Báo cáo khoa học: Acceleration of disulfide-coupled protein folding using glutathione derivatives potx

Acceleration of disulfide-coupled protein folding using glutathione derivatives Masaki Okumura 1,2 , Masatoshi Saiki 2,3 , Hiroshi Yamaguchi 1 and Yuji Hidaka 2 1 School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan 2 Graduate School of Science and Engineering, Kinki University, Osaka, Japan 3 Department of Applied Chemistry, Faculty of Engineering, Tokyo University of Science, Yamaguchi, Japan Introduction The formation of the correct disulfide bonds and the conversion of a protein into its native conformation are the result of reversible thiol(SH) ⁄ disulfide(SS) exchange reactions that occur during protein folding and are thermodynamically and kinetically related to the redox potential in the biological environment. Glu- tathione (c-Glu-Cys-Gly), one of the most abundant thiol compounds found in cells, plays a major role in the formation of disulfide bonds in proteins in the endoplasmic reticulum [1]. Oxidized glutathione (GSSG) functions as an oxidant in the formation of disulfide bonds in proteins and reduced glutathione (GSH) functions as a reducing agent that cleaves mis- bridged disulfide bonds in proteins, resulting in the formation of the thermodynamically stable conforma- tion of proteins in vivo [2]. Because of this, glutathione Keywords arginine; disulfide; folding; glutathione; uroguanylin Correspondence Y. Hidaka, Graduate School of Science and Engineering, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan Fax: +81 6 6723 2721 Tel: +81 6 6721 2332 E-mail: yuji@life.kindai.ac.jp (Received 20 October 2010, revised 18 January 2011, accepted 28 January 2011) doi:10.1111/j.1742-4658.2011.08039.x Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives – glutamyl-cysteinyl-argi- nine (ECR) and arginyl-cysteinyl-glycine (RCG) – and examined their abil- ity to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox sys- tem. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prou- roguanylin revealed that the folding velocity (K RCG = 3.69 · 10 )3 s )1 ) using reduced RCG ⁄ oxidized RCG was approximately threefold higher than that using reduced glutathione ⁄ oxidized glutathione. In addition, fold- ing experiments using only the oxidized form of RCG or glutathione indi- cated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in medi- ating the formation of native disulfide bonds in proteins. Abbreviation Arg-C, arginylendopeptidase C; ECR, glutamyl-cysteinyl-arginine; ECR ox , oxidized ECR; ECR red , reduced ECR; GSH, reduced glutathione; GSSG, oxidized glutathione; RCG, arginyl-cysteinyl-glycine; RCG ox , oxidized RCG; RCG red , reduced RCG. FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS 1137 is widely employed in studies of folding reactions of disulfide-containing proteins in vitro [3,4]. Generally, folding reactions of proteins that do not involve disulfide bond formation occur within a few minutes. However, disulfide-containing proteins require several hours to fold correctly, because disulfide- exchange reactions are usually the rate-determining step. During folding reactions, several types of mis- bridged intermediates are often observed, and proteins with the native conformation accumulate in a time- dependent manner, as a result of disulfide-exchange reactions [5]. Proteins in which cysteine residues are involved in folding are usually folded into the native conformation via the formation of complex disulfide intermediates in the presence of redox reagents. Cor- rect folding is attributed to the presence of disulfide intermediates, which are readily converted into the native conformation. Therefore, to achieve the native tertiary structure of target proteins, regulation of the formation of the proper folding intermediates is a criti- cal issue. A variety of redox reagents (GSH ⁄ GSSG, reduced dithiothreitol ⁄ oxidized dithiothreitol and cysteine ⁄ cystine) can be used in protein-folding experiments [6,7]. Recently, Beld et al. [8] reported that selenogluta- thione, in which the cysteine residue of glutathione is replaced with a selenocysteine, accelerated the folding of disulfide-containing proteins by regulating the disul- fide coupling reaction. Thus, glutathione, or derivatives thereof, have the potential to serve as disulfide- exchange reagents in protein-folding studies. However, the role of each of the amino acid residues of glutathi- one, except for the cysteine residue, in the disulfide- coupled folding reactions is not known in detail. In this study, to investigate the role of each amino acid residue of glutathione, a series of glutathione analogs – arginyl-cysteinyl-glycine (RCG) and glutamyl-cyste- inyl-arginine (ECR) – were prepared, and their ability to serve as a redox reagent in disulfide-exchange reac- tions was examined. Arginine has recently been employed in studies of protein folding [9]. It is thought that arginine prevents the formation of nonspecific aggregates during the folding reaction. Folding experi- ments using lysozyme in the presence of arginine indi- cated that arginine promoted the formation of its native conformation, compared with other amino acids [10], and that arginine effectively suppressed the aggre- gation of denatured lysozyme [11], resulting in an increase in folding yield [12]. Arginine has also been reported to stabilize the exposed hydrophobic area of single-chain Fv fragments during the folding reaction [13], and the addition of both GSSG and arginine resulted in an increase in folding recovery [14]. To examine this aspect further, an arginine residue was introduced into a glutathione molecule to increase the solubility of folding intermediates in which cross-disul- fide bond(s) are formed between the reagents and pro- teins. In this study, except for the cysteine residue, each amino acid residue of glutathione was systemati- cally replaced with an arginine residue. RCG and ECR were chemically synthesized and their participation in disulfide-coupled folding reac- tions of lysozyme and prouroguanylin, as model pro- teins containing disulfide bonds, were examined. Substituting a glutamic acid residue for an arginine residue in glutathione dramatically affected the ability of glutathione to function as a redox reagent for pro- tein folding, resulting in an improved folding efficiency of the correct tertiary structures of those proteins. Here, we present results which demonstrate that a positive charge on the redox molecule is preferred for accelerating disulfide-exchange reactions in protein folding. Results and Discussion Glutathione is a major reagent for regulating disulfide bond formation of naked proteins in the endoplasmic reticulum. It is also widely used to assist protein fold- ing in vitro [15,16]. GSH cleaves unfavorable disulfide bonds (solvent-exposed disulfide bonds) and GSSG promotes the formation of disulfide bonds in proteins, resulting in the formation of the native conforma- tion of a protein. Glutathione (c-GSH) contains a c-branched peptide bond between the glutamic acid and the cysteine residues. We first investigated whether the branched structure is required for disulfide- exchange reactions. For this purpose, a-glutamyl-cyste- inyl-glycine (a-GSH) was chemically synthesized and the folding of lysozyme was examined in the presence of a-GSH or c-GSH to estimate their reactivity. The folding recovery for lysozyme using a-GSH was similar to that of glutathione (Fig. S1). Therefore, RCG and ECR were designed as a-linked peptides and then syn- thesized. The folding of lysozyme has been studied extensively and it is known that the folding recovery of lyso- zyme decreases at high protein concentrations (> 0.1 mgÆmL )1 ) [17]. To estimate the ability of RCG [both reduced (RCG red ) and oxidized (RCG ox ) forms] and ECR [both reduced (ECR red ) and oxidized (ECR ox ) forms] to permit the disulfide-coupled folding of proteins, lysozyme was folded at high concentrations (0.1–1.6 mgÆmL )1 ). The molar ratios of the reduced and oxidized form of the reagents were adjusted to 2 : 1 in these experiments, because the molar ratio of Acceleration of disulfide-coupled protein folding M. Okumura et al. 1138 FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS GSH to GSSG in the endoplasmic reticulum ranges from 1 : 1 to 3 : 1 [2]. The fully reduced and the native form of lysozyme were eluted at positions correspond- ing to R and N, respectively, in Fig. 1. The HPLC peak for lysozyme folded in the correct conformation was assigned based on a co-elution experiment using native lysozyme [17,18]. The folding yields of lysozyme (cross-hatched bar in Fig. 2) in the presence of ECR red and ECR ox were similar to that for GSH ⁄ GSSG (open bar in Fig. 2) at the protein concentrations used in this study, indicating that the replacement of the glycine residue with an arginine residue had no significant effect on the reactivity of the redox reagent. On the other hand, the folding yields using RCG red and RCG ox (black bar in Fig. 2) were increased to 130%, 131%, 171% and 185%, compared with those using GSH ⁄ GSSG (open bar in Fig. 2) at concentrations of 0.2, 0.4, 0.8 and 1.6 mgÆmL )1 , respectively. The folding yield for the correct conformation of lysozyme was dramatically increased when RCG red ⁄ RCG ox was used at higher protein concentrations. The low folding recovery of lysozyme at high protein concentrations is thought to be caused by the formation of nonspecific aggregates. It therefore appears that the arginine resi- due introduced into the glutathione molecule prevented nonspecific aggregation of the folding intermediates at higher protein concentrations. However, the mecha- nism of the RCG-mediated folding remains to be examined. In order to further examine the effect of RCG red ⁄ RCG ox in protein folding, recombinant human prou- roguanylin was also examined as a model protein. Prouroguanylin contains six cysteine residues that par- ticipate in three intramolecular disulfide bonds in the native state (Cys41–Cys45, Cys74–Cys82 and Cys77– Cys85) and formation of the correct disulfide pairs is important for the complete folding recovery of the protein [19]. The folding reactions of prouroguanylin were examined using RCG red ⁄ RCG ox or GSH ⁄ GSSG, and the folding yields were estimated by HPLC analy- ses. The folding recovery of prouroguanylin using the typical GSH system was decreased at higher protein concentrations, as reported previously [19]. The RCG redox system resulted in improved folding yields at higher protein concentrations, compared with the GSH system, as shown in Fig. 3. When the RCG red ⁄ RCG ox redox system was used, the folding yields of the native conformation of prouroguanylin were increased to 114% and 143% at concentrations of 0.2 and 0.4 mgÆmL )1 , respectively, compared with that of GSH ⁄ GSSG. These results indicate that the RCG red ⁄ RCG ox system is an excellent redox system Retention time (min) 01020 30 N R Relative absorbance at 220 nm Fig. 1. HPLC profile of the folding of lysozyme. N and R represent the native and reduced forms, respectively. The proteins were eluted using a 20–60% linear gradient of CH 3 CN in 0.05% trifluoro- acetic acid, at a rate of 1% ⁄ min and a flow rate of 1.0 mLÆmin )1 , and monitored at 220 nm. Recovery of native conformation (%) Protein concentration (mg·mL –1 ) 100 80 60 40 20 0 0.1 0.2 0.4 0.8 1.6 Fig. 2. Folding yields of lysozyme in the presence of GSH ⁄ GSSG (open bars), reduced (ECR red ) and oxidized (ECR ox ) forms of ECR (cross-hatched bars), and reduced (RCG red ) and oxidized (RCG ox ) forms of RCG (shaded bars). 0.1 0.2 0.4 Recovery of native conformation (%) 100 80 60 40 20 0 Protein concentration (m g ·mL –1 ) Fig. 3. Folding yields of prouroguaylin in the presence of GSH ⁄ GSSG (open bars) and the RCG red ⁄ RCG ox (shaded bars). M. Okumura et al. Acceleration of disulfide-coupled protein folding FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS 1139 for the folding of prouroguanylin, as well as for the folding of lysozyme. Therefore, it can be concluded that RCG red ⁄ RCG ox serves as an effective redox sys- tem for disulfide-coupled protein folding. Arginine is thought to increase the folding recovery of proteins by suppressing the formation of aggregates. This effect was observed when Arg was used at a concentration of several hundred millimoles. However, our experiments were carried out at a concentration of only a few milli- molar concentrations of Arg. Therefore, it is likely that the efficient folding recovery using RCG is not the result of direct effects, such as stabilization of the hydrophobic surface of the denatured proteins. To address the question of how the RCG redox sys- tem increases the folding recovery of proteins and the nature of the mechanism responsible for this, it is nec- essary to characterize the folding intermediates. We hypothesized that RCG plays a role in the solubility of the denatured protein when the RCG is linked to fold- ing intermediates via disulfide bonds, and that the Arg residue in the cross-disulfide-linked RCG is important for the accumulation of proper folding intermediates in the native conformation. Generally, it is difficult to analyze folding intermediates of disulfide-containing proteins at each step of the conformational transition [20]. However, the folding intermediates of prourogu- anylin can be separated by HPLC. We therefore employed prouroguanylin as a model protein to ana- lyze the folding mechanism using RCG red ⁄ RCG ox . The distribution of folding intermediates of prouroguanylin produced using RCG red ⁄ RCG ox was compared with that of GSH ⁄ GSSG at several time-points of the fold- ing reactions, and the folding intermediates were sepa- rated by HPLC and analyzed using MALDI- TOF ⁄ MS. The folding products 0SH, 2SH, R and N, shown in Fig. 4, represent proteins with three disulfide bonds, including mis-bridged disulfide bonds, two disulfide bonds, no disulfide bonds and native disulfide bonds, respectively, as confirmed by MALDI-TOF ⁄ MS analy- ses. The folding reactions and the correct disulfide for- mation of prouroguanylin were complete within 24 h for both GSH ⁄ GSSG and RCG red ⁄ RCG ox . The reduced form (R in Fig. 4A,B) of prouroguanylin was present for a longer time in the reaction mixture that contained GSH ⁄ GSSG compared with the reaction mixture that contained RCG red ⁄ RCG ox . The formation and the reduction of mixed-disulfide bonds between GSH and proteins are reversible reactions. The forma- tion of the reduced form of proteins is predominant at the early stage of the folding reaction because an excess of reductant was present in the reaction mix- ture. The formation of the correct disulfide bond(s) then becomes predominant at the later stage in folding because the native disulfide bond is shielded by virtue of the fact that it is located in the interior of a protein molecule. In our experiment, the reduced form of prouroguanylin disappeared within 5 min (Fig. 4B) in the reaction mixture using RCG red ⁄ RCG ox but detect- able amounts were still present in the reaction mixture using GSH ⁄ GSSG (Fig. 4A) at that time-point. There- fore, these results indicate that the formation of intra- molecular disulfide bonds is rapid in the presence of RCG red ⁄ RCG ox and that the half life of folding inter- mediates with a low solubility becomes small, resulting in an improved folding recovery. In order to better understand the folding mechanism in the presence of RCG red ⁄ RCG ox , the folding inter- mediates were further analyzed in detail. The disulfide pairing of prouroguanylin was determined using a pre- viously reported method [21]. The intermediates 0SH and 2SH were observed at an early stage of the folding reaction (1 and 5 min in Fig. 4A and 1 min in Fig. 4B). To determine the positions of the disulfide bonds of the folding intermediates, the proteins were separated by HPLC and digested with the endoprotein- ase arginylendopeptidase C (Arg-C) [21]. The peptide fragment (Thr-Ile-Ala-uroguanylin) produced, includ- ing the mature region, was compared to chemically synthesized peptides with three different disulfide 1 min 5 min 2 h 24 h Refolding time Retention time Relative absorbance at 220 nm Relative absorbance at 220 nm R R R N 2SH 2SH 0SH 2SH 0SH 20 min AB 1 min 5 min 2 h 24 h Retention time R N 2SH 0SH 20 min N N N Refolding time Fig. 4. HPLC profiles of the reaction mix- tures of prouroguanylin in the presence of 2m M GSH ⁄ 1mM GSSG (A) and 2 mM RCG red ⁄ 1mM RCG ox (B). N, 2SH, 0SH and R, represent the positions of prouroguanylin with native disulfide pairing, with two thiols and two disulfide bonds, with three disulfide bonds and no thiol groups, and the fully reduced form of prouroguanylin, respec- tively. Acceleration of disulfide-coupled protein folding M. Okumura et al. 1140 FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS pairings using HPLC (data not shown), as described previously [21]. The results indicated that 0SH at 1 and 5 min in Fig. 4A and 0SH at 1 min in Fig. 4B cor- responded to prouroguanylin in which the disulfide bonds in the mature region were mis-bridged. The for- mation of native disulfide bonds of prouroguanylin was observed at 5 min in the presence of RCG red ⁄ RCG ox (Fig. 4B), but  20 min was required to accomplish this in the presence of GSH ⁄ GSSG (Fig. 4A). The mis-bridged disulfide species, 0SH and 2SH, were observed at an early stage in the folding reaction for both systems. These results suggest that the formation of the native disulfide bonds in prou- roguanylin is achieved via intermediates with mis-bri- ged disulfide bonds and that RCG red ⁄ RCG ox accelerates the formation of the correct disulfide pair- ings via disulfide-exchange reactions. Generally, the disulfide-exchange reaction is the rate-determining step in the disulfide-coupled folding of proteins and is catalyzed by a free thiol group. The kinetically trapped intermediates were observed at the early stage of the folding reaction because the local stability of each moiety of a protein molecule was pre- dominantly affected at the early stage in the folding reaction in vitro. The mis-bridged disulfide species then disappeared, followed by the formation of native disul- fide bonds. Therefore, the velocity of the disulfide- exchange reaction is important in achieving correct folding and folding recovery. The folded prouroguany- lin with the correct disulfide bonds was observed within 5 min in the folding reaction carried out in the presence of RCG red ⁄ RCG ox . This result indicates that RCG red ⁄ RCG ox accelerates the exchange reaction of disulfide bonds and effectively permits prouroguanylin to be converted into the native conformation. To further estimate the ability of the RCG redox sys- tem in the disulfide-coupled folding of proteins, the folding reaction was performed under anaerobic condi- tions using only RCG ox or GSSG. Under anaerobic conditions, RCG ox first oxidatively reacts with the thiol groups of proteins to form cross-disulfide bonds between RCG and proteins. The intramolecular disul- fide-exchange reaction mainly occurs under these condi- tions, because RCG red , as an initiator of the disulfide- exchange reaction, is absent, although trace amounts of RCG red exist as a side reaction product of the forma- tion of the cross-disulfide bond. Therefore, the intra- molecular disulfide-exchange reaction in the disulfide- coupled folding of proteins can be estimated by using only its oxidative form. The oxidative folding of prou- roguanylin was carried out in the presence of 1 mm GSSG or 1 mm RCG ox at room temperature for 48 h, and the folding products were analyzed by HPLC (Fig. 5A,B). To estimate the ratio of disulfide isomers of prouroguanylin, the mixtures of folding products (peaks a and b in Fig. 5A and B, respectively) were purified by HPLC and digested with the endoproteinase Arg-C to release the mature region (Thr-Ile-Ala-urogu- anylin), as described above. The digests were separated by HPLC (Fig. 5C,D) and analyzed by MALDI- TOF ⁄ MS. The mis-bridged disulfide peptide (I; Cys74– Cys85 and Cys77–Cys82) and the native disulfide pep- tide (N; Cys74–Cys82 and Cys77–Cys85) were observed in both cases, as shown in Fig. 5C,D. The ratios of I and N were  1 : 1 and 1 : 2 in the presence of GSSG and RCG ox , respectively. This indicates that RCG ox promotes the formation of the native conformation of prouroguanylin faster than that of GSSG and that this occurs via intramolecular disulfide-exchange reactions. Therefore, we conclude that the native conformation of proteins can be achieved effectively using RCG red ⁄ RC- G ox , which accelerates intramolecular disulfide- exchange reactions. To determine the kinetic parameters for the folding reaction using the RCG red ⁄ RCG ox system, CD mea- surements were carried out at 222 nm. The CD analysis Relative absorbance at 220 nm Retention time (min) N N I I a b 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 ABCD Fig. 5. HPLC profiles of the folding of prou- roguanylin in the presence of GSSG (A) and RCG ox (B) and Arg-C digests of peaks a (C) and b (D) in (A) and (B), respectively. N and I represent Thr-Ile-Ala-uroguanylin with native disulfide bonds and with mis-bridged disulfide bonds, respectively. M. Okumura et al. Acceleration of disulfide-coupled protein folding FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS 1141 of the prouroguanylin structure revealed that prou- roguanylin contains an a-helical structure (data not shown) and that the helical structure was formed within 10 min when using RCG red ⁄ RCG ox and within 30 min when using GSH ⁄ GSSG (Fig. 6). Importantly, these timescales corresponded to the formation of the native disulfide bonds (Fig. 4A,B), indicating that the forma- tion of the native disulfide bonds are closely related to the formation of an a-helical structure. Therefore, the folding kinetics can be estimated, based on the forma- tion of the a-helical structure. The molar ellipticity at 222 nm, obtained for each time-point, was used to cal- culate the rate constants, as previously described [22]. A curve-fitting method was applied to estimate the kinetic parameters, and the results indicated that the reaction followed single exponential kinetics. Therefore, the rate constants for the folding of prouroguanylin were calculated to be K gsh = 1.20 · 10 )3 s )1 and K rcg = 3.69 · 10 )3 s )1 (calculation using Kaleida Graph version 3.5) (i.e. the reaction rate in the case of RCG red ⁄ RCG ox was approximately three times faster than that of GSH ⁄ GSSG). Arginine has been employed in folding reactions of proteins to prevent the formation of nonspecific aggre- gates [9–12] and to improve folding recovery. On the other hand, arginine has also been reported to stabilize the exposed hydrophobic area of single-chain Fv frag- ments [13] and to increase the folding recovery of pro- teins [14]. The findings reported herein indicate that a novel compound, RCG, has the ability to accelerate the formation of the native disulfide bonds, thus resulting in an improved folding recovery. However, the role of the arginine residue of RCG in folding reactions appears to be different from that in the examples above. Arginine can suppress protein aggregation at concentrations that exceed  100 mm in such cases [12] but 1–2 mm RCG was used in our experiments. The electrostatic influence of local cysteine environments on disulfide-exchange kinetics was studied using several types of protein fragment [23]. The cysteine residue, when adjacent to a positively charged neighbor, reacted more rapidly, indicating that the adjacent positive charge accelerates disulfide formation and the exchange reaction [23]. The mixed-disulfide intermediates of RCG and prouroguanylin were produced at the first step in the folding of prouroguanylin, and the mixed- disulfide bond was then attacked by a free thiol anion (disulfide-exchange reaction). RCG, ECR and GSH possess net charges of +1, 0 and )1, respectively, at neutral pH without a thiol group of the cysteine resi- due. Local interactions between the positive (RCG) and the negative (thiol anion) charges may be favorable for the formation of disulfide bonds. Therefore, the arginine residue in RCG could accelerate the disulfide- exchange reaction by its positive charge and effectively mediate the construction of the native tertiary structure of proteins. However, the pI values of proteins do not appear to be important for the reaction. Two types of proteins were used in this study, based on electrostatic effects. The pI values for prouroguanylin and lysozyme are 5.5 and 11.0, respectively, indicating that prourogu- anylin and lysozyme exist in solution as a negatively charged protein and a positively charged protein, respectively. Therefore, the RCG system accelerates disulfide-exchange reactions, regardless of the net charge of the protein being studied. In addition, the ECR results were similar to that of GSH. ECR (net charge zero) possesses a negative charge at the side chain of the glutamic acid residue, and the arginine residue is positively charged. There- fore, it is possible that the negative charge of the glu- tamic acid residue offsets the acceleration effect of the positive charge of the arginine residue on disulfide-cou- pled folding. Arginine has been extensively studied as a protein- folding reagent because of its ability to suppress aggre- gation during the folding of proteins [10,12,24,25]. It is generally thought that arginine provides stability for the hydrophobic surfaces of folding intermediates. The findings herein indicate an alternative role of the argi- nine residue. The arginine residue in the glutathione analog efficiently improved folding recovery and accel- erated the disulfide-coupled folding of proteins. There are only a few examples of reagents for the disulfide- coupled folding of proteins. The disulfide-exchange reagent, RCG, and related analogs show promise for serving as a powerful tool for studies, not only of pro- tein folding but also for the effective recovery of the correct tertiary structure of target recombinant pro- teins from the denatured state, such as inclusion bodies in Escherichia coli cells. Refolding time (min) 0 –5 –10 –15 [θ] x 10 –3 deg·cm –2 ·dmol –1 a b 0 306090 Fig. 6. Molar ellipticity at 222 nm during the folding of prourogu- anylin. CD measurements were carried out in the presence of (a) GSH ⁄ GSSG or RCG red ⁄ RCG ox (b) at room temperature. Acceleration of disulfide-coupled protein folding M. Okumura et al. 1142 FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS In conclusion, replacement of the glutamic acid resi- due with an arginine residue in the glutathione mole- cule substantially improved its ability as a redox reagent for protein folding. The positive charge in close proximity to the cysteine residue of the redox molecule effectively accelerates disulfide-exchange reactions. Materials and methods Materials Glutathione, lysozyme and endoproteinase Arg-C were pur- chased from the Peptide Institute, Inc. (Osaka, Japan), Sei- kagaku Corporation (Tokyo, Japan) and Takara Bio Inc. (Kyoto, Japan), respectively. RCG and ECR were synthe- sized by Greiner Bio-One Co., Ltd. (Tokyo, Japan). All other chemicals and solvents used were of reagent grade. RP-HPLC The HPLC apparatus comprised an ELITE system (Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with an L-2400 detector and a D-2500 chromato-integrator. Proteins were separated by RP-HPLC using a Cosmosil 5C 18 -AR-II column (8 · 250 mm; Nacalai Tesque, Inc., Kyoto, Japan) or a Develosil UG-5 column (4.6 · 150 mm; Nomura Chemical Co., Ltd., Aichi, Japan). Folding yields were estimated by the HPLC peak area at 220 nm. MALDI-TOF ⁄ MS The molecular mass values of proteins were determined using a DALTONICS ultraflex spectrometer (Bruker Japan Co., Ltd) in the positive ion mode. Mass spectro- metric analyses of proteins and peptides were carried out in the linear or reflector modes using 3,5-dimethoxy-4-hy- droxycinnamic acid (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and a-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Co., Tokyo, Japan) as matrices, respec- tively. In a typical run, the lyophilized sample ( 0.1 nmol) was dissolved in 0.05% trifluoroacetic acid aq ⁄ 50% CH 3 CN (1 lL), mixed with 1 lL of a matrix solution (10 mgÆmL )1 ) and air-dried on the sample plate for MALDI-TOF ⁄ MS. CD measurements Denatured protein (2 nmol) was dissolved in 10 mm Tris ⁄ HCl (300 lL, pH 8.0) containing GSH ⁄ GSSG (final concentrations: 2 mm ⁄ 1mm) or RCG red ⁄ RCG ox (final con- centrations: 2 mm ⁄ 1mm). CD measurements were carried out in a Model J720WI CD spectrometer (JASCO Corpo- ration, Tokyo, Japan) at room temperature using a cuvette with 1 mm path-length. Folding reactions of lysozyme and prouroguanylin The reduction of lysozyme was carried out using a previ- ously described method, with minor modifications [11]. Briefly, lysozyme (5 mg) was dissolved in 0.1 m Tris ⁄ HCl (pH 8.3, 1 mL) containing 20 mm dithiothreitol and 8 m urea, and the solution was allowed to stand for 3 h at 40 °C. The reaction mixture was dialyzed against 10 mm HCl, lyophilized and stored at )20 °C until used. Folding reactions were carried out at several protein con- centrations (0.1–1.6 mgÆmL )1 of lysozyme; 0.1–0.4 mgÆmL )1 of prouroguanylin). The denatured ⁄ reduced proteins were dissolved in 0.1 m Tris ⁄ HCl (pH 8.0) and allowed to undergo folding in the presence of 2 mm reductant (GSH, RCG red or ECR red ) and 1 mm oxidant (GSSG, RCG ox or ECR ox ) at room temperature for 48 h, as described previ- ously [21]. All solutions used in the refolding experiments were flushed with N 2 gas, and the reactions were carried out in a sealed vial under an atmosphere of N 2 . The kinetic experiments were performed in the same buf- fer as described above. The reaction mixture (100-lL aliqu- ots) was removed at several time-points, quenched with an equivalent volume of 1 m HCl [26] and separated by RP-HPLC. The HPLC fractions were analyzed by MALDI-TOF ⁄ MS after lyophilization. Digestion with endoproteinase Arg-C The HPLC-purified proteins were dissolved in 0.1 m Tris ⁄ HCl buffer (pH 8.0) and digested with arginylendo- peptidase C at 37 °C for 16 h, as previously described [19,21]. 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Folding recoveries of lysozyme using c-GSH ⁄ GSSG (open bars) and a-GSH ⁄ GSSG (shaded bars). This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Acceleration of disulfide-coupled protein folding M. Okumura et al. 1144 FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS . Acceleration of disulfide-coupled protein folding using glutathione derivatives Masaki Okumura 1,2 , Masatoshi. widely employed in studies of folding reactions of disulfide-containing proteins in vitro [3,4]. Generally, folding reactions of proteins that do not involve