Formation Of Native Disulfide Bonds Research Articles

Enabling site-specific NMR investigations of therapeutic Fabusing a cell-free based isotopic labeling approach:application to anti-LAMP1 Fab.

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13C', 13Cα, 13Cβ resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs' higher order structures, dynamics and interactions, using solution-state NMR.

Introduction of a More Glutaredoxin-like Active Site to PDI Results in Competition between Protein Substrate and Glutathione Binding.

Proteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for PDI also in vivo, but at least for oxidation reactions, GSSG probably is not the major electron acceptor and PDI may not have evolved to react with glutathione with high affinity, but merely having adequate affinity for both glutathione and folding proteins/peptides. Glutaredoxins, on the other hand, have a high affinity for glutathione. They commonly have -CXFC- or -CXYC- active site, where the tyrosine residue forms part of the GSH binding groove. Mutating the active site of PDI to a more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the active site, as well as a decreased rate of the native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. This suggests that these variants lead to competition in binding between glutathione and folding protein substrates.

The Formation of Native Disulfide Bonds: Treading a Fine Line in Protein Folding.

The folding of proteins that contain disulfide bonds is termed oxidative protein folding. It involves a chemical reaction resulting in the formation of disulfide bonds and a physical conformational folding reaction that promotes the formation of the native structure. While the presence of disulfide bonds significantly increases the complexity of the folding landscape, it is generally recognized that native disulfide bonds help funnel the trajectory towards the final folded form. Here, we review the role of disulfide bonds in oxidative protein folding and argue that even structure-inducing native disulfide bond formation treads a fine line in the regeneration of disulfide-bond-containing proteins. The translation of this observation to protein misfolding related disorders is discussed.

Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories.

Escherichia coli is the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation. Proteins containing disulfide bonds have traditionally been made by targeting to the periplasm or by in vitro refolding of proteins made as inclusion bodies. More recently, systems for the production of disulfide-containing proteins in the cytoplasm have been introduced. However, it is unclear if these systems have the capacity for the production of disulfide-rich eukaryotic proteins. To address this question, we tested the capacity of one such system to produce domain constructs, containing up to 44 disulfide bonds, of the mammalian extracellular matrix proteins mucin 2, alpha tectorin, and perlecan. All were successfully produced with purified yields up to 6.5 mg/L. The proteins were further analyzed using a variety of biophysical techniques including circular dichroism spectrometry, thermal stability assay, and mass spectrometry. These analyses indicated that the purified proteins are most likely correctly folded to their native state. This greatly extends the use of E. coli for the production of eukaryotic proteins for structural and functional studies.

Applications of catalyzed cytoplasmic disulfide bond formation.

Disulfide bond formation is an essential post-translational modification required for many proteins to attain their native, functional structure. The formation of disulfide bonds, otherwise known as oxidative protein folding, occurs in the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasm of prokaryotes. While there are differences in the molecular mechanisms of oxidative folding in different compartments, it can essentially be broken down into two steps, disulfide formation and disulfide isomerization. For both steps, catalysts exist in all compartments where native disulfide bond formation occurs. Due to the importance of disulfide bonds for a plethora of proteins, considerable effort has been made to generate cell factories which can make them more efficiently and cheaper. Recently synthetic biology has been used to transfer catalysts of native disulfide bond formation into the cytoplasm of prokaryotes such as Escherichia coli. While these engineered systems cannot yet rival natural systems in the range and complexity of disulfide-bonded proteins that can be made, a growing range of proteins have been made successfully and yields of homogenously folded eukaryotic proteins exceeding g/l yields have been obtained. This review will briefly give an overview of such systems, the uses reported to date and areas of future potential development, including combining with engineered systems for cytoplasmic glycosylation.

Crystal structure of human anterior gradient protein 3.

Oxidative protein folding in the endoplasmic reticulum is catalyzed by the protein disulfide isomerase family of proteins. Of the 20 recognized human family members, the structures of eight have been deposited in the PDB along with domains from six more. Three members of this family, ERp18, anterior gradient protein 2 (AGR2) and anterior gradient protein 3 (AGR3), are single-domain proteins which share sequence similarity. While ERp18 has a canonical active-site motif and is involved in native disulfide-bond formation, AGR2 and AGR3 lack elements of the active-site motif found in other family members and may both interact with mucins. In order to better define its function, the structure of AGR3 is required. Here, the recombinant expression, purification, crystallization and crystal structure of human AGR3 are described.

Overexpression of the regulatory subunit of glutamate-cysteine ligase enhances monoclonal antibody production in CHO cells.

For decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate-cysteine ligase catalytic subunit (Gclc) and the glutamate-cysteine ligase modifier subunit (Gclm). The two genes were reconstructed from our RNA-Seq de novo assembly and then were functionally annotated. Once the sequences of the genes were confirmed using proteogenomics, a transiently expressed mAb was introduced into cell lines overexpressing either Gclc or Gclm. The new cell lines were compared for mAb production to the parental cell line and changes at the proteome level were measured using SWATH. As per our previous proteomics observations, overexpressing Gclm improved productivity, titer, and the frequency of high producer clones by 70%. In contrast, overexpressing Gclc, which produced a higher amount of GSH, did not increase mAb production. We show that GSH cannot be linked to higher productivity and that Gclm may be controlling other cellular processes involved in mAb production yet to be elucidated. Biotechnol. Bioeng. 2017;114: 1825-1836. © 2017 Wiley Periodicals, Inc.

Efficient production of (2)H, (13)C, (15)N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds.

BackgroundIn order to use most modern methods of NMR spectroscopy to study protein structure and dynamics, isotope-enriched protein samples are essential. Especially for larger proteins (>20 kDa), perdeuterated and Ile (δ1), Leu, and Val methyl-protonated protein samples are required for suppressing nuclear relaxation to provide improved spectral quality, allowing key backbone and side chain resonance assignments needed for protein structure and dynamics studies. Escherichia coli and Pichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. The P. pastoris system can be used to produce 13C, 15N-enriched and even 2H,13C, 15N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (δ1), Leu and Val methyl-protonated groups in P. pastoris are still unavailable. Glycosylation heterogeneity also provides challenges to NMR studies. E. coli expression systems are efficient for overexpressing perdeuterated and Ile (δ1), Leu, Val methyl-protonated protein samples, but are generally not successful for producing secreted eukaryotic proteins with native disulfide bonds.ResultsThe 33 kDa protein—Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using both P. pastoris and E. coli BL21 trxB (DE3) systems. Samples produced from both systems exhibit identical native disulfide bond formation and similar 2D NMR spectra, indicating similar native protein folding. The yield of 13C, 15N-enriched r27RCL produced using P. pastoris was 1.7 times higher that obtained using E. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced in P. pastoris exhibit O-glycosylation, while the protein samples produced in E. coli were not glycosylated. The specific activity of r27RCL from P. pastoris was ~1.4 times higher than that produced in E. coli.ConclusionsThese data demonstrate efficient production of 2H, 13C, 15N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic protein r27RCL with native disulfides using the E. coli BL21 trxB (DE3) system. For certain NMR studies, particularly efforts for resonance assignments, structural studies, and dynamic studies, E. coli provides a cost-effective system for producing isotope-enriched RCL. It should also be potential for producing other 2H, 13C, 15N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic proteins with native disulfide bonds.

Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli.

Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach) were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP) were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside.

Purification, crystallization and preliminary crystallographic investigation of FrnE, a disulfide oxidoreductase from Deinococcus radiodurans.

In prokaryotes, Dsb proteins catalyze the formation of native disulfide bonds through an oxidative folding pathway and are part of the cell machinery that protects proteins from oxidative stress. Deinococcus radiodurans is an extremophile which shows unparalleled resistance to ionizing radiation and oxidative stress. It has a strong mechanism to protect its proteome from oxidative damage. The genome of Deinococcus shows the presence of FrnE, a Dsb protein homologue that potentially provides the bacterium with oxidative stress tolerance. Here, crystallization and preliminary X-ray crystallographic analysis of FrnE from D. radiodurans are reported. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method with reservoir solution consisting of 100 mM sodium acetate pH 5.0, 10% PEG 8000, 15-20% glycerol. Diffraction data were collected on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 1.8 Å resolution at 100 K. The space group of the crystal was found to be P2₁22₁, with unit-cell parameters a=47.91, b=62.94, c=86.75 Å, α=β=γ=90°. Based on Matthews coefficient analysis, one monomer per asymmetric unit is present in the crystal, with a solvent content of approximately 45%.

The Thioredoxin Superfamily in Oxidative Protein Folding

The thioredoxin (Trx) superfamily proteins, including protein disulfide isomerases (PDI) and Dsb protein family, are major players in oxidative protein folding, which involves native disulfide bond formation. These proteins contain Trx folds with CXXC active sites and fulfill their physiological functions in oxidative cellular compartments such as the endoplasmic reticulum (ER) or the bacterial periplasm. The structure of the Trx superfamily protein PDI has been solved by X-ray crystallography and shown to be a flexible molecule, having a horseshoe shape with a closed reduced and an open oxidized conformation, which is important for exerting its catalytic activity. Atomic force microscopy revealed that PDI works as a placeholder to prevent early non-native disulfide bond formation and further misfolding. S-nitrosylation of the active site of PDI inhibits the PDI activity and links protein misfolding to neurodegenerative diseases like Alzheimer's and Parkinson's diseases. Electron transfer pathways of the oxidative protein folding show conserved Trx-like thiol-disulfide chemistry. Overall, mammalian cells have a large number of disulfide-containing proteins, the folding of which involves non-native disulfide bond isomerization. The process is sensitive to oxidative stress and ER stress. The correct oxidative protein folding is critical for the substrate protein stability and function, and protein misfolding is linked to, for example, neurodegenerative diseases. Further understanding on the mechanisms and specific roles of Trx superfamily proteins in oxidative protein folding may lead to drug development for the treatment of bacterial infection and various human diseases in aging and neurodegeneration.

Radically Different Thioredoxin Domain Arrangement of ERp46, an Efficient Disulfide Bond Introducer of the Mammalian PDI Family

The mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER.

Experimental and Computational Studies on the Dynamics and Flexibility of Protein Disulfide-Isomerase (PDI)

PDI is an abundant folding catalyst and chaperone, required for formation of native disulfide bonds in secretory proteins. PDI interacts with many redox partners in the oxidative protein folding pathway and acts on an immense range of unfolded and part-folded protein substrates. It is a multi-domain protein (a-b-b'-x-a'-c) comprising two redox-active thioredoxin-like domains (a, a'), two other domains structurally homologous to thioredoxin (b, b'), a linker (x) and an inessential C-terminal tail (c). Key sites are the redox-active motifs (-WCGHCK-) in a and a' domains, and a promiscuous non-covalent ligand-binding site in b' which can be gated by x. X-ray structures of PDI and its homologues provide static information, but evidence from NMR and intrinsic fluorescence suggests PDI is highly dynamic.We have explored PDI flexibility using FRET and computational techniques. Using yeast PDI, a very rapid method for predicting protein dynamics, based on FRODA and normal mode analysis, has been validated by a comparison of predictions with those from a full MD analysis. Both methods highlight inter-domain motion and show that the character of inter-domain motion at each domain boundary is different, with the x linker providing great freedom for twisting and tilting at the b'-a' interface. Two crystal structures show distinctive twist motion at the a-b interface which is predicted by the rapid method, but not by MD. Similar analyses are now underway on members of the human PDI family.Both theoretical analyses predict that PDI flexibility permits very significant change in the distances between functional sites. We are analysing this experimentally by single-molecule FRET techniques, with FRET probes attached to the redox-active sites and elsewhere. The data indicate that human PDI in solution can access a variety of conformations.

Disulfide Bonds are Allosteric Regulator of Mechanical Stability

Disulfide bonds are known to stabilize proteins against perturbations such as temperature or denaturants. Since mechanical force is the most common protein denaturant in vivo, there is increasing interest in the role that disulfide bonds have in the mechanical unfolding of proteins. For instance, disulfide bonds reduce the contour length of stretched proteins by limiting the extensibility up to the covalently linked cysteines. However very little is known about the effect of native disulfide bonds in the strength of structural clamps that determine mechanical stability. Here, we use single molecule atomic force spectroscopy to study the mechanical effects of disulfide bonding in immunoglobulin domains (Ig) that are constitutively under force. We observe that the formation of native disulfide bonds in immunoglobulin domains triggers drastic differences in the rupture forces, although the two cognate cysteines do not link together the β-strands of the mechanical clamp motif. In the case of bacterial gram-negative pilin FimH, disulfide increases the most probable unfolding force from 297 pN to 425 pN at 400nm/s. In contrast, the disulfide in the 69th immunoglobulin domain of human titin decreases the unfolding force at a pulling rate of 1200nm/s from 271 pN to 195 pN. We observe that both in titin and in the Fim pilus, the disulfide bonds are remarkably conserved along the entire stretched molecular architecture. Hence, many Igs in elastic segment of titin and in all Fim Igs are predicted to change mechanical stability depending of their oxidation state. Our results suggest that native disulfide bonds alter allosterically the transition state and modulate the internal flexibility of the stressed protein prior the breakage of its mechanical clamp motif.

Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4–PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Immuno EM–OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM)

In the atmospheric scanning electron microscope (ASEM), an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized the cytoskeletons formed by actin and tubulin, the chaperone PDI that catalyses native disulfide bond formation of proteins in the endoplasmic reticulum (ER) and the calcium sensor STIM1 that is integrated in ER membranes, using established cell lines. In particular, a dynamic string-like gathering of STIM1 was observed on the ER in Jurkat T cells in response to Ca(2+) store depletion. We have also visualized filamentous actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca(2+) signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework. Based on this work, ASEM correlative microscopy promises to allow the dynamics of various protein complexes to be investigated in the near future.

The H2O2-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol–disulfide milieu

Oxidative protein folding in the endoplasmic reticulum (ER) is associated with the formation of native disulfide bonds, which inevitably results in the formation of hydrogen peroxide (H2O2). Particularly in pancreatic β-cells with their high secretory activity and extremely low antioxidant capacity, the H2O2 molecules generated during oxidative protein folding could represent a significant oxidative burden. Therefore this study was conducted to elucidate the H2O2 generation during disulfide bond formation in insulin-producing RINm5F cells by targeting and specifically expressing the H2O2-sensitive biosensor HyPer in the ER (ER-HyPer). In addition the influence of overexpression of the H2O2-metabolizing ER-resident peroxiredoxin IV (PRDXIV) on H2O2 levels was examined. The ER-HyPer fluorescent protein was completely oxidized, whereas HyPer expressed in cytosol, peroxisomes, and mitochondria was prevalently in the reduced state, indicating a high basal H2O2 concentration in the ER lumen. These results could also be confirmed in non-insulin-producing COS-7 cells. Overexpression of PRDXIV in RINm5F cells effectively protected against H2O2-mediated toxicity; however, it did not affect the fluorescence signal of ER-HyPer. Moreover, the inhibition of de novo protein synthesis and the associated H2O2 generation by cycloheximide had no influence on the ER-HyPer redox state. Taken together, these findings strongly suggest that the H2O2-sensitive biosensor reflects exclusively the oxidative milieu in the ER and not the H2O2 concentration in the ER lumen.

Cell‐free expression of disulfide‐containing eukaryotic proteins for structural biology

We describe Escherichia coli based cell-free production of milligram quantities of eukaryotic proteins containing native disulfide bonds. Using a previously described expression system, we systematically investigated the influence of redox potential variation in the reaction mixture and the impact of adding disulfide bond catalysts on soluble protein production. It is then shown that the optimized reaction conditions for native disulfide bond formation can be combined with the use of N-terminal fusion constructs with the GB1 domain for increased expression yields. The resulting cell-free system is suitable for stable-isotope labeling and does not require chemical pretreatment of the cell extract to stabilize the redox potential. For the human doppel protein, the mouse doppel protein and mouse interleukin-22 we obtained 0.3-0.7 mg of purified native protein per milliliter of reaction mixture. Formation of disulfide bonds was validated using the Ellman assay, and native folding of the three proteins was monitored by NMR and CD spectroscopy.

Acceleration of Disulfide-Coupled Protein Folding by Positively Charged Glutathione Derivatives

Protein folding is a spontaneous process under appropriate condition and results in the formation of the native tertiary structure. However, disulfide-containing proteins require a redox system to form the correct disulfide pairings. Glutathione plays an important role in the formation of disulfide bonds in proteins in the endoplasmic reticulum. Because of this, it is also widely used as a redox reagent in the disulfide-coupled folding of proteins. However, the mechanism of the disulfide-coupled folding of proteins by glutathione is not completely understood. Therefore, to examine the glutathione mediated-folding of proteins containing disulfide bonds, a series of glutathione derivatives, Glu-Cys-Arg (ECR) and Arg-Cys-Gly (RCG) were prepared and their participation in protein folding was examined using lysozyme and prouroguanylin as model proteins.The folding recovery of lysozyme using the oxidized and reduced forms of RCG was higher than that of the typically used glutathione redox system, while the results for ECR were similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin indicated that the folding velocity using the RCG system was approximately 3 fold faster than that of the glutathione system. Furthermore, folding experiments using only the oxidized form of RCG revealed that prouroguanylin was converted into the native conformation more efficiently by RCG, compared to glutathione. We therefore conclude that the RCG system is superior to the glutathione system for protein folding because of its ability to accelerate the disulfide exchange reaction and its ability to mediate the formation of native disulfide bonds in proteins. In this paper, we report on the use of the positively charged glutathione molecule in disulfide-coupled protein folding, which results in an improved folding recovery and permits the formation of the correct tertiary structure of the target proteins.

Acceleration of disulfide‐coupled protein folding using glutathione derivatives

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of 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-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability 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 system. 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 prouroguanylin 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, folding experiments using only the oxidized form of RCG or glutathione indicated 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 mediating the formation of native disulfide bonds in proteins.