan et al. 2003; Sanchez and Kiefhaber 2003). Regardless of the outcome of this debate, the structural properties of these intermediates should provide important evidence about the folding of these larger proteins.

Creatine kinase (CK, ATP:creatine N-phosphotransferase, EC 2.7.3.2 [EC] ), a member of the subclass of guanidino-kinases, is widely distributed in tissues that require high energy. It catalyzes the reversible phosphoryl transfer from phosphocreatine to ADP and plays an important role in cellular energy metabolism in vertebrates (Watts 1973; Jacobus 1985). Extensive investigations have been carried out to understand the mechanism for the action of CK (Yao et al. 1984; Rosevear et al. 1987; Lin et al. 1994). In addition to its essential physiological functions, CK is a typical model for studying refolding of proteins because of several important properties, such as refolding spontaneously to its native conformation in vitro with restoration of enzymatic activity in the absence of any external assistance, as well as marked conformational changes of tertiary and secondary structure that occur during refolding. It is also a perfect model for studying intermediates during protein folding, as its folding as a larger dimeric protein is more complicated than that of small monomers and involves several intermediates. Recently, the refolding of rabbit muscle creatine kinase was extensively studied and a possible refolding model was proposed. In this model, several refolding intermediates were proposed to exist in the refolding process of CK. However, only one dimeric intermediate could be captured and characterized with the help of molecular chaperones and foldases (PPIases and PDIases) (Bickerstaff et al. 1980; Grossman 1984; Zhou and Tsou 1986; Yang et al. 1997, 1999; Li et al. 2001; Ou et al. 2001; Zhao et al. 2005). The existence of monomeric intermediate still needs to be evaluated. In this study, we investigated the refolding pathway of human muscle CK and found that the refolding of human muscle CK is a multiphasic process including fast and slow folding phases. An aggregation process competes with the proper folding pathway to decrease the levels of activity recovery. One intermediate was captured separately by GroEL and {alpha}s-casein. We studied the properties of this intermediate and proved that this intermediate was a monomer that resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during the refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregation by decreasing the concentration of free monomeric intermediates; whereas binding of {alpha}s-casein with this intermediate induces more aggregation.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Effect of enzyme concentration and temperature on the course of reactivation of guanidine chloride–denatured human muscle CK
It has been shown previously that rabbit muscle CK is completely inactivated by incubation in 6 M guanidine chloride for 2 h. The unfolding of the enzyme molecule is complete in the sense that all its buried SH groups are exposed and both the fluorescence and absorbance change reach completion (Yao et al. 1985). In our experiments, we treated human muscle CK with 6 M guanidine chloride for 1 or 2 h and with 3 M guanidine chloride for 1 h. The enzyme solutions were then diluted 60- and 30-fold, respectively, so that the final guanidine chloride concentration was identical. The results show that when guanidine chloride–denatured human muscle CK was diluted into renaturation buffer, gradual recovery of enzyme activity was observed. The courses of reactivation were the same in all three cases, indicating that human muscle CK had been completely inactivated by treatment with 3 M guanidine chloride for 1 h (data not shown).

Human muscle CK of different concentrations denatured in 3 M guanidine chloride was diluted 30-fold into the renaturation buffer, and the enzyme activity was measured versus time. Figure 1AGo shows that when the enzyme concentration changed between 0.19 and 3.0 µM, the process of reactivation was independent of enzyme concentration, indicating that the association of the monomer had no effect on the reactivation rate of the enzyme molecule.



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  		Figure 1. Study on the course of recovery of human muscle creatine kinase (CK) activity. (A) Effect of enzyme concentration on the course of recovery of human muscle CK activity. The guanidine-denatured enzyme was suitably diluted so that the final guanidine chloride concentration was always 0.1 M, whereas the enzyme concentrations (µM) were 0.19 ({square}), 0.76 (•), 3.0 ({circ}), 4.56 ({triangleup}), and 6.84 ({blacktriangleup}). Aliquots were taken at time intervals for activity measurements. (B) A semilogarithmic plot for the reactivation of human muscle CK at concentration of 3.0 µM. An{infty} in the vertical axis means the activity of human muscle CK measured after refolding for 24 h. In the vertical axis, "A" means the activity measured at any given time during the refolding process. (•) Experimental points; ({blacktriangleup}) points obtained by subtracting the contribution of the slow phase from data shown in the upper curve.

 
Figure 1BGo presents a semilogarithmic plot of the recovery of enzyme activity against time within the first 2 h at 3.0 µM enzyme concentration. It can be seen that the reactivation process contained a fast kinetic phase and a slow kinetic phase. The rate constant of the fast phase reaction was 1.40 ± 0.08 x 10–3 S–1, and the rate constant of the slow phase reaction was 0.58 ± 0.03 x 10–3 S–1.

As shown in Figure 1AGo, at 3.0 µM enzyme concentration, the reactivation yield after 2 h was ~70%. At enzyme concentrations >3.0 µM, the reactivation yield decreased sharply with increasing enzyme concentration. At 6.84 µM enzyme concentration, the reactivation yield after 2 h was only 10%. In addition, aggregation of human muscle CK molecules was observed when the enzyme concentration was >3.0 µM (data not shown).

Figure 2Go shows the effect of low and high temperatures on the course of reactivation of human muscle CK. At temperatures <20°C, an increase in temperature greatly accelerated the reactivation process of human muscle CK, with the final reactivation yield (after 24 h) of human muscle CK remaining unchanged. At temperatures between 20°C and 25°C, the temperature changes had no effect on the human muscle CK reactivation process (Fig. 2AGo). At temperatures >25°C, the final reactivation yield of human muscle CK decreased sharply with the increase in temperature (Fig. 2BGo), with little change in reactivation rate.



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  		Figure 2. Effect of temperature on the course of recovery of human muscle creatine kinase (CK) activity. (A) Effect of low temperature on the course of recovery of human muscle CK activity. The guanidine-denatured human muscle CK was diluted 30-fold into 10 mM Tris-HCl and 2 mM DTT buffer at different temperatures. The human muscle CK concentration was 3.0 µM. The temperatures were 25°C ({triangleup}), 20°C (•), 15°C ({triangledown}), 10°C ({square}), and 4°C ({circ}). (B) Effect of high temperature on the courses of recovery of human muscle CK activity. The temperatures were 20°C (•), 32°C ({triangleup}), 35°C ({triangledown}), and 37°C ({triangleup}). (C) Effect of temperature on aggregation of human muscle CK. The turbidity (apparent absorbance at 450 nm) was measured to show the aggregation extent of human muscle CK during refolding. The guanidine-denaturedenzymewasdiluted30-fold. The final enzyme concentration was 3.0 µM. The temperatures for the curves from bottom to top were 10°C, 25°C, 28°C, 30°C, 35°C, and 37°C.

 
Aggregation (turbidity) of human muscle CK while refolding at different temperatures was then examined by measuring the absorbance at 450 nm. As shown in Figure 2CGo, human muscle CK was prone to aggregation during refolding at temperatures >25°C. Aggregation increased strongly at higher temperature.

Effect of molecular chaperonin GroEL on the course of refolding of human muscle CK
When human muscle CK, denatured in 3 M guanidine chloride, was diluted 30-fold into a 10 mM Tris-HCl and 2 mM DTT buffer containing different concentrations of GroEL, the reactivation yield of human muscle CK decreased with increasing GroEL concentration (Fig. 3AGo). The reactivation yield of human muscle CK decreased from 70% to <5% as the molecular ratio of GroEL tetradecamer versus human muscle CK subunits increased from 0 to 0.5. When the molecular ratio was >0.5, the reactivation yield showed little change with increasing GroEL concentrations.



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  		Figure 3. Effect of GroEL on refolding of human muscle creatine kinase (CK). (A) Effect of GroEL concentration on the reactivation of denatured human muscle CK. The guanidine-denatured human muscle CK was diluted 30-fold into 10 mM Tris-HCl and 2 mM DTT buffer containing different concentrations of GroEL. The human muscle CK concentration was 1.5 µM. The GroEL concentrations (µM) were 0 (•), 0.375 ({square}), 0.75 ({circ}), 1.125 ({triangleup}), 1.5 ({triangledown}), and 2.25 ({blacktriangleup}). (B) Effect of GroEL concentration on the aggregation of human muscle CK upon dilution. Conditions for denaturation in guanidine chloride and refolding were as for Figure 3AGo. Turbidity (apparent absorbance at 450 nm) was measured 10 min after refolding. The human muscle CK concentration was 1.5 µM. The groEL concentrations (µM) were) 0 (1), 0.375 (2), 0.75 (3), 1.125 (4), and 1.5 (5).

 
Figure 3BGo shows the extent of human muscle CK aggregation (turbidity), in the presence of different concentrations of GroEL, measured as absorbance at 450 nm. The results showed that the extent of aggregation decreased with increasing GroEL concentrations in the renaturation buffer.

Samples of human muscle CK refolded in the presence of different GroEL concentrations were analyzed by native polyacrylamide gel electrophoresis (PAGE) (Fig. 4AGo). The results show that GroEL did not bind native human muscle CK; however, it bound human muscle CK in the refolding process. With increasing GroEL concentration, more and more free human muscle CK was bound by GroEL. The results of gel scanning at 595 nm are shown in Figure 4BGo. The content of free human muscle CK decreased linearly with the increase of the molecular ratio of GroEL tetradecamer versus human muscle CK subunits. When the molecular ratio reached 0.5, the content of free human muscle CK decreased to zero, which indicates that all human muscle CK molecules were bound by GroEL. The results above suggest that human muscle CK formed stable complexes with GroEL during the refolding process and that each GroEL tetradecamer bound two human muscle CK subunits.



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  		Figure 4. Analysis of binding between human muscle creatine kinase (CK) and GroEL. (A) Native polyacrylamide gel electrophoresis of human muscle CK, GroEL, and the complexes of GroEL with human muscle CK. Guanidine-denatured human muscle CK was diluted 30-fold in the presence of 0, 0.375, 0.75, 1.125, 1.5, and 1.875 µM GroEL (lanes 4–9, respectively) and then separated on native polyacrylamide gel. The final human muscle CK concentration was 1.5 µM. Lane 1 is for native human muscle CK; lane 2, native GroEL; and lane 3, the mixture of native human muscle CK and GroEL. The native human muscle CK and GroEL concentrations were all 1.5 µM. (B) Gel scanning results of lanes 4–8 in Figure 5AGo. The wavelength was fixed on 595 nm. The molecular ratio of GroEL tetradecamer vs. human muscle CK subunits was recorded on the abscissa. The ratio of the peak area of free human muscle CK un each lane vs. that of native human muscle CK in lane 3 was recorded on the ordinate. (C) Effects of GroEL and GroEL•CK complex on the reactivation of denatured and reduced lysozyme. Reactivation of 200 µM denatured and reduced lysozyme was triggered by dilution to 2 µM in the presence of different concentrations of GroEL (•) or GroEL•CK complex ({blacksquare}).

 
The binding profiles of GroEL with human muscle CK subunits were further examined by checking the ability of their complexes to bind a stoichiometric amount of denatured and reduced lysozyme. The results (Fig. 4CGo) show that unlike GroEL itself, the complexes of GroEL and human muscle CK did not bind denatured and reduced lysozyme, which indicates that both ends of the GroEL tetradecamer were occupied by CK subunits.

The intrinsic tryptophan fluorescence of GroEL-bound human muscle CK was measured to detect its tertiary structure. The GroEL component interferes only minimally with the analysis because it lacks tryptophan residues. The fluorescence of human muscle CK changed markedly as the protein unfolded: The emission maximum shifted from 334 nm to 355 nm, accompanied by an increase in the fluorescence intensity. The emission maximum of refolded human muscle CK was also 334 nm, indicating full renaturation of the unfolded human muscle CK. The emission maximum of GroEL-bound human muscle CK was 341 nm, between the fluorescence maxima of the native and the unfolded proteins (Fig. 5AGo). Apparently, the tryptophan residues in GroEL-bound human muscle CK were already in a more hydrophobic environment than was the completely unfolded protein, as indicated by the 70% blueshift of the emission maximum. The structure of GroEL-bound human muscle CK was less compact than that of native human muscle CK, as indicated by the redshift of 7 nm. The fluorescence emission of ANS (1-anilino-8-naphthalene sulfonate) is known to increase when the dye binds to the hydrophobic regions of a protein (Stryer 1965). Figure 5BGo shows the ANS fluorescence of free and GroEL-bound human muscle CK. The GroEL-stabilized human muscle CK showed strong ANS fluorescence compared with that of the native or urea-unfolded human muscle CK or with the GroEL tetradecamer alone. The results of fluorescence and ANS fluorescence indicate that GroEL stabilizes unfolded human muscle CK as a flexible tertiary structure with an internal hydrophobic core, as in the "molten globule" state.



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  		Figure 5. Trp fluorescence and ANS fluorescence of free and GroEL-bound human muscle creatine kinase (CK). The final human muscle CK concentrations were all 1.5 µM. The concentration of GroEL was 1.5 µM. (A) Trp fluorescence of (1) native human muscle CK, (2) refolded human muscle CK, (3) GroEL-bound human muscle CK refolding intermediate, and (4) human muscle CK unfolded in 3M guanidine chloride. (B) ANS fluorescence of (1) free ANS, (2) human muscle CK unfolded in 3M guanidine chloride, (3) native human muscle CK, (4) GroEL, (5) refolded human muscle CK, (6) the complex of GroEL with native human muscle CK, and (7) GroEL-bound human muscle CK refolding intermediate. The concentration of ANS was 25 µM.

 
Effect of {alpha}s-casein on the course of refolding of human muscle creatine kinase
Figure A shows the changes of the reactivation yields of human muscle CK with the changes of {alpha}s-casein concentration. The reactivation yields decreased as the {alpha}s-casein concentration increased. The existence of {alpha}s-casein also resulted in the aggregation of human muscle CK of low concentration in the refolding process. Figure 6BGo shows the extent of human muscle CK aggregation in the presence of different concentrations of {alpha}s-casein measured as absorbance at 450 nm. The extent of aggregation increased with increasing {alpha}s-casein concentrations.



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  		Figure 6. Effect of {alpha}s-casein on refolding of human muscle creatine kinase (CK). (A) Effect of {alpha}s-casein concentration on the reactivation of denatured human muscle CK. The guanidine-denatured human muscle CK was diluted 30-fold into 10 mM Tris-HCl and 2 mM DTT buffer containing different concentrations of {alpha}s-casein. The human muscle CK concentration was 1.5 µM. The {alpha}s-casein concentrations (µM) were 0 (•), 0.75 ({square}), 1.5 ({blacktriangleup}), 2.25 ({circ}), 3.0 ({triangleup}), and 3.75 ({blacksquare}). (B) Effect of {alpha}s-casein concentration on the aggregation of human muscle CK upon dilution. Conditions for denaturation in guanidine chloride and refolding were as in A. Turbidity (apparent absorbance at 450 nm) was measured 10 min after refolding. The human muscle CK concentration was 1.5 µM. The {alpha}s-casein concentrations (µM) were 0 (1), 0.75 (2), 1.5 (3), 2.25 (4), 3.0 (5), and 3.75 (6).

 
After guanidine-denatured human muscle CK was diluted 30-fold into renaturation buffer containing {alpha}s-casein, the mixed solution was centrifuged to remove the precipitate. The supernatant was then subjected to size-exclusion chromatography. The results of size-exclusion chromatography are shown in Figure 7AGo. By comparing the results with the standard size-exclusion chromatography curves of native human muscle CK and {alpha}s-casein, we found that the molecular weight of the protein in peak I was the same as that of native human muscle CK and that the molecular weight of the protein in peak III was the same as that of {alpha}s-casein. Peak II contained a new component whose molecular weight was between that of native human muscle CK and {alpha}s-casein. Samples in the three peaks were then analyzed by using SDS-PAGE (Fig. 7BGo). The results of SDS-PAGE clearly proved that peak I contained only native human muscle CK and peak III contained only {alpha}s-casein. However, the component in peak II can be separated into human muscle CK monomer and {alpha}s-casein by SDS treatment. Because the molecular weight of this component is less than that of native human muscle CK dimer, as shown by the result in Figure 7AGo, the component is likely composed of one human muscle CK monomer and one {alpha}s-casein molecule. The amount of each protein in lane 5 was quantitated by gel scanning at 595 nm for each band and comparing with purified {alpha}s-casein and human muscle CK standards (data not shown). The estimated molar ratio between the two proteins in lane 5 is 1.12. This result also proves that one {alpha}s-casein molecule binds to one human muscle CK monomer.



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  		Figure 7. Analysis of binding between human muscle creatine kinase (CK) and {alpha}s-casein. (A) Size-exclusion chromatography elution profile of human muscle CK refolded in renaturation buffer containing {alpha}s-casein. The human muscle CK concentration was 1.5 µM. The {alpha}s-casein concentrations was 2.25 µM. (B) SDS-PAGE result of each peak in A. Samples were low-molecular-weight marker, native human muscle CK, {alpha}s-casein, samples in peak I, samples in peak II, and samples in peak III from left to right.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Correct protein folding depends on many factors, such as protein concentration, pH, temperature, ionic strength, and redox environment. In this study, we found that unlike rabbit muscle CK, unfolded human muscle CK only refolded in a reducing environment (in the presence of 2 mM DTT). We think the difference in requirement of a reducing environment is caused by a difference in the protein structure of the two enzymes. We believe that the cysteine side chains of human muscle CK are susceptible to oxidative damage during the refolding process; therefore, human muscle CK must refold in a reducing environment to prevent such damage.

Our results showed that similar to rabbit muscle CK, the reactivation process of human muscle CK in the first 2 h contained a fast kinetic phase and a slow kinetic phase (Fig. 1BGo). In addition, a very slow final phase was also observed; there was a small increase in enzyme activity after 24 h (data not shown). The refolding kinetics of human muscle CK, determined by fluorescence measurement, also confirmed the above findings (data not shown). These results indicate that the refolding process consisted of at least two first-order reactions. Considering there may be very fast phases in the early refolding process that cannot be detected by the normal activity and fluorescence measurements, we propose that the total refolding process can be divided into several stages and that certain intermediates exist in the refolding process.

The results in Figure 1AGo show that when the enzyme concentration was low (<3.0 µM), the changes of concentration had no effect on the reactivation processes. This suggests that the dimerization of monomeric intermediate is not the rate-limiting process in the refolding pathway (Artigues et al. 1997; Fan et al. 1998). However, when the enzyme concentration was high (>3.0 µM), the changes of concentration affected the reactivation processes significantly. The yield of reactivation decreased with increasing human muscle CK concentration, accompanied by detectable aggregation. We conclude that, taken together, aggregation takes place in the folding process of human muscle CK when the enzyme concentration is high (>3.0 µM) and thus partially prevents the recovery of the native structure. Protein aggregation occurs during protein folding in vivo as well as in vitro. Aggregation of refolding intermediates usually competes with the correct folding process as an off-pathway process and thus results in decrease of reactivity (Jeannine 1996). Similar to other proteins, unfolded human muscle CK has two possible fates in the refolding process: It either folds properly to form the active dimer structure or forms aggregates of intermediates. When the enzyme concentration is low during refolding, the probability of intermediate aggregation is very low and most intermediates undergo the proper folding process to form the active enzyme molecules. When the enzyme concentration is high, the probability of intermediate aggregation increases, which decreases the final reactivity of human muscle CK.

The patterns of reactivation of human muscle CK at different temperatures further confirm the existence of aggregation during the refolding process. Usually the change in temperature only affects the reactivation rate by altering the accessibility to activation energy and the solvent viscosity (Jacob and Schmid 1999). Our results showed that at temperatures <25°C, the changes of temperature only affected the reactivation rate of human muscle CK and had no effect on the final reactivation yields after 24 h. However, at temperatures >25°C, the reactivation rate only changed a little but the reactivation yields of human muscle CK decreased with the increase of temperature. The poor yield of active enzyme at higher temperatures was directly proportional to the formation of large aggregated species. Aggregate formation is usually a temperature-dependent process. As shown in Figure 2CGo, aggregation (turbidity of the solution) increased significantly with increasing temperature.

Molecular chaperones have been shown to play an essential role in assisting the folding of nascent peptides to form biologically functional proteins by binding with the folding intermediate to avoid kinetic traps and, consequently, by suppressing aggregation of the substrate (Horwich et al. 1999; Hartl and Hayer-Hartl 2002). GroEL is an important molecular chaperone that has been studied extensively. It is generally accepted that GroEL assists the correct folding of denatured proteins of eukaryotic cells in vitro by preventing aggregation (Buchner et al. 1991; Martin et al. 1991; Guise et al. 1996; Persson et al. 1996; Ranson et al. 1998). In this experiment, we studied the effect of GroEL on the refolding process of human muscle CK to see whether it can prevent aggregation during refolding. The results showed that the existence of GroEL in refolding solutions decreased the reactivation yields of human muscle CK in a concentration-dependent manner. The existence of GroEL also decreased the turbidity of the refolding system, indicating that GroEL prevented aggregation of human muscle CK during refolding process. These results showed that GroEL could act as a protector against aggregation but also acted as an inhibitor of reactivation during CK refolding. One possible explanation is that stable GroEL–CK complexes formed; thus, the reactivation and aggregation yields decreased at the same time. The results of PAGE further confirmed the existence of complexes consisting of GroEL and the refolding intermediates of human muscle CK.

By analyzing the PAGE results and the time course of reactivation for human muscle CK during refolding in solutions containing different concentrations of GroEL, we found that one GroEL tetradecamer can bind with two human muscle CK subunits. Thus two possibilities exist: One GroEL tetradecamer can bind either with one dimeric intermediate or two monomeric intermediates. In the next experiment, we examined the binding profiles of GroEL with human muscle CK intermediate by checking the ability of the complex to bind a stoichiometric amount of denatured and reduced lysozyme (Li and Wang 1999; Song et al. 2000). The complex of GroEL and human muscle CK could not bind denatured and reduced lysozyme, suggesting that GroEL bound with two monomeric folding intermediates to form a stable 1:2 complex with one substrate on each end of the GroEL double rings and left no space for denatured and reduced lysozyme.

The fluorescence and ANS fluorescence of this GroEL-bound intermediate showed that it contained a relatively compact, but flexible, tertiary structure with much hydrophobic surface exposure. These characteristics are also the typical conformational properties of the molten globule in the folding pathway of proteins. Martin et al. (1991) previously reported that the conformation of GroEL-bound polypeptides resembles that of the molten globule or "compact intermediate," an early folding state that rapidly interconverts with the fully unfolded form. It contains all or part of the secondary structure of the protein in conjunction with a relatively compact, but flexible, tertiary structure with an internal "molten" hydrophobic core. The GroEL-stabilized intermediate of human muscle CK satisfies these criteria. Therefore, we suggest that the inactive monomeric folding intermediate that binds with GroEL is a kind of molten globule–like intermediate. These results also suggested that the hydrophobic interaction between GroEL and the monomeric intermediate is the major driving force of the binding.

In the presence of {alpha}s-casein, the reactivation yields of human muscle CK decreased sharply with increasing concentration of {alpha}s-casein. It is clear that the addition of {alpha}s-casein at high concentrations prevents correct refolding of human muscle CK. At the same time, turbidity of the refolding system increased with increasing concentration of {alpha}s-casein, indicating that the existence of {alpha}s-casein in the refolding process of human muscle CK can induce more aggregation. The interaction between {alpha}s-casein and human muscle CK during its refolding was examined by size-exclusion chromatography and SDS-PAGE of the supernatant of the refolding system. The results showed that one {alpha}s-casein molecule could capture one monomeric refolding intermediate of human muscle CK to form a relatively stable complex. Caseins belong to a family of phosphoproteins synthesized in the mammary gland and secreted as large colloidal aggregates, termed micelles (Ginger and Grigor 1999). Caseins, including the {alpha}-, {beta}-, and {kappa}-caseins, were demonstrated to have less ordered secondary and tertiary structures in comparison with typical globular proteins (Swaisgood 1993). Since {alpha}s-casein exposes a great number of hydrophobic residues in water solutions so as to possess a large hydrophobic surface (Waxman and Goldberg 1986; Ostoa-Saloma et al. 1990) and the monomeric intermediate of human muscle CK is a molten globule–like intermediate that also contains much hydrophobic surface, we suggest that {alpha}s-casein binds the monomeric intermediates of human muscle CK primarily through hydrophobic interaction. Interestingly, both as-casein and {beta}-casein have been reported to have chaperone-like activity by preventing protein aggregation induced by thermal as well as nonthermal stresses through hydrophobic interaction with unfolding proteins (Bhattacharyya and Das 1999; Zhang et al. 2005). However, {alpha}s-casein does not serve as a chaperone in our experiments. The possible reason is that the monomeric intermediate of human muscle CK possesses more hydrophobic surface than do the normal unfolding proteins. Therefore, hydrophobic interaction between the monomeric intermediate of human muscle CK and {alpha}s-casein is relatively strong. After forming a complex with {alpha}s-casein, the monomeric intermediate of human muscle CK could not be released easily from the complex for subsequent refolding to the native conformation. As a result, {alpha}s-casein did not facilitate the refolding of human muscle CK. It also did not prevent aggregation of human muscle CK during its refolding process. On the contrary, the existence of {alpha}s-casein induced more aggregation. The mechanism of the {alpha}s-casein–induced aggregation is still unclear and requires further study.

Combining all the results we obtained in this experiment, we propose that the total refolding process of human muscle CK can be divided into stages and that several intermediates are present in this process. When the protein concentration and environment temperature are high, aggregation process competes favorably with the proper folding process. A monomeric refolding intermediate "I" in the early stage of refolding can be captured by the molecular chaperonin GroEL, and the aggregation process is then prevented. {alpha}s-Casein binds with this monomeric refolding intermediate and induces more aggregation. By interpreting these results, we propose that the monomeric refolding intermediates are prone to interact with each other or other proteins inside cells by hydrophobic interactions. Such interactions among the intermediates may be the reason for aggregation of human muscle CK during refolding.

Folding of proteins is a very complicated process. At present, no general model has been proposed to illustrate the folding mechanism of all proteins. Here we studied the reactivation and aggregation of human muscle CK during the refolding process to show some basic principles for folding of this special protein. We also examined the effect of GroEL and {alpha}s-casein on its folding to mimic the complicated interactions among proteins during the process of protein folding inside living cells. A monomeric intermediate was captured and characterized for the first time. Although these observations are too preliminary to extrapolate to in vivo conditions, these findings shed light on the folding mechanism of normal proteins. Further studies of human muscle CK refolding, especially studies of the early stages of the refolding process and the effects of protein folding catalysts and chaperones, will lead to a better understanding of how such proteins fold in their intracellular environment.



   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Materials
Human muscle CK was prepared as described by Ritter et al. (1981). The chaperonin GroEL was purified from the Escherichia coli overproducing strain pOF39-JM101, as described by Li et al. (2001). Purified human muscle CK and GroEL were homogeneous on SDS-PAGE. {alpha}s1-Casein, ANS, and guanidine chloride (ultra pure grade) were obtained from Sigma. Creatine and ATP were purchased from Fluka. DTT was from Promega. All other chemicals were local products of analytical grade.

Determination of protein concentration
The concentrations of GroEL and creatine kinase were determined by the absorbance at 280 nm with coefficients E0.1%1cm = 0.25 (Bochkareva et al. 1992) and E0.1%1cm = 0.88 (Yao et al. 1984), respectively.

Unfolding and refolding of human muscle CK
The enzyme was denatured for 1 h at 25°C in 3 M guanidine chloride dissolved in 10 mM Tris-HCl and 2 mM DTT buffer (pH 8.0). In the refolding studies, the enzyme denatured as described above was diluted 30-fold into renaturation buffer (10 mM Tris-HCl, 2 mM DTT at pH 8.0) without urea for activity measurements. The CK activity measurements were then carried out as described by Yao et al. (1982).

Fluorescence spectra measurements
Fluorescence spectra measurements were made with a Hitachi 850 spectrofluorometer using an excitation wavelength of 295 nm. For the ANS-binding fluorescence spectra, the excitation wavelength was 380 nm.

Size-exclusion chromatography measurement
Size-exclusion chromatography was carried out with a Superdex 200 HR 10/30 column on an Amersham Pharmacia Biotech ÄKTA HPLC system. Each collected fraction was concentrated and used for SDS-PAGE.

PAGE measurements
Native PAGE was carried out on a gel, consisting of 3.75% polyacrylamide for the stacking gel and 6.5% polyacrylamide for the separating gel, at a constant voltage of 160 V for 90 min using a Bio-Rad Mini-PROTEAN cell. The SDS-PAGE was carried out on a gel, consisting of 3.75% polyacrylamide for the stacking gel and 12.5% polyacrylamide for the separating gel, at a constant voltage of 160 V for 60 min. All gels were stained with Coomassie brilliant blue R-250.

Identification of binding between GroEL–CK complexes and lysozyme
The complexes of GroEL–CK were obtained by 30-fold dilution of 45 µM denatured human muscle CK into 10 mM Tris-HCl and 2 mM DTT buffer (pH 8.0) containing 1.5 µM GroEL and incubation for 60 min at 25°C. Binding between GroEL–CK complexes and lysozyme was determined according to the method of Li and Wang (1999) with minor modification.


   Footnotes
 
3 These authors contributed equally to this work. Back

4 Present address: Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Back


   Acknowledgments
 
This work was supported by funds from the China Natural Science Foundation, National Key Basic Research Specific Foundation of China Grants G1999075607 and 30221003, and the 985 Project of Tsinghua University.


   References
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 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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