Native disulfide bond formation in proteins

Native disulfide bond formation is critical for the proper folding of many proteins. Recent studies using newly identified protein oxidants, folding catalysts, and mutant cells provide insight into the mechanism of oxidative protein folding in vivo. This insight promises new strategies for more efficient protein production.     

Statistics of disulfide bond formation in proteins

A new set of statistical expressions describing the reformation of disulfide bonds from SH groups is proposed. The results of the statistical calculations of disulfide bond reformation are discussed in terms of protein folding.     

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system

Background  

Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.     

Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.     

The disulfide bond formation (Dsb) system

In oxidative folding of proteins in the bacterial periplasmic space, disulfide bonds are introduced by the oxidation system and isomerized by the reduction system. These systems utilize the oxidizing and the reducing equivalents of quinone and NADPH, respectively, that are transmitted across the cytoplasmic membrane through integral membrane components DsbB and DsbD. In both pathways, alternating interactions between a Cys-XX-Cys-containing thioredoxin domain and other regulatory domain lead to the maintenance of oxidized and reduced states of the specific terminal enzymes, DsbA that oxidizes target cysteines and DsbC that reduces an incorrect disulfide to allow its isomerization into the physiological one. Molecular details of these remarkable biochemical cascades are being rapidly unraveled by genetic, biochemical, and structural analyses in recent years.     

Intracellular manipulation of disulfide bond formation in rotavirus proteins during assembly.

Rotavirus undergoes a unique mode of assembly in the rough endoplasmic reticulum (RER) of infected cells. Luminal RER proteins undergo significant cotranslational and posttranslational modifications, including disulfide bond formation. Addition of a reducing agent (dithiothreitol [DTT]) to rotavirus-infected cells did not significantly inhibit translation or disrupt established disulfide bonds in rotavirus proteins but prevented the formation of new disulfide bonds and infectious viral progeny. In DTT-treated, rotavirus-infected cells, all vp4, vp6, and ns28 epitopes but no vp7 epitopes were detected by immunohistochemical staining with a panel of monoclonal antibodies. When oxidizing conditions were reestablished in DTT-treated cells, intramolecular disulfide bonds in vp7 were rapidly and correctly established with the restoration of antigenicity, although prolonged DTT treatment led to the accumulation of permanently misfolded vp7. Electron microscopy revealed that cytosolic assembly of single-shelled particles and budding into the ER was not affected by DTT treatment but that outer capsid assembly was blocked, leading to the accumulation of single-shelled and enveloped intermediate subviral particles in the RER lumen.     

Reversible disulfide bond formation of intracellular proteins probed by NMR spectroscopy

Reversible oxidation of amino acids within intracellular proteins leads to local and/or global conformational changes in protein structure. Thus, the enzymatic activity or binding properties of a protein might be regulated by local changes in a cell's redox potential, mediated by the availability of reducing/oxidizing equivalents. Whereas it is well established that intracellular pools of oxidizable groups compensate for oxidative stress, far less is known about the molecular mechanisms that accompany transient and reversible oxidation of cytoplasmic proteins. Therefore, the intrinsic redox properties of proteins amenable to reversible oxidation need to be determined. Here we describe the application of NMR spectroscopy to derive the redox properties of intracellular proteins. As exemplified for thioredoxin 1, the Tnk-1 kinase SH3 domain, and the hSH3(N) domain of the T cell protein ADAP, the conformational changes associated with disulfide bond formation can be followed directly upon titration with different ratios of reduced to oxidized glutathione. Redox potentials can be measured accurately in homogeneous solutions and define the conditions under which regulatory oxidation of the respective protein may occur in the living cell.     

Multi-hour translation of mRNA in a cell-free system

In this study, we demonstrate that mRNA molecules can serve as an efficient template for cell-free translation through a combination of methods to protect them from nucleolytic digestion. Removal of major endonucleases activity from cell extract, the addition of a stemloop structure at the 3??-end of the mRNA and continuous reloading of ribosomes onto mRNA were found to be crucial for maintaining the functional integrity of mRNA during cell-free synthesis. When these three approaches were combined, mRNA-directed protein synthesis continued over 15 h, leading to the production of 2.6 mg/mL of encoded protein. The methods for direct translation of mRNA presented herein will provide a useful option for deciphering genetic information, including the fields of mRNA display and materialization of metagenomic information.     

Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ_D) of ～200 µsec for Rhodamine‐labeled IgG, corresponding to a hydrodynamic radius (R_H) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ_D under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation‐dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169–177. © 2014 Wiley Periodicals, Inc.     

An intersubunit disulfide bond prevents in vitro aggregation of a superoxide dismutase-1 mutant linked to familial amytrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS) is linked to over 90 point mutations in superoxide dismutase-1 (SOD1), a dimeric metalloenzyme. The postmortem FALS brain is characterized by SOD1 inclusions in the motor neurons of regions in which neuronal loss is most significant. These findings, together with animal modeling studies, suggest that aggregation of mutant SOD1 produces a pathogenic species. We demonstrate here that a mutant form of SOD1 (A4V) that is linked to a particularly aggressive form of FALS aggregates in vitro, while wild-type SOD1 (WT) is stable. Some A4V aggregates resemble amyloid pores formed by other disease-associated proteins. The WT dimer is significantly more stable than the A4V dimer, suggesting that dimer dissociation may be the required first step of aggregation. To test this hypothesis, an intersubunit disulfide bond between symmetry-related residues at the A4V dimer interface was introduced. The resultant disulfide bond (V148C-V148C') eliminated the concentration-dependent loss of enzymatic activity of A4V, stabilized the A4V dimer, and completely abolished aggregation. A drug-like molecule that could stabilize the A4V dimer could slow the onset and progression of FALS.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Cooperative disulfide bond formation in apamin.

Apamin is being studied as a model for the folding mechanism of proteins whose structures are stabilized by disulfide bonds. Apamin consists of 18 amino acid residues and forms a stable structure consisting of a C-terminal alpha-helix and two reverse turns. This structure is stabilized by two disulfide bonds connecting Cys-1 to Cys-11 and Cys-3 to Cys-15. We used glutathione and dithiothreitol as reference thiols to measure the stabilities of the two disulfide bonds as a function of urea concentration and temperature in order to understand what contributes to the stability of the native structure. The results demonstrate modest contributions from secondary structure to the overall stability of the two disulfide bonds. The equilibrium constants for disulfide bond formation between the fully reduced peptide and the native structure with two disulfide bonds at 25 degrees C and pH 7.0 are 0.42 M2 using glutathione and 2.7 x 10(-5) using dithiothreitol. The equilibrium constant decreases by a factor of approximately 4 in 8 M urea and decreases by a factor of 3 between 0 and 60 degrees C. At least three one-disulfide intermediates are found at low concentrations in the equilibrium mixture. Using glutathione, the equilibrium constants for forming the one-disulfide intermediates with respect to the reduced peptide are approximately 0.025 M. The second disulfide bond forms with an equilibrium constant of approximately 17 M. Thus, apamin folding is very cooperative, but the native structure is only modestly stabilized by urea- or temperature-denaturable secondary structure.     

Efficient disulfide bond formation in virus-like particles

Virus-like particles (VLPs) consist of a virus's outer shell but without the genome. Similar to the virus, VLPs are monodisperse nano-capsules which have a known morphology, maintain a high degree of symmetry, and can be engineered to encapsidate the desired cargo. VLPs are of great interest for vaccination, drug/gene delivery, imaging, sensing, and material science applications. Here we demonstrate the ability to control the disulfide bond formation in VLPs by directly controlling the redox potential during or after production and assembly of VLPs. The open cell-free protein synthesis environment, which has been reported to produce VLPs at yields comparable or greater than traditional in vivo technologies, was employed. Optimal conditions for disulfide bond formation were found to be VLP dependent, and a cooperative effect in the formation of such bonds was observed.     

Enhancing multiple disulfide bonded protein folding in a cell-free system

A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction.     

Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.     

Pathways of disulfide bond formation in Escherichia coli

Disulfide bond formation is required for the correct folding of many secreted proteins. Cells possess protein-folding catalysts to ensure that the correct pairs of cysteine residues are joined during the folding process. These enzymatic systems are located in the endoplasmic reticulum of eukaryotes or in the periplasm of Gram-negative bacteria. This review focuses on the pathways of disulfide bond formation and isomerization in bacteria, taking Escherichia coli as a model.