Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase Subhankar Paul 1,2 , Shashikala Punam 1 and Tapan K. Chaudhuri 1 1 Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, India 2 Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, India The protein folding problem remains one of the key unsolved mysteries in biology [1,2]. Despite huge research exercises and much recent advancement, it is still unclear exactly how a disordered polypeptide chain spontaneously folds into a uniquely structured, biologically active protein molecule [3–6]. In his work on the refolding of ribonuclease, Anfin- sen [7] concluded that the unique tertiary structure of a protein is determined by its amino acid sequence, and that the protein recovers its complete native struc- ture when the denaturing stress is withdrawn, indicat- ing that the unfolding and refolding of proteins is a reversible phenomenon. The general validity of this conclusion was later proved to be wrong as a number of proteins, such as subtilisin E [8], a-lytic protease [9] and carboxypeptidase Y [10], failed to refold correctly from the unfolded state. During refolding, many pro- teins formed aggregates of misfolded proteins whereas Keywords chemical chaperone-assisted refolding; GroEL; GroES; MalZ; protein aggregation Correspondence S. Paul, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela 769008, India Fax: +91 661 2462999 Tel: +91 661 2462284 E-mail: subhankar_paul@rediffmail.com, spaul@nitrkl.ac.in T. K. Chaudhuri, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Fax: +91 11 2658 2282 Tel: +91 11 2659 1012 E-mail: tapan@dbeb.iitd.ac.in (Received 4 August 2007, revised 27 Sep- tember 2007, accepted 1 October 2007) doi:10.1111/j.1742-4658.2007.06122.x In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escher- ichia coli protein, was studied in the presence of glycerol, dimethylsulfox- ide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glyc- erol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemi- cal chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemi- cal chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL ⁄ GroES-assisted refolding of denatured protein, the yield of cor- rectly folded protein from glycerol- or proline-assisted spontaneous refold- ing process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and mole- cular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chap- erones, or the combination of both chaperones, could be used in the effi- cient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance. Abbreviations EG, ethylene glycol; GdnHCl, guanidine hydrochloride; MalZ, maltodextrin glucosidase; TMAO, trimethylamine-N-oxide. 6000 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS some refolded to a non-native conformation [11,12]. The conformation of the refolded protein depends not only on the nature of the unfolding and refolding con- ditions, but also on the molecular mass of the protein. Low molecular mass proteins showed a tendency to fold reversibly [13]. For comparatively high molecular weight proteins, misfolding and aggregation is a commonly observed phenomenon in their folding pathways [14–17]. Efficient refolding of proteins and prevention of their aggrega- tion during folding has a huge importance in recombi- nant protein production and in finding cures for several genetic disorders. Correct folding in vitro or in vivo competes with unproductive side reactions such as mis- folding or aggregation [18]. Aggregation of proteins during folding, both in vitro and in vivo, is known to lead to poor native protein yields as well as the onset of several age-related diseases [19]. Hence, there is a grow- ing interest in developing strategies to prevent protein aggregation for enhancing protein–refolding yields and designing new drugs countering many protein-misfold- ing diseases. Several attempts have been made in this direction with successes as well as failures [20–22]. In vitro, osmolytes, such as the small molecules beta- ine, proline, trehalose, glycerol, dimethylsulfoxide, trimethylamine-N-oxide (TMAO) and ethylene glycol (EG), have been reported to protect native proteins from heat denaturation and favor the formation of native protein oligomers [23–29]. They serve as stabiliz- ers of proteins and cell components against the dena- turing effect of ionic strength. Furthermore, these osmolytes behave as ‘chemical chaperones’ by promot- ing the correct refolding of proteins in vitro and in the cell and by protecting native proteins from heat dena- turation. Some osmolytes behave as chemical chaper- ones by promoting the correct folding of unfolded protein in vitro and in vivo [26,27,30–32]; for example, proline behaves as a protein folding chaperone [26]. In the present study, we investigated the effect of a few osmolytes ⁄ chemical chaperones, such as glycerol, dimethylsulfoxide, TMAO, trehalose, EG and proline, on the refolding of a Escherichia coli protein maltodex- trin glucosidase (MalZ), a 69 kDa monomeric protein responsible for the degradation of maltodextrins to maltose by eliminating one glucose residue from the reducing end at each time. We also aimed to compare the efficiency of osmolyte-mediated refolding with the most popular cellular chaperones, GroEL and GroES, in the assisted refolding of MalZ in vitro. It has recently been reported that, in the presence of ATP, GroEL and GroES assist the folding of aggregation prone recombinant MalZ in vivo and the same study also demonstrated that, when MalZ was refolded from a guanidine hydrochloride (GdnHCl) denatured state, GroEL, GroES and ATP together recovered the MalZ activity substantially [33] and significantly increased the refolding yield of MalZ. The present study demon- strates that all the osmolytes ⁄ chemical chaperones, with the exception of EG, enhance the extent of refold- ing of MalZ significantly over the spontaneous yield and the recovery of folded MalZ in glycerol and pro- line-assisted refolding was comparable to the GroEL ⁄ GroES-assisted recovery of MalZ. It is also demon- strated that chemical chaperones ⁄ osmolytes protected MalZ from thermal denaturation, as well as denatur- ation induced by chaotropic agents such as urea. It is further observed that the combined application of chemical chaperones and the molecular chaperones GroEL ⁄ GroES yielded a higher recovery of refolded MalZ than the yield obtained through their individual assistance. Results Deactivation of MalZ by urea Denaturation of proteins using high concentrations of chaotropic agents (e.g. GdnHCl or urea) is a well known technique that is normally used to study the unfolding of proteins. Such chemically induced dena- turation of proteins results in a gradual loss of their secondary and tertiary structures. In the present study, 8 m urea and 20 mm dithiothreitol in 20 mm sodium phosphate buffer solution pH 7.0 was used to denature MalZ to ensure the complete denaturation of the enzyme. Deanturation of MalZ was monitored by the complete loss of MalZ activity (Fig. 1A) and the loss of relative tryptophan fluorescence intensity (Fig. 1B) with increasing concentration of urea. Effect of protein concentration on refolding yield For in vitro refolding of MalZ, correct folding com- petes kinetically with misfolding as well as aggregation. Unproductive aggregation may primarily originate from hydrophobic interactions of unfolded polypeptide chains as second or higher order processes. Aggrega- tion of MalZ is concentration dependent as observed from the experimental results (Fig. 2). It has been reported previously that the refolding yield of many proteins depends on the protein concentration [34,35]. It was observed that there was a drastic reduction in the spontaneous refolding yield of MalZ with increased concentration of the protein, with negligible refolding observed beyond the MalZ concentration of 50 lgÆmL )1 (Fig. 2). S. Paul et al. Chaperone-assisted folding of maltodextrin glucosidase FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 6001 Folding of MalZ When urea denatured MalZ was diluted into 20 mm sodium phosphate buffer, pH 7.0, the recovery of refolded protein was almost negligible; however, the extent of refolding was enhanced in the presence of reducing agent dithiothreitol in the dilution buffer at a specific concentration. Subsequently, the yield of refolded protein was found to be significantly increased by the addition of MgCl 2 in the refolding buffer. The optimal concentration of the reductant dithiothreitol was found to be 5 mm (Fig. 3B) and that of the Mg +2 ion was 1 mm (Fig. 3A). Dilution of the denatured MalZ into the renaturing buffer containing these two inorganic cofactors restored approximately 10% of the activity of the recombinant enzyme maltodextrin glucosidase. Table 1 shows the effect of concentration of various osmolytes (i.e. glycerol, dimethylsulfoxide, TMAO, tre- halose, EG and proline in the concentration range 1–9 m), depending on their solubility, on the refolding yield of MalZ at 30 °C. All osmolytes led to an initial increase in the refolding yield, followed by a decrease at higher concentrations, but EG, which is known as a destabilizer of protein conformation, showed almost no improvement in the refolding yield of MalZ over the spontaneous yield, with a maximum refolding yield of 12% at 2 m concentration. For dimethylsulfoxide, there was a sharp decline in the yield of refolding beyond 3 m, with very little refolding being obtained at 8 m (Table 1). Glycerol, which has long been known to stabilize the native structure of proteins against chemical and thermal denaturation, also led to a grad- ual increase in the refolding yield, and a very high refolding yield of 61% was obtained at 2 m, followed by a decrease of the yield. It was not possible to use glycerol at concentrations higher than 8 m due to high viscosity of the solutions. Effect of temperature on the refolding yield To observe the effect of temperature on the refolding yield in the chemical as well as molecular chaperone- 0 20 40 60 80 100 A B [Urea] 0 100 200 300 400 500 600 700 [Urea] 02468 02468 Relative fluotrescence intensity of MalZ Relative MalZ activity Fig. 1. Unfolding of MalZ (5 lM) was carried out by the addition of urea at pH 7.0 in 20 m M sodium phosphate buffer containing 20 m M dithiothreitol. (A) MalZ samples were incubated for 4 h at 30 °C with different concentrations of urea in the range 0–8 M. MalZ enzymatic activity with increasing concentration of urea was measured and plotted against the concentration of urea. Percent- age of residual activity was expressed relative to activity obtained from same amount of native protein. (B) MalZ samples were incu- bated for 4 h at 30 °Cin8 M urea and in 20 mM dithiothreitol. Dif- ferent concentrations of urea were used for equilibrium unfolding of MalZ. Relative intrinsic fluorescence emission was measured at 346 nm for all the samples at the k Ex max 279 nm. The excitation and emission band pass were 5 nm and 7.5 nm, respectively, and the scan rate was 60 nmÆmin )1 . 0 102030405060 0 5 10 15 20 25 30 % Relative activity of MalZ MalZ conc. µg/µL Fig. 2. Effect of protein concentration on the spontaneous refolding yield of MalZ at 30 °C. Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al. 6002 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS assisted in vitro refolding of MalZ, the protein was denatured by 8 m urea and incubated for 4 h at room temperature. Refolding reactions were carried out at various temperatures (10, 15, 20, 25, 30, 35 and 40 °C) by diluting the denatured protein solution with 20 mm sodium phosphate buffer, pH 7.0, containing respective chemical chaperones or GroEL ⁄ GroES ⁄ ATP. The spontaneous refolding was measured to be approxi- mately 13% at 25 °C (Table 2). Contrary to spontane- ous refolding, chemical chaperones and E. coli molecular chaperones GroEL ⁄ GroES increased the refolding yield of MalZ to a better extent. Glycerol, proline and GroEL ⁄ GroES particularly increased the recovery of active MalZ significantly at higher tempera- ture. Although GroEL ⁄ GroES-mediated recovery of folded MalZ at a temperature of 40 °C was found to be highest (approximately 66%), the yield of folded MalZ, assisted by glycerol and proline, was also close to the recovery obtained by GroELS-assisted refolding (Table 2). Although the spontaneous refolding of MalZ was maximum at 20 °C (approximately 16%), this yield was much less than the chemical chaperones or GroEL ⁄ GroES assisted refolding yield under the same conditions. The spontaneous refolding yield was reduced drastically with increasing temperature and, at 40 °C, it was only 3%. Osmolyte-induced protection of MalZ activity in vitro To examine how various cosolvents protect the native structure of MalZ, an increasing concentration of urea, in the range 0–8 m, was used to denature MalZ and the denaturation was monitored by measuring the loss of biological activity of MalZ. Optimized concentration of different chemical chaperones in the denaturation mixture and, in every case, their effect on protecting the biological activity of the protein was monitored. The control refolding experiment of MalZ was car- ried out in absence of any osmolyte. It was observed Table 1. The effect of different concentrations (M) of chemical chaperones on the refolding yield of MalZ at 30 °C. The data are an average of at least three independent observations with a maximum percentage error of ± 5%. Chaperone concentration (M) Refolding yield of MalZ (%) in presence of following chemical chaperones Glycerol Dimethylsulfoxide TMAO Trehalose EG Proline 0111111111111 1362924421258 2614530331131 3423549221128 4323240211026 525283816921 6152122–518 7111518–517 86118––9 9––7––6 0 2 4 6 8 10 12 14 A B [MgCl 2 ] (m M ) 0 2 4 6 8 10 12 14 0246810 % Refolding yield of MalZ % Refolding yields of MalZ [dithiothreitol] (mM) 0 2 4 6 8 10 12 14 16 Fig. 3. Determination of the optimum concentrations of dithiothrei- tol and MgCl 2 for the in vitro refolding of MalZ. (A) The denatured solution of MalZ in 8 M urea was diluted 100-fold into 20 mM sodium phosphate, pH 7.0, containing various concentrations of MgCl 2 . (B) The renaturation was carried out with 1 mM MgCl 2 and various concentrations of dithiothreitol. The enzyme solutions were incubated at room temperature for 4 h, and the activity assay was performed as described by Tapio et al. [41]. S. Paul et al. Chaperone-assisted folding of maltodextrin glucosidase FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 6003 that all the polyols protected the MalZ from denatur- ation and deactivation to certain extent (Fig. 4). Pro- line and glycerol, among all the osmolytes, exhibited the highest degree of protection to the MalZ activity. For example, at 4 m urea, when the activity of MalZ is almost negligible, glycerol and proline helped the pro- tein to retain approximately 40% of its initial activity. Although dimethylsulfoxide, TMAO, trehalose and EG have lower protection ability than glycerol and proline, they provided a fair level of protection towards the MalZ activity. Chemical chaperones enhance the refolding yield of MalZ in vitro MalZ was denatured by 8 m urea and dithiothreitol and incubated at 30 °C for 4 h. Urea denatured MalZ was refolded by 100-fold dilution with refolding buffer (20 mm sodium phosphate buffer, pH 7.0, containing 5mm dithiothreitol and 1 mm MgCl 2 ). Chemical chap- erone-mediated refolding was carried out by diluting denatured MalZ with the refolding buffer containing the desired concentration of various chemical chaper- ones. To monitor spontaneous refolding, denatured MalZ (10 lm) was diluted directly into refolding buffer (20 mm sodium phosphate, pH 7.0, containing 5 mm dithiothreitol and 1 mm MgCl 2 ). Molecular chaperone- assisted refolding was carried out by diluting the dena- tured MalZ with the refolding buffer containing GroEL (in such a manner that the final concentration of MalZ and GroEL was 0.1 lm in the solution). After 10 min of incubation at 30 °C, ATP (5 mm final con- centration) and GroES (0.2 lm final concentration) were added to the refolding mixture. The negative con- trol for all these experiments comprise of the buffer containing 0.08 m urea, which corresponds to the residual concentration of urea in the refolding mixture, and the positive control was the buffer containing 0.1 lm native MalZ protein and 0.08 m urea. Refold- ing mixtures were withdrawn at different time intervals and MalZ activity was assayed for different samples at different time intervals up to 10 h. The percentage recovery of MalZ activity in different samples was calculated after considering the equivalent amount of native MalZ activity as 100%. The spontaneous refolding of MalZ was approxi- mately 11% (Table 3). When the refolding reaction was carried out in the presence of various chemical chaperones, a significant enhancement of refolding yield was observed over the spontaneous refolding yield, with EG being the exception. For all cases, there was a gradual increase of refolding yield with time and, after 5 h of refolding, no increase in yield was observed. The use of glycerol and proline resulted in the most significant improvement of refolding recovery over the spontaneous one (61% and 58% refolding yield, respectively). However, the common trend for 02468 0 20 40 60 80 100 [urea] ( M) % Relative MalZ activity Fig. 4. Deactivation of MalZ (5 lM) by increasing concentration of urea in the presence of an optimized concentration of TMAO (.), glycerol (m), dimethylsulfoxide (d), trehalose (r), EG (+) or proline (·), or in absence of any osmolytes (j), at 30 °C. Table 2. Percent of refolding compared to native MalZ, which was treated in the same way as the denatured protein at the respective tem- peratures. Temperature (°C) Spontaneous The effect of different concentrations ( M) of chemical chaperones on the percentage refolding yield of MalZ at 30 °C Dimethylsulfoxide Glycerol TMAO Trehalose EG Proline GroEL ⁄ GroES 10 09 17 22 18 15 07 20 21 15 15 21 25 23 21 10 26 35 20 16 28 37 29 25 11 31 44 25 13 42 44 44 35 12 47 52 30 11 45 61 49 42 14 58 55 35 9 51 65 50 42 15 68 58 40 3 51 62 50 40 12 64 66 Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al. 6004 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS chemical chaperone-mediated refolding was found to be slower than the GroEL ⁄ GroES mediated refolding process. By contrast, GroEL and GroES-assisted refolding was faster, reaching the maximum refolding yield (55%) within 1 h, with no further enhancement in refolding recovery being observed after that (Table 3). For example, the maximum recovery of MalZ activity in the chemical chaperone-mediated pro- cess (proline) was only 18% after 15 min, whereas GroELS-assisted refolding produced a recovery of 34% after the same refolding period (Table 3). Combined application of chemical and molecular chaperones provides higher refolding of MalZ The combined effect of chemical and molecular chap- erones on the refolding yield of MalZ over their indi- vidual contribution was also investigated. It was observed that their joint application produced a rela- tively higher yield of refolded MalZ for all cases, except the combination of EG and GroELS. It has already been demonstrated that chemical as well as molecular chaperones assist individually in the refolding of MalZ in vitro by preventing its aggrega- tion, and produce a substantial amount of the physio- logically active form of the protein. Urea-denatured MalZ was allowed to refold in the presence of chemi- cal chaperones, GroEL, GroES and ATP and the refolding was monitored by withdrawing the refolding mixtures at different time intervals followed by a MalZ activity assay. The recovered MalZ activity was com- pared with the control where 0.08 m urea was present in native MalZ sample. The percentage of refolding of MalZ was calculated and the result was compared with the extent of refolding achieved from the processes mediated by both types of chaperones (Fig. 5). In each case, it was observed that the recovery of refolded MalZ was substantial and that the GroEL⁄ GroES- assisted refolding and recovery at the earlier stage of dilution was relatively higher (Table 4). By contrast, chemical chaperone based refolding was initially slow and increased gradually (Table 3). When the yield of osmolyte-assisted refolding of MalZ was analyzed after 5 h of refolding, it was observed that the highest incre- ment of recovery was achieved in the presence of dimethylsulfoxide when used together with GroEL and GroES (Fig. 6). Glycerol, TMAO and proline also enhanced the recovery of MalZ to a good extent when used in the presence of GroEL and GroES. However, EG, as an exception, could not improve the recovery of folded MalZ even in the presence of GroEL and GroES. Table 3. The dependence of the refolding yield of MalZ from the urea-denatured state on refolding time in the presence of an optimized concentration of osmolytes at 30 °C. Time (min) Spontaneous Chaperone(s) used Glycerol Dimethylsulfoxide TMAO Trehalose EG Proline GroELS 109 2 6 5 4 089 15 15 11 12 10 11 14 09 18 34 30 11 23 25 20 26 09 37 44 60 11 37 34 34 33 10 45 55 120 11 40 39 42 37 12 50 55 300 11 52 42 44 41 12 55 55 600 11 61 45 49 42 12 58 55 1200 11 61 45 49 42 12 58 55 0 10 20 30 40 50 60 70 80 % Refolding Yield of MalZ ELS -+ -+ -+ -+ -+ -+ MalZ ++ ++ ++ ++ ++ ++ ++ -+ Glycerol ++ dimethylsulfoxide ++ TMAO ++ Trehalose ++ Proline ++ EG ++ Fig. 5. Histogram showing the reconstitution of urea denatured MalZ (0.1 l M) after 5 h of refolding in the presence of chemical or molecular chaperone as well as in the presence of both chaper- ones. The extent of refolding was expressed as the percent of activity recovered compared to the same amount of native MalZ. Refolding was initiated by 100-fold dilution of denatured MalZ into refolding buffer (20 m M sodium phosphate, pH 7.0, 1 mM MgCl 2 , 5m M dithiothreitol) containing the respective chemical or molecular chaperone system, as well as both chaperones. S. Paul et al. Chaperone-assisted folding of maltodextrin glucosidase FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 6005 Chemical chaperones protect MalZ from denaturation and irreversible aggregation during thermal stress MalZ loses its native conformation and undergoes aggregation during incubation at 42 °C. We investi- gated whether osmolytes such as glycerol and dimeth- ylsulfoxide could protect MalZ against thermal aggregation in vitro. The native MalZ was incubated at 42 °C in the absence or presence of optimized concentrations of chemical chaperones and its activity was measured at different time intervals. It was observed that the chemical chaperones reduced the aggregation of MalZ efficiently (Fig. 7). The highest degree of thermoprotection of MalZ was achieved throughout the whole incubation period in the presence of 1 m of proline, which can be best com- pared with glycerol-assisted protection because glycerol also protected the denaturation of MalZ significantly (Fig. 7). All of the chemical chaperones that were used in the studies, with the exception of EG, protected MalZ from thermal stress to a different extent. Discussion Six different chemical chaperones (glycerol, dimethyl- sulfoxide, TMAO, EG, trehalose and proline) were studied to investigate whether they assist in the refolding of a 69 kDa E. coli monomeric protein maltodextrin glucosidase. It was previoudly reported that GroEL, in the presence of its cochaperonin GroES, assists in the folding of MalZ in vivo and, in the same study, it was also demonstrated that GroEL, GroES and ATP together led to the sub- stantial recovery of folded MalZ upon refolding from a GdnHCl denatured state in vitro [33]. In the Table 4. The effect on refolding of MalZ by the combined application of chemical chaperones and cellular chaperones when protein was unfolded by 8 M urea at 30 °C. Time (min) Spontaneous Chaperone(s) used GroEL + glycerol GroEL + dimethylsulfoxide GroEL + TMAO GroELS + trehalose GroEL +EG GroEL + proline GroELS 1 09 10 11 9 10 11 12 15 15 11 30 28 31 19 13 26 34 30 11 39 34 37 28 14 34 44 60 11 47 44 45 36 14 46 55 120 11 54 52 50 43 14 56 55 300 11 65 60 55 47 14 61 55 600 11 71 62 61 48 14 68 55 1200 11 71 62 61 48 14 68 55 0 5 10 15 20 25 30 35 40 16.7% 17.2% 14.3% 24.5% 38% 28% ProlineEGtrehaloseTMAODMSOGlycerol Increase of MalZ refolding yield Fig. 6. An optimized concentration of different chemical chaper- ones was used along with GroEL ⁄ GroES in the assisted refolding of MalZ at 30 °C. The increment of refolding yield of MalZ, due to combined application of chemical chaperones and GroEL ⁄ GroES over the yield where chemical chaperones were solely used as folding aids during refolding, was calculated and expressed as a percentage. DMSO, dimethylsulfoxide. 0 20 40 60 80 100 0 5 10 15 20 25 30 Time (min) % Relative MalZ activity Fig. 7. Thermoprotection of MalZ by glycerol, dimethylsulfoxide and TMAO. Native MalZ was incubated in buffer (j), 3 M glycerol (d), 3 M dimethylsulfoxide (m), 3 M TMAO (.), proline (·), EG (+) and Trehalose (r)at42°C. At different time intervals, aliquots were withdrawn and enzymatic assay of MalZ was carried out. Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al. 6006 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS present study, chaperone-like activity was observed for all of the above-mentioned osmolytes, except EG, in the refolding of urea denatured MalZ. GroEL, GroES and ATP-mediated refolding of urea denatured MalZ was also performed and it was observed that the refolding yield of MalZ was enhanced by five-fold over the spontaneous yield, which was only 11% at 30 °C (Table 3). The above-mentioned observations indicate that MalZ was unable to fold itself properly in vitro. Proba- bly, the nascent polypeptide forms aggregate during its refolding process and hence it requires the assistance of GroEL and GroES in its productive folding. We were particularly interested to use a few osmolytes, com- monly known as ‘chemical chaperones’, in the refold- ing studies of MalZ from its urea denatured state, to examine whether those osmolytes could enhance the in vitro folding of MalZ. Our findings demonstrated that the above-mentioned osmolytes acted as chaper- ones in the refolding of MalZ and enhanced the in vitro refolding yield of MalZ significantly, with the exception of EG, which did not improve the refolding yield of MalZ over the spontaneous one. Among all the osmolytes used, glycerol, which is commonly known to be a strong stabilizer of the native structure of proteins, led to a maximum recovery of active MalZ during the refolding process at a concentration of 2 m (Table 1). However, the refolding yield of MalZ was reduced beyond 2 m con- centration, suggesting that, in the osmolyte-mediated refolding of MalZ, factors other than thermodynam- ics also contribute significantly. A similar result was observed in the proline-assisted refolding of MalZ. Proline, at a lower concentration, has also been reported to be an excellent protein folding chaperone because it plays a chaperone-like role in the refold- ing of many proteins [26,36–38]. Experimental evi- dences have suggested that proline inhibits protein aggregation not only by binding to folding inter- mediate(s) and trapping the folding intermediate(s) into enzymatically inactive, ‘aggregation-insensitive’ state(s), but also by accelerating the hydrophobic collapse of creatine kinase to a packed protein [38,39]. It has also been reported that, at higher con- centrations (> 1.5 m), proline forms loose, higher- order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphi- pathic character. Formation of higher-order aggre- gates is believed to be crucial for proline to function as a protein folding aid [39]. In the present study, we also observed that, unlike glycerol, which even at concentrations of 3–4 m led to a good recovery of refolded protein, low concentrations of proline pro- duced the highest refolding recovery, whereas, at a higher concentration (> 1 m), the refolding yield reduced drastically (Table 1). The reduction in the efficiency of refolding might have been due to the formation of a higher-order molecular aggregate ⁄ supramolecular assembly that stabilizes protein aggregates. Other osmolytes (e.g. dimethylsulfoxide, TMAO and trehalose) also provided a good yield of refolded MalZ (Table 1) under similar conditions. Unlike glycerol and dimethylsulfoxide, TMAO had an optimum concentration of 3 m where the yield of refolded MalZ was maximum. Unfortunately, EG could not improve the reconstitution yield of MalZ over spontaneous refolding. Osmolyte-induced refolding efficiency was also monitored at different temperatures in the range 10)40 °C (Table 2). With the exception of EG, all other osmolytes and GroEL ⁄ GroES demonstrated their ability to protect MalZ from thermal denatur- ation and rescued a substantial amount of active MalZ compared to spontaneous recovery. GroEL and GroES are heat shock proteins known to pre- vent other cellular proteins from thermal denatur- ation. Glycerol and proline have also been reported in many cases to prevent protein aggregation against thermal stress and to stabilize the native structure of proteins in solution. Indeed, GroEL ⁄ GroES showed the highest protection by rescuing 66% of active MalZ molecules from denaturation, whereas glycerol and proline, among all osmolytes, provided compara- ble protection (> 60%) against thermal denatur- ation. Dimethylsulfoxide and TMAO produced approximately 50% recovery, and trehalose offered the lowest protection of approximately 40% at 40 °C. EG did not show any protection. We were also interested to investigate the effect of the combined application of osmolytes and GroELS on the refolding efficiency of MalZ. Interestingly, for all cases except EG, the yield was enhanced during the chemical and molecular chaperone-assisted refolding compared to the yield obtained from individual chap- erone-assisted refolding (Fig. 5). This was most likely due to osmolytes assisting in the local folding of GroEL-bound non-native protein molecules. It was also determined that the extent of refolding due to GroEL ⁄ GroES alone was approximately 60% at 30 °C. This signifies that the remaining approximately 40% of MalZ molecules must have formed aggregates. The other possibility is that those MalZ molecules not captured by GroEL, or that received no productive assistance from GroEL, would be taken care of by osmolytes and that is why there was a significant increase of refolding yield when both osmolytes and S. Paul et al. Chaperone-assisted folding of maltodextrin glucosidase FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 6007 GroELS were used together. As shown in Fig. 5, we also observed that the percent increase of refolding yield of MalZ in dimethylsulfoxide and GroELS- assisted folding was maximum among all cases, except for trehalose where it was lowest. Perhaps the viscos- ity factor is important here as trehalose has the highest viscosity, and this might have prevented GroEL from binding to unfolded MalZ molecules in solution. To observe the effect of chemical chaperone-medi- ated thermoprotection on the activity of MalZ, the MalZ solution temperature was increased to 42 °C. We observed that there was gradual loss of activity with time in the absence of any osmolytes in solution and, after 15 min of incubation time, complete biological activity of the protein was lost. However, when various osmolytes were added to the MalZ solution and incu- bated at the higher temperature, the enzymatic activity was protected significantly, even after 30 min (Fig. 7). In conclusion, polyol osmolytes, glycerol, dimethyl- sulfoxide, TMAO, trehalose, EG and proline assist in the productive refolding of MalZ considerably. There was a good correlation between the increase in the refolding yield in the presence of osmolytes and its ability to suppress protein aggregation. The decrease in the refolding yield at high osmolyte concentrations suggests that, in addition to the thermodynamic con- siderations, kinetic factors also are important during refolding in the cosolvents. There should be a cut-off viscosity of the refolding medium in the presence of the respective osmolyte that may lead to productive folding of MalZ. GroEL ⁄ GroES and osmolytes in combination enhanced the refolding yield of MalZ to the higher level. The findings also suggest that small organic molecules such as osmolytes can be used as effective agents in preventing protein aggregation and in the therapy of several aggregation-related debilitat- ing diseases when applied together with molecular chaperones such as GroEL and GroES. Experimental procedures Materials TG1, M15 and BL21 E. coli strains were used for the expres- sion and purification of MalZ, GroEL and GroES, respec- tively. The plasmid pCS19MalZ containing (His)6malZ was generous gift from W. Boos (University of Konstanz, Germany) and the plasmid pACYCEL, containing the groEL gene, and pET22dES, containing the groES gene, were a gift from A. L. Horwich (Yale University, CT, USA). Urea, glycerol, TMAO, dimethylsulfoxide, trehalose, ethylene glycol, proline and p-nitrophenyl-d-maltoside were purchased from Sigma Chemical Company (St Louis, MO, USA) and the disodium salt of ATP was purchased from Sisco Research Laboratories Pvt. Ltd (New Delhi, India). Dithiothreitol and MgCl 2 were purchased from Merck (New Delhi, India). All other reagents used were of analytical grade. Purification of MalZ, GroEL and GroES MalZ and GroEL were overexpressed in TG1 and M15 E. coli cells, respectively, and GroES was overexpressed in BL21 E. coli cells. Plasmids pCS19MalZ, pACYCEL and pET22dES were used for overexpression of MalZ, GroEL and GroES, respectively. Chaperones were purified as previ- ously described [39,40]. Cells were disintegrated in French press and lysates were centrifuged at 23 500 g for 45 min. Supernatant was separated and applied for chromato- graphic process in an AKTA FPLC system (Pharmacia, USA). GroEL was purified using FFQ anion-exchange chromatography (Pharmacia, USA), GroES was purified using FFSP cation-exchange chromatography (Pharmacia). After the FPLC purification process, affigel blue treatment was performed on GroEL to remove bound substrate pro- teins and, for GroES, differential precipitation by lowering the pH of the protein mixture was applied to eliminate other proteins before the FPLC purification process. MalZ purification was a single step process using Ni-chelating col- umn HisTrap HP (Pharmacia). Denaturation of MalZ MalZ was unfolded in the 20 mm sodium phosphate buffer, pH 7.0, containing 8 m urea and 20 m m dithiothreitol. The denaturation was confirmed by observing the complete loss of enzyme activity of MalZ in solution and also from the maximum change in the intensity of intrinsic tryptophan fluorescence emission. Tryptophan fluorescence studies Steady state fluorescence was recorded on a LS55 lumines- cence spectrofluorimeter (Perkin-Elmer, New Delhi, India). Intrinsic tryptophan fluorescence spectra were recorded by exciting the samples at 279 nm with excitation and emission slit widths set at 5.0 and 7.5 nm, respectively. The emission spectra were recorded in the range 300–400 nm. Baseline corrections were carried out using buffer without protein in all the cases. Refolding of MalZ MalZ (10 lm) was denatured in 20 mm sodium phosphate buffer, pH 7.0, containing 8.0 m urea and 20 mm dithio- erythritol and incubated for 4 h at 30 °C. Refolding experi- ments were carried out by diluting the denatured protein Chaperone-assisted folding of maltodextrin glucosidase S. Paul et al. 6008 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 100-fold into the refolding buffer (20 mm sodium phos- phate, pH 7.0, 1 mm MgCl 2 and 5 mm dithiothreitol). The enzyme concentration during refolding was 0.1 lm. In case of cosolvent-assisted refolding, various cosolvents, such as glycerol, dimethylsulfoxide and TMAO, were added at the desired concentrations to the refolding buffer into which the denatured protein was diluted. Refolding mixtures were incubated at the desired temperatures until completion of the reaction. The extent of refolding was calculated from the recovered activity relative to the activity of the same amount of native protein. Acknowledgements The authors thank DST, Government of India, and MHRD, Government of India, for financial support. We also thank Professor Winfried Boos, University of Konstanz, Germany, for the gift of plasmids encoding His-tagged MalZ and Professor Arthur Horwich, Yale University, CT, USA, for providing the plasmids for expressing GroEL and GroES. References 1 Gierasch LM & King J (1990) Protein Folding. Ameri- can Association for the Advancement of Science, Washington, DC. 2 Creighton TE (1992) Protein Folding . Freeman, New York, NY. 3 Kim PS & Baldwin RL (1990) Intermediates in the fold- ing reactions of small proteins. Annu Rev Biochem 59, 631–660. 4 Nall BK & Dill KA (1991) Conformations and Forces in Protein Folding . American Association for the Advance- ment of Science, Washington, DC. 5 Merz KM Jr & LeGrand SM (1994) The Protein Folding Problem and Tertiary Structure Prediction. Birkhauser, Boston, MA. 6 Dill KA, Bromberg S, Yue KZ, Fiebig KM, Yee DP, Thomas PD & Chan HS (1995) Principles of protein folding ) a perspective from simple exact models. Protein Sci 4, 561–602. 7 Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181, 223–230. 8 Ikemura I, Takagi H & Inauye M (1987) Requirement of pro-sequence for the production of active subtilisin in Escherichia coli. J Biol Chem 262, 7859–7864. 9 Silen JL & Agard DA (1989) The alpha-lytic protease pro-region does not require a physical linkage to acti- vate the protease domain in vivo. Nature 341, 462–464. 10 Winther JR & Sorensen P (1991) Propeptide of carboxy- peptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity. Proc Natl Acad Sci USA 88, 9330–9334. 11 Matthews CB (1993) Pathways of protein folding. Annu Rev Biochem 62, 653–683. 12 Rudon RW & Bedows E (1997) Assisted protein fold- ing. J Biol Chem 272, 3125–3128. 13 Finkelstein AV & Ptitsyn OB (1987) Why do globular proteins fit the limited set of folding patterns? Prog Biophys Mol Biol 50, 171–190. 14 Speed MA, Wang DIC & King J (1996) Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition. Nat Biotechnol 14, 1287. 15 Speed M, King J & Wang DIC (1997) Polymerization mechanism of polypeptide chain aggregation. Biotechnol Bioeng 54, 333–343. 16 Cleland JL & Wang DIC (1990) Refolding and aggre- gation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis. Biochemistry 29, 11072–11078. 17 Plomer JJ & Gafni A (1993) Renaturation of glucose-6- phosphate dehydrogenase from Leuconostoc mesentero- ides after denaturation in 4 M guanidine hydrochloride: kinetics of aggregation and reactivation. Biochim Bio- phys Acta 1163, 89–96. 18 Jaenicke R (1998) Protein self-organization in vitro and in vivo: partitioning between physical biochemistry and cell biology. Biol Chem 379, 237–243. 19 Dobson CM (2003) Protein folding and misfolding. Nature 426, 884–890. 20 De Bernardez Clark E, Schwarz E & Rudolph R (1999) Inhibition of aggregation side reactions during in vitro protein folding. Methods Enzymol 309, 217–237. 21 Mason JM, Kokkoni N, Stott K & Doig AJ (2003) Design strategies for anti-amyloid agents. Curr Opin Struct Biol 13, 526–532. 22 Hammarstrom P, Wiseman RL, Powers ET & Kelly JW (2003) Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science 299, 713–716. 23 da Costa MS, Santos H & Galinski EA (1998) An over- view of the role and diversity of compatible solutes in Bacteria and Archaea. Adv Biochem Eng Biotechnol 61, 117–153. 24 Arakawa T & Timasheff SN (1985) The stabilization of proteins by osmolytes. Biophys J 47, 411–414. 25 Caldas T, Demont-Caulet N, Ghazi A & Richarme G (1999) Thermoprotection by glycine betaine and choline. Microbiology 145, 2543–2548. 26 Samuel D, Kumar TK, Ganesh G, Jayaraman G, Yang PW, Chang MM, Trivedi VD, Wang SL, Hwang KC, Chang DK et al. (2000) Proline inhibits aggregation during protein refolding. Protein Sci 9 , 344–352. 27 Singer MA & Lindquist S (1998) Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of treha- lose. Trends Biotechnol 16, 460–468. S. Paul et al. Chaperone-assisted folding of maltodextrin glucosidase FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS 6009 [...].. .Chaperone-assisted folding of maltodextrin glucosidase S Paul et al 28 Chen BL & Arakawa T (1996) Stabilization of recombinant human keratinocyte growth factor by osmolytes and salts J Pharmacol Sci 85, 419–426 29 De Sanctis G, Maranesi A, Ferri T, Poscia A, Ascoli F & Santucci R (1996) Influence of glycerol on the structure and redox properties of horse heart cytochrome c... Mishra S & Chaudhuri TK (2007) The 69 kDa Escherichia coli maltodextrin glucosidase does not get encapsulated underneath GroES during GroEL ⁄ GroES assisted folding FASEB J 21, 2874–2885 34 Weissman JS, Hohl CM, Kovalenko O, Kashi Y, Chen S, Braig K, Saibil HR, Fenton WA & Horwich AL (1995) Mechanism of GroEL action: productive release 6010 35 36 37 38 39 40 41 of polypeptide from a sequestered position... (1997) Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL Nature 388, 792–798 Buchner J, Schmidt M, Fuchs M, Jaenicke R, Rudolph R, Schmid FX & Kiefhaber T (1991) GroE facilitates refolding of citrate synthase by suppressing aggregation Biochemistry 30, 1586–1591 Zhi W, Landry SJ, Gierasch LM & Srere PA (1992) Renaturation of citrate synthase: influence of denaturant and... Biochem Mol Biol Int 46, 509–517 Fisher MT (2006) Proline to the rescue Proc Natl Acad Sci USA 103, 13265–13266 Tapio S, Yeh F, Shuman HA & Boos W (1991) The malZ gene of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase J Biol Chem 266, 19450–19458 FEBS Journal 274 (2007) 6000–6010 ª 2007 The Authors Journal compilation ª 2007 FEBS ... denaturant and folding assistants, Protein Sci 1, 522–529 Ou WB, Park Y-D & Hai-Meng Zhou (2002) Effect of osmolytes as folding aids on creatine kinase refolding pathway Int J Biochem Cell Biology 34, 136–147 Kumat TKS, Samuel D, Jayaraman G, Srimathi T & Yu C (2006) The role of proline in the prevention of aggregation during protein folding in vitro Biochem Mol Biol Int 46, 509–517 Fisher MT (2006) Proline... chaperones interfere with the formation of scrapie prion protein EMBO J 15, 6363–6373 31 Yang DS, Yip CM, Huang TH, Chakrabartty A & Fraser PE (1999) Manipulating the amyloid-beta aggragation pathway with chemical chaperones J Biol Chem 274, 32970–32974 32 Voziyan PA & Fisher MT (2000) Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity . of Escherichia coli, a member of the maltose regulon, encodes a maltodextrin glucosidase. J Biol Chem 266, 19450–19458. Chaperone-assisted folding of maltodextrin. combination of both chaperones, could be used in the effi- cient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying