Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems

International Journal of Biological Macromolecules 51 (2012) 845–858

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Effects of osmolytes on human brain-type creatine kinase folding in dilute solutions and crowding systems Yong-Qiang Fan a , Jinhyuk Lee b,c , Sangho Oh b , Hong-Jian Liu a , Chang Li d , Yu-Shi Luan a , Jun-Mo Yang e , Hai-Meng Zhou f , Zhi-Rong Lü f,∗ , Yu-Long Wang f,∗∗ a

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, PR China Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea c Department of Bioinformatics, University of Sciences and Technology, Daejeon 305-350, Republic of Korea d School of Life Science, Tsinghua University, Beijing 100084, PR China e Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710, Republic of Korea f Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, PR China b

a r t i c l e

i n f o

Article history: Received 3 July 2012 Received in revised form 26 July 2012 Accepted 27 July 2012 Available online 3 August 2012 Keywords: Human brain-type creatine kinase Macromolecular crowding Protein folding Kinetics Aggregation

a b s t r a c t The effects of osmolytes on the unfolding and refolding process of recombinant human brain-type creatine kinase (rHBCK) were comparatively, quantitatively studied in dilute solutions and macromolecular crowding systems (simulated by 100 g/L polyethylene glycol 2000), respectively. The results showed that the osmolytes, including glycerol, sucrose, dimethylsulfoxide, mannitol, inositol, and xylitol, could both protect the rHBCK from denaturation induced by 0.8 M GdnHCl and aid in the refolding of denaturedrHBCK in macromolecular crowding systems. When we examined the effects of sucrose and xylitol on the parameters of residual activity, reaction kinetics and intrinsic fluorescence of rHBCK during unfolding, it was found that the protecting effects of osmolytes in a macromolecular crowding system were more significant compared with those in a dilute solution, which resulted in more residual activities, protected the conformational changes and greatly decreased the rates of both the fast and slow tracks. Regarding the effects of glycerol, sucrose and mannitol on the denatured-rHBCK refolding parameters of refolding yield, reaction kinetics and aggregation, the results indicated that the osmolytes could alleviate the aggregation of rHBCK during refolding in both dilute solutions and macromolecular crowding systems, and the refolding yields and reaction rates under macromolecular crowding environment could be increased by the addition of osmolytes, though higher yields were obtained in the dilute solution. For further insight, osmolyte docking simulations and rHBCK denaturation were conducted successfully and confirmed our experimental results. The predictions based on the docking simulations suggested that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites on rHBCK, and the higher number of interactions with rHBCK by osmolytes than guanidine may be one of the causes of rHBCK refolding. In brief, the additive effects of the exclusive volume effect from the macromolecular crowding system and the osmophobic effects from the osmolytes resulted in better performance of the osmolytes in a macromolecular crowding system, which also led to a better understanding of protein folding in the intracellular environment. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In vitro folding of proteins has been extensively investigated, usually in dilute solution systems with low protein

Abbreviations: CK, creatine kinase; HBCK, human brain-type creatine kinase; rHBCK, recombinant HBCK; GdnHCl, guanidine hydrochloride; PEG 2000, polyethylene glycol 2000; CT DNA, calf thymus DNA; max , maximum emission wavelength. ∗ Corresponding author. Tel.: +86 573 82582999; fax: +86 573 82570020. ∗∗ Corresponding author. Tel.: +86 573 82582636; fax: +86 573 82582636. E-mail addresses: [email protected] (Z.-R. Lü), [email protected] (Y.-L. Wang). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.07.026

concentrations [1]. However, this environment differs from that encountered within living cells, which contain high concentrations (on the order of 50–400 g/L) of various soluble and insoluble macromolecules, including protein, nucleic acids, ribosomes, and carbohydrates (polysaccharides) [2–4]. The intracellular environment, described as “crowding” or “macromolecular crowding,” has a significant effect on biochemical reactions [5], the mechanism of which is usually explained by the excluded volume effect theory [6]. This theory predicts that estimates of reaction rate and equilibrium prepared using dilute solutions differ by orders of magnitude from estimates based on the same reactions carried out in a macromolecular crowding situation [7]. A variety of macromolecular crowding

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agents that simulate the intracellular environment, including dextran, ficoll, polyethylene glycol, nucleic acid, and different proteins, have been used to aid research on the effects of volume exclusion [2,8,9]. Osmolyte, a “chemical chaperone,” has been proposed to rescue defective proteins and to protect native proteins from proteotoxic intracellular environments [10]. Chemically, almost all the osmolytes used by organisms can be classified into three major groups: polyols (mannitol, glycerol, sorbitol, inositol, pinitol, sugar, and sugar derivatives), free amino acids (glycine, alanine and proline) and their derivatives (taurine and ␤-alanine), and methyl ammonium compounds [11,12]. These osmolytes have effects on the solvent’s affinity for hydrophobic surfaces of enzymes and protein stability and solubility [13]. It is assumed that osmolytes were derived through natural selection [14]. Therefore, there is no reason to expect different osmolytes to have the same mechanism for protecting protein because the relevant environmental stresses vary [15]. Creatine kinase (CK) is an attractive model for studying protein folding, which is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphocreatine to adenosine triphosphate (ATP) [16,17]. Several types of CK, each with its own unique characteristics, have been shown to be expressed in various tissues. The muscle and brain types of CK are the most common, and three different isoenzymes, including CK-MM (the muscle-type homodimer), CK-BB (the brain-type homodimer) and CK-MB (the muscle plus brain-type heterodimer), originate from these two common types [18]. It has been reported that CK is important for the study of many serious diseases, such as Alzheimer’s disease, Parkinson’s disease and psoriasis [19–22]. Therefore, it is quite meaningful to understand the characteristics of CK folding in an intracellular environment. CK is completely unfolded in either 6 M urea or 3 M guanidine hydrochloride (GdnHCl) after 1 h [23] and usually can be reactivated by a 50-fold dilution of the concentrated denatured enzyme solution into the refolding buffer [24]. During the past few decades, the influence of osmolytes on the folding of proteins in dilute solutions, including xylanase [25], aminoacylase [24], rabbit muscle CK [13], and arginine kinase [26], has been well documented. There have also been reports on the effects of macromolecular crowding on the refolding of CK-MM [2,9]. However, the effects of osmolytes on human brain-type creatine kinase (HBCK) in either dilute solutions or macromolecular crowding systems have not been studied. In the present study, we investigated the effects of osmolytes on the folding (unfolding and refolding) of recombinant HBCK (rHBCK) in dilute solutions and macromolecular crowding systems, respectively. When the effects of macromolecular crowding agents on the refolding of denatured-rHBCK were examined, it was reported that polyethylene glycol 2000 (PEG 2000) at a concentration of 100 g/L has a comparatively strong exclusive volume effect [27]. Therefore, 100 g/L PEG 2000 was used as a model crowding agent to stimulate a macromolecular crowding environment in this study. The equilibrium and kinetics of the folding of rHBCK in the presence of osmolytes were quantitatively studied in both dilute solutions and macromolecular crowding systems. In addition, the intrinsic fluorescence spectra of unfolding rHBCK were measured, and the aggregation of rHBCK during refolding was also examined. The goal of this investigation was to develop an understanding of the effects of different osmolytes on the folding of proteins in order to examine the differences of protein folding in dilute solutions and macromolecular crowding systems and also to reinforce the growing appreciation for the need to understand the in vitro cellular process.

2. Materials and methods 2.1. Materials PEG 2000, creatine, magnesium acetate, thymol blue, dithiolthreitol (DTT), and GdnHCl were all purchased from Sigma. The other chemicals were local products of the highest analytical grade. All reagent solutions were prepared in a 10 mM Tris–HCl buffer (pH 8.0). 2.2. Enzymes The HBCK gene has been cloned into the pET21b expression vector, expressed in Escherichia coli BL21 (Promega) and purified according to the previous reports of this laboratory [28]. The purified rHBCK was shown to be homologous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme concentration was determined using the Bradford assay with BSA as the standard protein [29]. 2.3. Unfolding of rHBCK at a low concentration of GdnHCl Inactivation of rHBCK was carried out by incubation of protein dissolved in 10 mM Tris–HCl (pH 8.0) with 0.8 M GdnHCl in the absence and presence of osmolytes in dilute solutions and macromolecular crowding systems at 25 ◦ C for 30 min, respectively. The final concentration of rHBCK was 0.2 mg/mL for most experiments unless otherwise noted. PEG 2000 at a concentration of 100 g/L was introduced to simulate macromolecular crowding for all of the experiments in this study. 2.4. Refolding of fully denatured rHBCK The enzyme was incubated in a 10 mM Tris–HCl buffer (pH 8.0) containing 3 M GdnHCl and 1 mM EDTA at 25 ◦ C for 1 h. Refolding was initiated by a rapid 50-fold dilution of the concentrated, denatured rHBCK solution into the refolding buffer containing 10 mM Tris–HCl buffer (pH 8.0), 1 mM EDTA, 1 mM DTT, and different concentrations of osmolytes in dilute solutions and macromolecular crowding systems, respectively. All refolding experiments were carried out at 25 ◦ C. 2.5. Enzyme activity and aggregation measurements Samples were assayed for enzyme activity at different intervals after initiation of the unfolding and refolding process. The rHBCK activity was measured by the pH-colorimetry method [30]. The substrate was composed of 24 mM creatine, 4 mM ATP, 5 mM magnesium acetate, 0.01% thymol blue, and 5 mM glycine–NaOH (pH 9.0). The reaction system contained 1 mL of substrate and 10 ␮L enzyme solution. Proton generation was followed by monitoring the change in absorbance of the indicator at 597 nm at 25 ◦ C. All enzyme activities were normalized against and expressed as a percentage of 0.2 mg/mL solution of native rHBCK in 10 mM Tris–HCl buffer (pH 8.0). All experiments were carried out using a Helios Gamma spectrophotometer (Thermo Spectronic, England). Aggregation of rHBCK during the refolding in both the absence and presence of osmolytes in dilute solution and a macromolecular crowding system was followed by turbidity measurement at 400 nm using a Helios Gamma spectrophotometer (Thermo Spectronic, England) at 25 ◦ C. The aggregation was also detected by measuring the protein concentration in the supernatant of rHBCK aggregation. Refolding of rHBCK was performed as previously described at 25 ◦ C for 4 h. The supernatants were harvested by centrifugation (13,000 rpm for 30 min at 10 ◦ C) and measured for enzyme concentration and activity. All enzyme concentrations

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were normalized against and expressed as a percentage of supernatant of native rHBCK in the refolding buffer. In all experiments, blanks were subtracted to correct for absorbance of the buffer components (including osmolytes and the crowding agent). 2.6. Intrinsic fluorescence assay The intrinsic fluorescence was measured on a Hitachi F2500 fluorescence spectrophotometer with a 1 cm path-length cuvette; the excitation and emission wavelengths were 280 nm and 300–400 nm, respectively. All the resultant spectra were collected after unfolding for 12 h at 25 ◦ C in a 10 mM Tris–HCl buffer (pH 8.0). The measurements were repeated at least three times. 2.7. Determination of kinetic constants The time-courses of rHBCK denaturation with 0.8 M GdnHCl in the presence or absence of osmolytes in dilute solutions and macromolecular crowding systems were fitted well to first-order biphasic kinetics using OriginLab Origin 8.5 software [31]: A = A1 e−k1 t + (100 − A1 )e−k2 t . Here, A is the activity remaining; A1 , the percentage of denaturation in the fast phase; and k1 and k2 , apparent constants of the fast and the slow phase, respectively. The time-courses of reactivation of fully denatured rHBCK in the presence or absence of osmolytes in dilute solutions and macromolecular crowding systems, respectively, were fitted well to exponential equations for first-order reactions (Y = Ymax (1 − e−kt ) in monophasic and Y = Ymax,1 (1 − e−k1 t ) + Ymax,2 (1 − e−k2 t ) in biphasic kinetics) using OriginLab Origin 8.5 software [2,32]. Here, Y is the refolding yield measured at the time t. For first-order reactions in monophasic kinetics, Ymax represents the maximal refolding yield of rHBCK, and k is the rate constant. For first-order reactions in biphasic kinetics, Ymax,1 and Ymax,2 represent the maximal refolding yields for the fast track and slow track of rHBCK refolding, respectively, while k1 and k2 are the rate constants for the fast track and the slow track, respectively. The limiting value of the enzymatic activity (Alim ) is given by (Ymax,1 + Ymax,2 ). Each kinetic constant was calculated as an average of at least three repetitions. 2.8. Docking simulations of osmolytes and denaturant with rHBCK Autodock Vina [33] was used to perform docking simulations. Several types of osmolytes (glycerol, sucrose, dimethylsulfoxide (DMSO), mannitol, inositol, and xylitol) and denaturant (guanidine) were used as ligands. Their structures were drawn manually and imported to the docking simulation. X-ray crystal structure (PDB ID: 3DRE) was used to model rHBCK. In the docking simulation, we identified pocket sites where the ligands could bind. Each ligand was put in the Calpha positions (total of 89) in the pocket site, and then the docking simulations were performed. The generated ligands were grouped into several clusters. The ligands within 2 A˚ from each other were grouped into the same cluster. In each cluster, we measured the number of structures and the minimal/binding energy. 3. Results 3.1. Effects of osmolytes on the activity of native rHBCK Effects of eight kinds of osmolytes on the activity of native rHBCK were examined in the macromolecular crowding system, respectively, as shown in Fig. 1. Two different concentrations for each osmolyte (except inositol and mannitol) were tested. As a result of the solubility problem, inositol and mannitol were used at concentrations of 200 g/L only. However, the concentrations all reached

Fig. 1. Effects of PEG 2000 and different concentrations of osmolytes on the activity of native rHBCK. The macromolecular crowding system was simulated by 100 g/L PEG 2000.

the concentration upper limit value for each respective osmolyte used in this study. The residual activities of rHBCK were measured after 1 h incubation at 25 ◦ C in both the absence and the presence of osmolytes. The results (Fig. 1) showed that the effects of osmolytes, including proline, glutamine and 400 g/L glycerol, on the activity of native rHBCK were significant compared with the control samples in the absence of osmolytes, while greater than 80% of the activity could be retained for other osmolytes after 1 h incubation. Therefore, osmolytes, including xylitol, DMSO, glycerol (<200 g/L), inositol, sucrose, and mannitol, were employed in the following experiments. 3.2. Effects of osmolytes on rHBCK denaturation in a macromolecular crowding system We examined the effects of osmolytes, including glycerol, sucrose, DMSO, mannitol, inositol and xylitol, on the rHBCK denatured by 0.8 M GdnHCl in a macromolecular crowding system, respectively, as shown in Fig. 2. The results indicated that the inactivation of rHBCK was very fast in the macromolecular crowding system and became almost fully inactivated after denaturing for 30 min. However, the presence of osmolytes protected the rHBCK from denaturing to some extent compared with the control (only 100 g/L PEG 2000 presented) in the absence of osmolytes. Among the six kinds of osmolytes, samples tested in the presence of sucrose (400 g/L) and xylitol (400 g/L) retained more activity than those of the other four osmolytes, resulting in 43% and 24% residual activities, respectively. Therefore, different concentrations of sucrose and xylitol were employed for the kinetics analysis of rHBCK inactivation in the following experiments. 3.3. Effects of osmolytes on inactivation kinetics of rHBCK in a dilute solution and a macromolecular crowding system The time-courses of rHBCK inactivated by 0.8 M GdnHCl that contained 100–400 g/L sucrose or xylitol in dilute solutions and macromolecular crowding systems are given in Figs. 3 and 4, respectively, both of which were fitted well to first-order biphasic kinetics with the kinetic parameters shown in Table 1. The results (Figs. 3 and 4) showed the protective effects of sucrose and xylitol by investigating the kinetics of rHBCK inactivation. In the presence of osmolytes, the residual activities of the GdnHCl-denatured

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Table 1 Kinetic parameters of rHBCK unfolding in the absence and presence of osmolytes in dilute solutions and macromolecular crowding systems, respectively. Kinetic parameters were given by the fitted exponential equations as described in Materials and Methods. The macromolecular crowding system was simulated by 100 g/L PEG 2000. Data are expressed as mean ± S.D. (n = 3). Osmolyte concentration (g/L)

Absent Xylitol 100 200 300 400 Sucrose 100 200 300 400

Dilute solution

Macromolecular crowding system

A1 (%)

k1 (s−1 )

k2 (×10−3 s−1 )

A1 (%)

k1 (s−1 )

k2 (×10−3 s−1 )

49 ± 3

0.61 ± 0.02

20.2 ± 1.4

49 ± 2

3.42 ± 0.54

16.8 ± 0.2

49 48 47 43

± ± ± ±

6 4 6 2

0.84 0.88 1.41 0.78

± ± ± ±

0.02 0.03 0.04 0.02

22.6 20.1 13.9 18.0

± ± ± ±

0.7 1.0 2.6 1.6

49 47 41 38

± ± ± ±

3 1 6 1

1.11 1.3 0.61 0.65

± ± ± ±

0.05 0.02 0.05 0.10

12.1 7.8 14.4 3.6

± ± ± ±

5.0 6.7 2.3 1.3

47 47 40 39

± ± ± ±

1 1 2 2

0.66 0.50 0.45 0.40

± ± ± ±

0.03 0.02 0.02 0.02

15.2 8.7 3.0 1.4

± ± ± ±

2.9 5.5 1.0 0.5

48 43 38 24

± ± ± ±

7 4 9 1

0.74 0. 42 0. 34 0.32

± ± ± ±

0.02 0.01 0.01 0.03

7.9 2.1 1.0 0.4

± ± ± ±

0.8 1.7 0.2 0.1

Fig. 2. Effects of different osmolytes on the inactivation of rHBCK induced by 0.8 M GdnHCl for 30 min at 25 ◦ C in the macromolecular crowding system. The concentrations of glycerol, sucrose, DMSO, and xylitol were 400 g/L, and those for mannitol and inositol were 200 g/L. The macromolecular crowding system was simulated by 100 g/L PEG 2000. Data with error bars are expressed as the mean ± S.D. (n = 3).

rHBCK were higher than those of the controls (without osmolytes in a dilute solution and a macromolecular crowding system); with the increasing concentrations of osmolytes, the extent of rHBCK inactivation became gradually lower. Data in Fig. 3 suggested that the protective effects of sucrose in the macromolecular crowding system were better than those at the same concentration in the dilute solution, and Fig. 4 also supported the same conclusions when we examined the effects of xylitol in a dilute solution and a macromolecular crowding system. The kinetics parameters in Table 1 indicated that the value of A1 decreased as the concentrations of osmolytes were increased, and osmolytes could decrease the inactivation rate constants (k1 and k2 ) to a greater extent in the macromolecular system. Compared with their own controls, the values of k1 and k2 both decreased when xylitol was present in a macromolecular crowding system. In contrast, those values did not change significantly when xylitol was present in a dilute solution at the same concentration. For sucrose, the k1 and k2 values both decreased significantly in the macromolecular crowding system, and only k2 values decreased in dilute solutions. However, compared with their own controls, the presence of 400 g/L sucrose decreased the values of k1 and k2 to 9% and 2% in the macromolecular crowding system, respectively, while those values were 66% and 7% for the dilute solution. These results also suggested that the protective effects of sucrose were

Fig. 3. Kinetic inactivation time courses of rHBCK induced by 0.8 M GdnHCl in the absence and presence of sucrose under dilute solutions and macromolecular crowding systems, respectively. (A) rHBCK was incubated with 0.8 M GdnHCl for 30 min in the absence () and presence of 100 g/L (䊉), 200 g/L (), 300 g/L (), and 400 g/L () sucrose in a dilute solution. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.98476 (), 0.97624 (•), 0.95766 (), 0.98821(), and 0.99846 (), respectively. (B) rHBCK was incubated with 0.8 M GdnHCl for 30 min in the absence () and presence of 100 g/L (), 200 g/L (), 300 g/L (), and 400 g/L (♦) sucrose in a macromolecular crowding system. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.97991 (), 0.97857 (), 0.99706 (), 0.97569 (), and 0.98451 (♦), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The points are the experimental data, and solid curves (—) were obtained from the calculated values of parameters. Data are the means of at least three independent experiments.

better than those of xylitol regardless of whether they occurred in a dilute solution or a macromolecular crowding system. 3.4. Effects of osmolytes on intrinsic fluorescence spectra of unfolding rHBCK in a dilute solution and a macromolecular crowding system The rHBCK was denatured with 0.8 M GdnHCl for 12 h in either a dilute solution or a macromolecular crowding system containing different concentrations of osmolytes. The intrinsic fluorescence

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and the macromolecular crowding system. The peak position for rHBCK unfolded in 0.8 M GdnHCl with increasing xylitol concentration was from 332 to 334.5 nm in the dilute solution and from 332 to 333 nm in the macromolecular crowding system, as shown in Fig. 6. The osmolytes in the macromolecular crowding system were better at preventing conformational changes of partially unfolded rHBCK than those in the dilute solution, especially for xylitol. The results also showed that the osmolyte concentrations played an important role in the rHBCK unfolding process. 3.5. Effects of osmolytes on rHBCK reactivation in a macromolecular crowding system

Fig. 4. Kinetic inactivation time courses of rHBCK induced by 0.8 M GdnHCl in the absence and presence of xylitol under dilute solutions and macromolecular crowding systems, respectively. (A) rHBCK was incubated with 0.8 M GdnHCl for 30 min in the absence () and presence of 100 g/L (䊉), 200 g/L (), 300 g/L (), and 400 g/L () xylitol in a dilute solution. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.98476 (), 0.97602 (•), 0.96466 (), 0.95432 (), and 0.97295 (), respectively. (B) rHBCK was incubated with 0.8 M GdnHCl for 30 min in the absence () and presence of 100 g/L (), 200 g/L (), 300 g/L (), and 400 g/L (♦) xylitol in a macromolecular crowding system. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.97991 (), 0.97715 (), 0.96602 (), 0.95024 (), and 0.93379 (♦), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The points are the experimental data, and solid curves (—) were obtained from the calculated values of parameters. Data are the mean of at least three independent experiments.

spectra were shown in Figs. 5 and 6, and the results indicated that osmolytes could decrease the red shift of the maximum rHBCK’s peak. The results (Fig. 5) showed that, in 0.8 M GdnHCl, the red shift of rHBCK’s maximum peak was from 332 to 337.5 nm in the dilute solution and from 332 to 336.5 nm in the macromolecular crowding system. Increasing sucrose concentration prevented conformational changes of partially unfolded rHBCK so that its peak position was located at 332–335 nm in both the dilute solution

The effects of osmolytes, including glycerol, sucrose, DMSO, mannitol, inositol and xylitol, on the refolding of denaturedrHBCK by 3 M GdnHCl in a macromolecular crowding system were examined, respectively, as shown in Fig. 7. The results indicated that the rHBCK reactivation yields increased compared to selfrenatured rHBCK in the macromolecular crowding system when osmolytes were added to the renaturation system. Among all of the six types of osmolytes, samples in the presence of glycerol (150 g/L) and mannitol (75 g/L) resulted in a greater reactivation yield than those of other four osmolytes, giving 48% and 49% reactivation yields after refolding for 1 h, respectively. In addition, sucrose performed the worst among all of the tested osmolytes. Therefore, glycerol, mannitol and sucrose were employed for the kinetics analysis of rHBCK refolding in the following experiments. 3.6. Effects of osmolytes on refolding kinetics of rHBCK in a dilute solution and a macromolecular crowding system The refolding time-courses of denatured-rHBCK by 3 M GdnHCl that contained glycerol (50–200 g/L), sucrose (50–200 g/L) and mannitol (25–100 g/L) in a dilute solution and a macromolecular crowding system are given in Figs. 8–10, respectively, which were fitted well to first-order reactions with the kinetic parameters shown in Tables 2 and 3. For those in a dilute solution, the refolding of rHBCK in the presence of osmolytes followed the biphasic first-order mechanism except in the case of 100 g/L mannitol (Figs. 8A, 9A and 10A). As shown in Fig. 8B, the reaction adhered to monophasic first-order kinetics when 100 g/L PEG 2000 were used to simulate the macromolecular crowding system. However, the refolding of rHBCK changed from a monophasic to a biphasic first-order reaction when the osmolytes were added into the macromolecular crowding reactivation system. Whether in a dilute solution or a macromolecular crowding system, osmolytes affected the rHBCK reactivation in a

Fig. 5. Intrinsic fluorescence emission spectra of unfolded rHBCK induced by 0.8 M GdnHCl in the absence and presence of sucrose in dilute solutions and macromolecular crowding systems, respectively. (A) Native rHBCK was added to the unfolding buffer containing 0.8 M GdnHCl and 0 g/L (curve 2), 100 g/L (curve 3), 200 g/L (curve 4), 300 g/L (curve 5), and 400 g/L (curve 6) sucrose in a macromolecular crowding system. (B) Native rHBCK was added to the unfolding buffer containing 0.8 M GdnHCl and 0 g/L (curve 2), 100 g/L (curve 3), 200 g/L (curve 4), 300 g/L (curve 5), and 400 g/L (curve 6) sucrose in a dilute solution. The intrinsic fluorescence emission spectrum of native rHBCK is labeled as curve 1. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The inset plots show the maximum wavelength of rHBCK in the presence of sucrose with various concentrations, and the data of the abscissa axis were correlated with the numbers for the curves.

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Fig. 6. The intrinsic fluorescence emission spectra of unfolded rHBCK induced by 0.8 M GdnHCl in the absence and presence of xylitol in dilute solutions and macromolecular crowding systems, respectively. (A) Native rHBCK was added to the unfolding buffer containing 0.8 M GdnHCl and 0 g/L (curve 2), 100 g/L (curve 3), 200 g/L (curve 4), 300 g/L (curve 5), and 400 g/L (curve 6) xylitol in the macromolecular crowding system. (B) Native rHBCK was added to the unfolding buffer containing 0.8 M GdnHCl and 0 g/L (curve 2), 100 g/L (curve 3), 200 g/L (curve 4), 300 g/L (curve 5), and 400 g/L (curve 6) xylitol in a dilute solution. The intrinsic fluorescence emission spectrum of native rHBCK is labeled as curve 1. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The inset plots show the maximum wavelength of rHBCK in the presence of sucrose with various concentrations, and the data of the abscissa axis were correlated with the numbers for the curves.

concentration-dependent manner, though there were some differences between these two systems. For those in the dilute solution, the data in Figs. 8A, 9A and 10A and Table 2 showed that the maximum refolding yields increased by adding low concentrations of

glycerol (<100 g/L), sucrose (<100 g/L) and mannitol (<50 g/L) into the refolding buffer. However, with increasing concentrations of glycerol (>100 g/L), sucrose (>100 g/L) and mannitol (>50 g/L) in the reactivation system, the enzymatic activity gradually decreased.

Fig. 7. Effects of osmolytes with different concentrations on the reactivation of 3 M GdnHCl-denatured rHBCK for 1 h at 25 ◦ C in a macromolecular crowding system. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The data with error bars are expressed as the mean ± S.D. (n = 3).

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Table 2 Kinetic parameters of rHBCK refolding in the absence and presence of osmolytes in dilute solutions. Kinetic parameters were given by the fitted exponential equations as described in Materials and Methods. Data are expressed as mean ± S.D. (n = 3). Osmolyte concentration (g/L)

Ymax,1 (Ymax ) (%)

k1 (k) (×10−3 s−1 )

Ymax,2 (%)

Absent Glycerol 50 100 150 200 Sucrose 50 100 150 200 Mannitol 25 50 75 100a

33 ± 3

11.1 ± 0.2

33 ± 3

a

k2 (×10−3 s−1 ) 3.7 ± 0.8

Ymax,1 + Ymax,2 (%) 66 ± 6

64 23 16 6

± ± ± ±

9 4 9 5

6.0 61.0 24.8 18.6

± ± ± ±

0.3 5.2 3.4 1.0

14 59 39 47

± ± ± ±

7 4 9 6

0.8 3.3 4.2 5.2

± ± ± ±

0.6 0.4 0.2 0.5

78 82 55 53

± ± ± ±

16 8 18 11

19 18 43 29

± ± ± ±

8 3 4 3

17.6 49.9 13.2 21.9

± ± ± ±

3.6 6.7 1.0 1.1

43 62 32 41

± ± ± ±

8 3 4 3

3.2 3.3 1.9 1.8

± ± ± ±

0.2 0.6 0.1 0.2

62 80 75 70

± ± ± ±

16 6 8 6

38 47 7 52

± ± ± ±

5 8 6 2

9.7 6.8 38.7 3.5

± ± ± ±

0.5 0.6 1.9 0.4

28 ± 4 32 ± 7 61 ± 6 –

1.2 ± 0.4 1.9 ± 0.1 5.0 ± 0.5 –

66 ± 9 79 ± 15 68 ± 12 –

Reaction was monophasic.

Table 3 Kinetic parameters of rHBCK refolding in the absence and presence of osmolytes in macromolecular crowding systems. Kinetic parameters were given by the fitted exponential equations as described in Materials and Methods. The macromolecular crowding system was simulated by 100 g/L PEG 2000. Data are expressed as mean ± S.D. (n = 3). Osmolyte concentration (g/L) a

Absent Glycerol 50 100 150 200 Sucrose 50 100 150 200 Mannitol 25 50 75 100 a

Ymax,1 (Ymax ) (%) 19 ± 1

k1 (k) (×10−3 s−1 ) 3.2 ± 0.2

Ymax,2 (%) –

k2 (×10−3 s−1 ) –

Ymax,1 + Ymax,2 (%) –

8 21 17 13

± ± ± ±

4 2 2 2

55.4 24.5 32.9 40.0

± ± ± ±

2.0 1.4 1.4 1.1

29 23 30 26

± ± ± ±

4 2 2 2

4.0 1.9 2.7 4.3

± ± ± ±

0.3 0.2 0.4 0.7

37 44 47 39

± ± ± ±

8 4 4 4

12 8 15 12

± ± ± ±

3 2 2 7

10.0 20.1 13.8 5.6

± ± ± ±

2.4 4.8 0.5 0.1

19 23 19 9

± ± ± ±

1 19 2 6

1.2 1.7 1.6 1.5

± ± ± ±

0.4 0.1 0.8 0.2

31 31 34 21

± ± ± ±

4 21 4 13

17 13 27 15

± ± ± ±

8 2 3 8

6.3 14 9.5 13.6

± ± ± ±

0.1 0.5 0.5 0.2

18 29 23 21

± ± ± ±

7 2 3 8

1.4 1.5 1.1 2.9

± ± ± ±

0.3 0.1 0.4 0.9

35 41 50 36

± ± ± ±

15 4 6 16

Reaction was monophasic.

For those in the macromolecular crowding system, osmolytes at all tested concentrations had the potential to increase the refolding yields compared to that of self-renatured rHBCK in the presence of a crowding agent only. However, glycerol, sucrose and mannitol (with respective concentrations of 150 g/L, 150 g/L and 75 g/L) produced the best results compared with the other three concentrations for each osmolyte. Glycerol and mannitol performed the best among all tested osmolytes in the dilute solution and macromolecular crowding system, increasing the refolding yield by 24% and 163%, respectively, compared to their own controls. Data in Tables 2 and 3 suggest that the effects of osmolytes on the reaction rate are concentration dependent, and we noticed that osmolytes assisted rHBCK refolding with an even higher efficiency under crowding conditions compared to those in dilute solutions. The rate constant of the fast track in a dilute solution was greatly affected by the presence of osmolytes, while the rate constant of the slow track was only slightly changed (Table 2). The rates of the fast track of rHBCK refolding were increased from 11.1 ± 0.2 to 61.0 ± 5.2, 49.9 ± 6.7 and 38.7 ± 1.9 × 10−3 s−1 when glycerol (100 g/L), sucrose (100 g/L) and mannitol (75 g/L), respectively, were added to the dilute solution. For those in the macromolecular crowding system, data in Figs. 8B, 9B and 10B and Table 3 indicated that the reaction rates were accelerated by the presence of osmolytes. For example, the presence of 50 g/L glycerol in a macromolecular crowding system resulted in a maximum fast track rate of 55.4 ± 2.0 × 10−3 s−1 and a slow track rate of 4.0 ± 0.3 × 10−3 s−1

compared with the rate constant of 3.2 ± 0.2 × 10−3 s−1 for the monophasic reaction of rHBCK refolding in the presence of 100 g/L PEG 2000 only. Under the macromolecular crowding condition, the rate differences of the fast track among the three types of osmolytes were significant, whereas those for the rates of the slow track were not.

3.7. Effects of osmolytes on aggregation during rHBCK refolding in a dilute solution and a macromolecular crowding system When denatured rHBCK induced by 3 M GdnHCl was diluted into the buffer at 25 ◦ C, it aggregated immediately. The results in Figs. 11–13 suggest that aggregation was inhibited by the presence of osmolytes in a concentration-dependent manner, whether in a dilute solution or a macromolecular crowding system. Data in Fig. 11A and B show that the aggregation in a macromolecular crowding system was even more serious than that in a dilute solution. The presence of osmolytes alleviated the aggregation, and sucrose showed the best inhibiting effect on the aggregation during rHBCK refolding compared with those of the other two osmolytes. As the sucrose concentration increased from 0 to 200 g/L, the aggregation of rHBCK refolding was decreased from 13% to 2% in a dilute solution and from 68% to 15% in the macromolecular crowding system, respectively (Fig. 12C and D). The results for aggregation obtained from the measurement of the absorbance correlated well

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Fig. 8. Kinetic time courses for refolding of 3 M GdnHCl-denatured rHBCK in the absence and presence of glycerol in dilute solutions and macromolecular crowding systems at 25 ◦ C, respectively. (A) Refolding of denatured-rHBCK in the absence () and presence of 50 g/L (䊉), 100 g/L (), 150 g/L (), and 200 g/L () glycerol in a dilute solution. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.99671 (), 0.99324 (䊉), 0.99848 (), 0.99464 (), and 0.99522 (), respectively. (B) Refolding of denatured-rHBCK in the absence () and presence of 50 g/L (), 100 g/L (), 150 g/L (), and 200 g/L (♦) glycerol in a macromolecular crowding system. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.95761 (), 0.99403 (), 0.9976 (), 0.99878 (), and 0.99927 (♦), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The points are the experimental data, and the solid curves (—) were obtained from the calculated values of parameters. Data are the mean of at least three independent experiments.

with those obtained from measuring the protein concentration in the supernatant of rHBCK aggregation. 3.8. Docking simulations of osmolytes and denaturant with rHBCK We identified the binding sites and the predicted binding energies of the denaturant (Fig. 14) and six osmolytes (Figs. 15 and 16) with rHBCK. In the figures, we tabulated the number of ligand structures in the same groups with the lowest and average energy values. The lowest energy structures from each ligand were superimposed on one rHBCK protein structure (Fig. 17), indicating that the denaturant ligand shared common binding sites with six osmolytes. The deactivation of guanidine may have been blocked by osmolytes because they share common binding sites in rHBCK. The larger number of osmolytes interactions with rHBCK compared to guanidine may be one possible cause of refolding of rHBCK. 4. Discussion CK is an important enzyme in cellular energy metabolism, the folding of which has been studied extensively in vitro under dilute experimental conditions [34–36]. However, the fact that protein folding occurs in a physical environment simulated by crowding agents, including polymers, small molecules and simple sugars,

Fig. 9. Kinetic time courses for refolding of 3 M GdnHCl-denatured rHBCK in the absence and presence of sucrose in dilute solutions and macromolecular crowding systems at 25 ◦ C, respectively. (A) Refolding of denatured-rHBCK in the absence () and presence of 50 g/L (䊉), 100 g/L (), 150 g/L (), and 200 g/L () sucrose in a dilute solution. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.99671 (), 0.99671 (䊉), 0.99905 (), 0.99828 (), and 0.99762 (), respectively. (B) Refolding of denaturedrHBCK in the absence () and presence of 50 g/L (), 100 g/L (), 150 g/L (), and 200 g/L (♦) sucrose in a macromolecular crowding system. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.95761 (), 0.99078 (), 0.98956 (), 0.99549 (), and 0.99354 (♦), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The points are the experimental data, and solid curves (—) were obtained from the calculated values of parameters. Data are the mean of at least three independent experiments.

still remains of great interest for researchers [37,38], especially for the folding of HBCK in crowding environments. Usually, the agents used for mimicking the crowding environments should be inert to the target proteins. BSA, calf thymus DNA, ficoll 70, PEG 2000, and dextran 70 have been used as macromolecular crowding agents in studies on the refolding of CK-MM [2,9]. Recently, PEG 2000 was found to have a strong volume exclusive effect on the folding of rHBCK when we examined the effects of macromolecular crowding, with single or mixed macromolecular crowding agents [27]. Therefore, 100 g/L PEG 2000 were used to simulate the macromolecular crowding system in this study, which is a similar concentration as that found in the cytoplasm. Cellular osmolytes are produced in cells in response to osmotic shock and have an important roles in other functions, including enhancing the degree of crowding in the intracellular medium, balancing intracellular osmotic pressure, compensating perturbation of macromolecules, and acting as chemical chaperones [39]. Therefore, many works related to the effects of osmolytes on protein folding have been reported. As most osmolytes are produced in cells, it is necessary to examine the effects of these osmolytes on the activity of the target protein. In this study, we found that glycerol moieties with high concentrations (400 g/L), namely proline and glutamine, had greater effects on the activity of rHBCK, though they have been used for the study of CK-MM folding [13,40]. These findings indicate that some differences exist among different types

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Fig. 10. Kinetic time courses for refolding of 3 M GdnHCl-denatured rHBCK in the absence and presence of mannitol in dilute solutions and macromolecular crowding systems at 25 ◦ C, respectively. (A) Refolding of denatured-rHBCK in the absence () and presence of 25 g/L (䊉), 50 g/L (), 75 g/L (), and 100 g/L () mannitol in a dilute solution. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.99671 (), 0.99508 (䊉), 0.99951 (), 0.99825 (), and 0.98741 (), respectively. (B) Refolding of denatured-rHBCK in the absence () and presence of 25 g/L (), 50 g/L (), 75 g/L (), and 100 g/L (♦) mannitol in a macromolecular crowding system. The data were fitted well to exponential equations for first-order reactions, and the correlation constants for fittings were 0.95761 (), 0.99354 (), 0.99717 (), 0.99593 (), and 0.9926 (♦), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The points are the experimental data, and solid curves (—) were obtained from the calculated values of parameters. Data are the mean of at least three independent experiments.

of CK, and the crowding agents should be examined carefully to make sure they are inert to the target proteins. It has been reported that osmolytes could prevent proteins from denaturing and contribute to the refolding of denatured protein in vitro [13,41,42] in dilute solutions. However, PEG 2000 has been found to have a protective effect against inactivation of protein induced by a low concentration of GdnHCl (unpublished) and to greatly perturb the refolding of rHBCK [27]. Therefore, osmolytes used for kinetics analysis were selected based upon their performances in the macromolecular crowding system only; we did not consider those in the dilute solution. This study examined the effects of osmolytes, mainly including sucrose and xylitol, on the equilibrium of rHBCK unfolding in dilute solution and macromolecular crowding conditions, respectively. Osmolyte concentrations are very important in rHBCK unfolding. At lower GdnHCl concentrations (0.8 M), osmolytes showed a marked ability to protect rHBCK from denaturing, and the protective effects in the macromolecular crowding system were stronger than those in the dilute solution when osmolytes with the same concentrations were used. The osmolytes could decrease the rates of the slow track (k2 ), but they had little effect on those of the fast track (k1 ) when they were used in dilute solutions. In contrast, in the macromolecular crowding system, osmolytes significantly decreased the values of both k1 and k2 . The multiphase courses of CK inactivation at lower GdnHCl concentrations indicated the presence of

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partly active intermediates during denaturation [43]. Also, it has been shown that osmolytes increase the concentration of GdnHCl required to unfold the phosphoglycerate kinase [44]. In dilute solution, the presence of osmolytes may play an important role in stabilizing some unfolding intermediates, thereby protecting the activity and conformation of rHBCK and affecting the reaction kinetics. According to Minton’s crowding theory [7], by excluding a part of the available volume, macromolecules reduce the conformational entropy, resulting in increases in the free energy of a solution and chemical potential of all molecules present in that solution. The main result is to favor the formation of a state that excludes the smallest volume relative to all other macromolecules present. Though there was also no protecting effect of the macromolecular crowding system in the absence of osmolytes on the denaturation of rHBCK induced by 0.8 M GdnHCl, it can be concluded that more intermediates should exist when rHBCK is unfolded in a macromolecular crowding environment (data not shown). With the increasing concentrations of osmolytes in the macromolecular crowding system, the exclusive volume effect was amplified, and the protecting effects of osmolytes on the unfolding intermediates were also more significant in a crowding environment. Therefore, it can be concluded that the exclusive volume effect of the macromolecular crowding agent and the protective effects of osmolytes with a homogeneous distribution are additive. The additive protecting effects could explain the differences of rHBCK inactivation conducted in a dilute solution and a macromolecular crowding system. In this study, we also investigated the differences of effects of osmolytes on the refolding parameters of denatured-rHBCK in a dilute solution and a macromolecular crowding system, including refolding yield, rates (both fast track and slow track refolding) and amount of aggregation. The results (Figs. 8 and 11) showed that the refolding of rHBCK in a macromolecular crowding system induced more serious aggregation and suppressed its reactivation compared with that in a dilute solution. However, the osmolytes used in these two systems can bate the aggregation and increase the final refolding yields in a concentration-dependent manner. The manner in which newly synthesized amino acids transform themselves into a folded protein depends on both the intrinsic properties of the amino acid sequence and multiple contributing influences from the crowded cellular milieu [45]. Protein molecules, however, all have a finite tendency either to misfold or to fail to maintain a correctly folded status under some circumstances [46]. Therefore, correct folding and misfolding/aggregation is a competitive process during protein folding in intracellular environments. The crowding theory indicates that association reactions are highly favored under crowding conditions. The self-association of monomeric subunits for assembly of dimeric molecules is one kind of association reaction. The macromolecular crowding agents can favor both the correct folding and misfolding of monomeric subunits, which is a competing relationship. Under a macromolecular crowding system simulated by 100 g/L PEG 2000, the effect of aggregation dominates, contributing to the loss of enzymatic activity. With the increasing concentrations of osmolytes in a macromolecular crowding system, the exclusive volume effect formed by macromolecules could be reduced by the osmophobic effects of osmolytes, which could promote the correct folding of protein, even though the degree of crowding for the system would be increased. In addition, the much higher viscosity in the presence of high concentrations of osmolytes likely blocks the interactions between protein molecules, thus preventing the formation of precipitates. It has been proposed that the coating of proteins with a hydration shell around charged or polar groups to prevent self-binding is one possible strategy for suppressing aggregation and for stabilizing proteins, and the osmolytes may enhance their stabilities through rehydration effects [47]. Therefore, osmolytes could increase the refolding yields of rHBCK

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Fig. 11. Effects of glycerol on the aggregation of 3 M GdnHCl-denatured rHBCK during refolding in dilute solutions and macromolecular crowding systems at 25 ◦ C, respectively. (A and B) Aggregation of denatured rHBCK during refolding in the absence and presence of glycerol determined by monitoring the turbidity at 400 nm in a dilute solution (A) and a macromolecular crowding system (B), respectively. (C and D) Aggregation of denatured-rHBCK during refolding in the absence and presence of glycerol detected by measuring the protein concentrations and activities for the supernatants of rHBCK aggregation in a dilute solution (C) and macromolecular a crowding system (D), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The data with error bars indicate the mean ± S.D. (n = 3).

and alleviate its aggregation in these ways in both dilute solutions and macromolecular crowding systems. Osmolytes likely increase the refolding yields to a greater extent in a macromolecular crowding system, although high yields were also obtained in dilute solutions. The refolding yields were not always elevated with increasing concentrations of osmolytes (Figs. 11–13), and this may

be because the osmolytes also stabilize some intermediates with no activity. CK is a homo-dimeric protein, and the refolding model has been well established [48]. According to the model, there are two ratelimiting steps for the refolding of CK: the dimerization of the two monomers and the adjustment of the conformation of a dimeric,

Fig. 12. Effects of sucrose on the aggregation of 3 M GdnHCl-denatured rHBCK during refolding in dilute solutions and macromolecular crowding systems at 25 ◦ C, respectively. (A and B) Aggregation of denatured-rHBCK during refolding in the absence and presence of sucrose determined by monitoring the turbidity at 400 nm in a dilute solution (A) and a macromolecular crowding system (B), respectively. (C and D) Aggregation of denatured-rHBCK during refolding in the absence and presence of sucrose detected by measuring the protein concentrations and activities for the supernatants of rHBCK aggregation in dilute solutions (C) and macromolecular crowding systems (D), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The data with error bars indicate the mean ± S.D. (n = 3).

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Fig. 13. Effects of mannitol on the aggregation of 3 M GdnHCl-denatured rHBCK during refolding in dilute solutions and macromolecular crowding systems at 25 ◦ C, respectively. (A and B) Aggregation of denatured-rHBCK during refolding in the absence and presence of mannitol determined by monitoring the turbidity at 400 nm in a dilute solution (A) and a macromolecular crowding system (B), respectively. (C and D) Aggregation of denatured-rHBCK during refolding in the absence and presence of mannitol detected by measuring the protein concentrations and activities for the supernatants of rHBCK aggregation in dilute solutions (C) and macromolecular crowding systems (D), respectively. The macromolecular crowding system was simulated by 100 g/L PEG 2000. The data with error bars indicate the mean ± S.D. (n = 3).

partially folded intermediate to fold completely. The presences of osmolytes also have an exclusive volume effect, which could favor the dimerization of the two monomers. As chemical chaperones, osmolytes could significantly affect the correct folding. This process may explain why osmolytes accelerated the fast track of the rHBCK refolding in both dilute solutions and macromolecular crowding systems. It also has been reported that osmolytes facilitate the recovery of conformation of CK-BB dimers and stabilize their secondary and tertiary conformations to the native state [13,49]. This explains the present result that they contributed to the slow track of rHBCK refolding as well. As the refolding rates for the fast track and slow track are equal to each other in the presence of high concentrations of mannitol, the refolding of rHBCK could change from biphasic to monophasic first-order reactions in dilute solutions.

Furthermore, to explain why osmolytes could prevent rHBCK from the inactivation induced by GdnHCl and help the refolding of denatured-rHBCK, we conducted docking simulation assays of osmolytes and denaturant with rHBCK. The docking simulations were successful with significant scores for every osmolyte and the denaturant, and they predicted that the deactivation of guanidine may be blocked by osmolytes because they share common binding sites in rHBCK. Among all of the docking simulations for osmolytes, sucrose produced the greatest number of structures and the lowest energy, which indicated that rHBCK might contain multiple sucrose binding sites, and the interaction between sucrose and rHBCK may be stronger than those of other osmolytes. This notion is supported by the fact that sucrose exhibited the strongest protective effect of rHBCK against inactivation induced by 0.8 M GdnHCl. However,

Fig. 14. Binding position of guanidine with rHBCK protein structure. In each inset table, the first row represents the number of structures, and the second and third rows indicate the average energy and the lowest energy (kcal/mol) in the same group, respectively. The best energy cluster is shaded in yellow. In the figure, the ligands in the same group are drawn in the same color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 15. Binding positions of (A) glycerol, (B) mannitol and (C) inositol with rHBCK protein structure. In each inset table, the first row represents the number of structures, and the second and third rows indicate the average energy and the lowest energy (kcal/mol) in the same group, respectively. The best energy cluster is shaded in yellow. In the figure, the ligands in the same group are drawn in the same color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the solution viscosity has to be considered in addition to the interaction between osmolytes and rHBCK. As sucrose produces the highest viscosity of any tested osmolyte with the same concentration and greatly disturbs the dimerization of the two monomers during the refolding of rHBCK, it did not produce a better result when it was used to aid in the refolding of rHBCK. Although factors of macromolecular crowding agents were not considered when we

conducted the docking simulation, it can be concluded from the results that a macromolecular crowding system could enhance the interactions of osmolytes with rHBCK; in other words, the effects of osmolytes and exclusive volume effects could be additive. In conclusion, we have comparatively investigated the effects of osmolytes on the folding (unfolding and refolding) of rHBCK in dilute solutions and macromolecular crowding systems,

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Fig. 16. Binding positions of (A) DMSO, (B) xylitol and (C) sucrose with rHBCK protein structure. In each inset table, the first row represents the number of structures, and the second and third rows indicate the average energy and the lowest energy (kcal/mol) in the same group, respectively. The best energy cluster is shaded in yellow. In the figure, the ligands in the same group are drawn in the same color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

respectively. When we examined the unfolding parameters (residual activity, reaction kinetics and intrinsic fluorescence) of rHBCK induced by 0.8 M GdnHCl in both the absence and presence of osmolytes, we found that the protective effects of osmolytes in macromolecular crowding systems were more significant than

those in dilute solutions, resulting in more residual activities and decreasing the rates of both the fast and slow tracks. In our analysis of denatured rHBCK refolding parameters (refolding yields, reaction kinetics and aggregation) in the absence and presence of osmolytes, the results showed that the osmolytes

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Y.-Q. Fan et al. / International Journal of Biological Macromolecules 51 (2012) 845–858 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

Fig. 17. Best result of each ligand. The lowest energy structure in each ligand is drawn in the corresponding colors. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

[16] [17] [18]

could alleviate the aggregation of rHBCK refolding in both dilute solutions and macromolecular crowding systems and also that the refolding yields and reaction rates in a macromolecular crowding environment could be increased by the addition of osmolytes, although higher yields were obtained in dilute solutions. The docking simulations of osmolytes and denaturant with rHBCK will help us to more clearly and accurately understand the interactions of osmolytes with rHBCK. The prediction made by the docking simulations confirmed our experimental results. As a result of the exclusive volume effect, the mechanisms for folding of rHBCK in these two systems may be different. However, the exclusive volume effect and the effects of chemical chaperones resulted in better osmolyte performance in macromolecular crowding systems, which also led to a better understanding of protein folding in the intracellular environment.

[19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]

Acknowledgments Dr. Zhi-Rong Lü was supported by a grant from the project supported by Zhejiang Provincial Natural Science Foundation of China (Grant No. LY12C05001). Dr. Jun-Mo Yang was supported by a grant of the Korea Health 21 R&D Project (Ministry of Health, Welfare and Family Affairs, Republic of Korea, 01-PJ3-PG6-01GN12-0001) and a grant from Samsung Biomedical Research Institute (GL1-B2-1811). Dr. Jinhyuk Lee was supported by a grant from Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program and the Korean Ministry of Education, Science and Technology (MEST) under grant number 2012R1A1A2002676. Dr. Hai-Meng Zhou was supported by a grant from the project supported by Zhejiang leading team of Science & Technology innovation (Team No. 2010R50019). References [1] Y. Wang, H. He, S. Li, Biochemistry (Moscow) 75 (2010) 648–654. [2] F. Du, Z. Zhou, Z.Y. Mo, J.Z. Shi, J. Chen, Y. Liang, Journal of Molecular Biology 364 (2006) 469–482.

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