Disulfide Bridges In Proteins Research Articles

Recent advancements in disulfide bridge characterization: Insights from mass spectrometry.

Disulfide bridges (DSB) play an important role in stabilizing three-dimensional structures of biopharmaceuticals, single purified proteins, and various cyclic peptide drugs that contain disulfide in their structures. Incorrect cross-linking known as DSB scrambling results in misfolded structures that can be inactive, immunogenic, and susceptible to aggregation. Very few articles have been published on the experimental annotation of DSBs in proteins and cyclic peptide drugs. Accurate characterization of the disulfide bond is essential for understanding protein confirmation. Characterizing DSBs using mass spectrometry (MS) involves the chemical and enzymatic digestion of samples to obtain smaller peptide fragments, in both reduced and nonreduced forms. Subsequently, these samples are analyzed using MS to locate the DSB, either through interpretation or by employing various software tools. The main challenge in DSB analysis methods using sample preparation is to obtain a sample solution in which nonnative DSBs are not formed due to high pH, temperature, and presence of free sulfhydryl groups. Formation of nonnative DSBs can lead to erroneous annotation of disulfide bond. Sample preparation techniques, fragmentation methods for DSB analysis, and contemporary approaches for DSB mapping using this fragmentation were discussed. This review presents the latest advancement in MS-based characterization; also a critical perspective is presented for further annotation of DSBs using MS, primarily for single purified proteins or peptides that are densely connected and rich in cysteine. Despite significant breakthroughs resulting from advancements in MS, the analysis of disulfide bonds is not straightforward; it necessitates expertise in sample preparation and interpretation.

High-Density Immobilization of TCEP on Silica Beads for Efficient Disulfide Reduction and Thiol Alkylation in Peptides.

Tris-(2-carboxyethyl)phosphine (TCEP) linked to agarose beads is widely used for reducing disulfide bridges in proteins and peptides. The immobilization of TCEP on beads allows efficient removal after reduction to prevent its reaction with alkylating reagents and thus interference with conjugation reactions. However, a limitation of agarose TCEP is its relatively low reduction capacity per milliliter of wet beads (about 15 μmol/ml), making it unsuitable for the reduction of disulfides from molecules at millimolar concentrations. In this work, we tested the immobilization of TCEP to a range of different solid supports and found that conjugation to silica gel offers TCEP beads with about 8-fold higher reduction capacity (129±16 μmol/ml wet beads). We show that it allows reducing disulfide-cyclized peptides at millimolar concentrations for subsequent cyclization by bis-electrophile linker reagents. Given the substantially higher reduction capacity, the robust performance in different solvents, the low cost of the silica gel, and the ease of functionalization with TCEP, the silica gel-TCEP is suited for reducing disulfide bridges in essentially any peptide and is particularly useful for reducing peptides at higher concentrations.

Association of Abnormal Disulfide Bond Formation with Disease Development and Progression

As intermolecular and intramolecular disulfide bridges in proteins play a vital role in the stability of the final protein structure, the disruption of disulfide bridges in proteins may lead to disease development and progression. Therefore, understanding the association of abnormal protein disulfide bond formation with disease development and progression can be useful for developing novel drugs for various diseases. Considering that disulfide-linked protein folding involves redox reactions in the endoplasmic reticulum, this process may be affected by oxidative stress. We hypothesized that oxidative stress-related diseases may be induced by abnormal protein disulfide bond formation. This review introduces the association of abnormal protein disulfide bond formation with disease development and progression.

A Single-Molecule Strategy to Capture Non-native Intramolecular and Intermolecular Protein Disulfide Bridges.

Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.

Značaj tiol-disulfidne ravnoteže u SARS-CoV-2 infekciji

Studies of the molecular mechanisms regarding interaction of different viruses with receptors on the host cell surface have shown that the viral entry depends on the specific relationship between free thiol (SH) groups and disulfides on the virus surface, as well as the thiol disulfide balance on the host cell surface. The presence of oxidizing compounds or alkylating agents, which disturb the thiol-disulfide balance on the surface of the virus, can also affect its infectious potential. Disturbed thiol-disulfide balance may also influence protein-protein interactions between SARS-CoV-2 protein S and ACE2 receptors of the host cell. This review presents the basic mechanisms of maintaining intracellular and extracellular thiol disulfide balance and previous experimental and clinical evidence in favor of impaired balance in SARS-CoV-2 infection. Besides, the results of the clinical application or experimental analysis of compounds that induce changes in the thiol disulfide balance towards reduction of disulfide bridges in proteins of interest in COVID-19 infection are presented.

Residual Dipolar Couplings for Resolving Cysteine Bridges in Disulfide-Rich Peptides.

Disulfide bridges in proteins are formed by the oxidation of pairs of cysteine residues. These cross-links play a critical role in stabilizing the 3D-structure of small disulfide rich polypeptides such as hormones and venom toxins. The arrangement of the multiple disulfide bonds directs the peptide fold into distinct structural motifs that have evolved for resistance against biochemical and physical insults. These structural scaffolds have, therefore, proven to be very attractive in bioengineering efforts to develop novel biologics with applications in health and agriculture. Structural characterization of small disulfide rich peptides (DRPs) presents unique challenges when using commonly applied biophysical methods. NMR is the most commonly used method for studying such molecules, where the relatively small size of these molecules results in highly precise structural ensembles defined by a large number of distance and dihedral angle restraints per amino acid. However, in NMR the sulfur atoms that are involved in three of the five dihedral angles in a disulfide bond cannot be readily measured. Given the central role of disulfide bonds in the structure of these molecules, it is unclear what the inherent resolution of such NMR structures is when using traditional NMR methods. Here, we use an extensive set of long-range residual dipolar couplings (RDCs) to assess the resolution of the NMR structure of a disulfide-rich peptide. We find that structures based primarily on NOEs, yield ensembles that are equivalent to a crystallographic resolution of 2-3 Å in resolution, and that incorporation of RDCs reduces this to ~1-1.5 Å resolution. At this resolution the sidechain of ordered amino acids can be defined accurately, allowing the geometry of the cysteine bridges to be better defined, and allowing for disulfide-bond connectivities to be determined with high confidence. The observed improvements in resolution when using RDCs is remarkable considering the small size of these peptides.

Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor.

Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment.

Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii.

The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (k cat/K m) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.

S. cerevisiae whole-cell based capacitive biochip for the detection of toxicity of different forms of carbon nanotubes

A whole-cell based capacitive biochip (WCB) was employed for detecting nanotoxicity of different types of carbon nanotubes (CNTs). The WCB was made of arrays of capacitors that were functionalized with living S. cerevisiae (yeast) cells through coupling on their cell-surface protein disulfide bridges. Cells-on-chip were exposed to varying concentrations of single-walled (SW), multi-walled (MW) and double-walled (DW) CNTs. Dynamic cell-surface charge distributions as a result of cell-CNT interactions on chip were measured as change in relative capacitance under the applied AC-frequency. The WCB response provided a direct relationship between the integrity of cells-on-chip and their strong interaction with CNTs by adsorption/adhesion. Cellular damages imposed by CNTs was determined based on the magnitude of changes in relative capacitance against different types and concentrations of CNTs. Increasing toxicity experienced by cells-on-chip followed the order DWCNTs<MWCNTs<SWCNTs suggesting that cells were severely affected by SWCNTs followed by MWCNTs and DWCNTs. The above results were further validated through cell viability tests and fluorescence assays using quantum dot conjugated cells that enabled determination of the responses at the interface of cell-membranes against different types of CNTs. The developed WCB can be extended to high-throughput screening of toxic nanomaterials (NMs) in food and environmental samples.

Kinetic Characterization of Na,K-ATPase Inhibition by the Acetaminophen Metabolite N-Acetylbenzoquinoneimine

N-acetylbenzoquinoneimine (NABQI) is a toxic metabolite of the common analgesic acetaminophen (APAP). NABQI is an electrophilic intermediate formed via the oxidation of APAP within the cytochrome P450 system. Within the normally recommended low-dose use of APAP, NABQI is a minor metabolite which is either quickly reduced back to APAP or conjugated to Glutathione (GSH) producing an innocuous by-product. However, with overdose or prolonged high-dose usage of acetaminophen, GSH levels can become depleted and the bioactive NABQI is thought to form adducts with proteins and oxidize protein sulfhydryls producing intra- and intermolecular disulfide bridges in proteins. In this work we investigated the effect of NABQI on purified kidney Na,K-ATPase to see if the clinical renal insufficiencies seen in APAP overdose may be linked to inhibition of the Na,K-ATPase. Our work has shown that NABQI does indeed inhibit the Na,K-ATPase in a dose dependent (IC50 = 19.8 ± 2.9 μM) and irreversible manner. Interestingly, brief storage of NABQI at -20°C eliminates the irreversible effects of the compound, and leads to a product that remains a potent reversible inhibitor of the Na,K-ATPase (IC50 = 58.7 ± 19.5 μM). Further, the reversible inhibition produced by stored NABQI competes with para-nitrop.

Redox Modulation Matters: Emerging Functions for Glutaredoxins in Plant Development and Stress Responses.

Glutaredoxins (GRXs) are small ubiquitous glutathione (GSH)-dependent oxidoreductases that catalyze the reversible reduction of protein disulfide bridges or protein-GSH mixed disulfide bonds via a dithiol or monothiol mechanism, respectively. Three major classes of GRXs, with the CPYC-type, the CGFS-type or the CC-type active site, have been identified in many plant species. In spite of the well-characterized roles for GRXs in Escherichia coli, yeast and humans, the biological functions of plant GRXs have been largely enigmatic. The CPYC-type and CGFS-type GRXs exist in all organisms, from prokaryotes to eukaryotes, whereas the CC-type class has thus far been solely identified in land plants. Only the number of the CC-type GRXs has enlarged dramatically during the evolution of land plants, suggesting their participation in the formation of more complex plants adapted to life on land. A growing body of evidence indicates that plant GRXs are involved in numerous cellular pathways. In this review, emphasis is placed on the recently emerging functions for GRXs in floral organ development and disease resistance. Notably, CC-type GRXs have been recruited to participate in these two seemingly unrelated processes. Besides, the current knowledge of plant GRXs in the assembly and delivery of iron-sulfur clusters, oxidative stress responses and arsenic resistance is also presented. As GRXs require GSH as an electron donor to reduce their target proteins, GSH-related developmental processes, including the control of flowering time and the development of postembryonic roots and shoots, are further discussed. Profiling the thiol redox proteome using high-throughput proteomic approaches and measuring cellular redox changes with fluorescent redox biosensors will help to further unravel the redox-regulated physiological processes in plants.

N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide, a molecule for radiohalogenation of proteins and peptides

In this work a new coupling reagent, N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide, for radiohalogenation has been synthesized and characterized. The reagent is intended to either be attached to reduced disulfide bridges of proteins (making the halogenation site-specific) or to free terminal cysteine groups on peptides. The new reagent was also shown to be easily halogenated with inactive bromine and iodine as well as 125I and 211At, indicating potential use within targeted radiotherapy.

Facilitating Protein Disulfide Mapping by a Combination of Pepsin Digestion, Electron Transfer Higher Energy Dissociation (EThcD), and a Dedicated Search Algorithm SlinkS

Disulfide bond identification is important for a detailed understanding of protein structures, which directly affect their biological functions. Here we describe an integrated workflow for the fast and accurate identification of authentic protein disulfide bridges. This novel workflow incorporates acidic proteolytic digestion using pepsin to eliminate undesirable disulfide reshuffling during sample preparation and a novel search engine, SlinkS, to directly identify disulfide-bridged peptides isolated via electron transfer higher energy dissociation (EThcD). In EThcD fragmentation of disulfide-bridged peptides, electron transfer dissociation preferentially leads to the cleavage of the S-S bonds, generating two intense disulfide-cleaved peptides as primary fragment ions. Subsequently, higher energy collision dissociation primarily targets unreacted and charge-reduced precursor ions, inducing peptide backbone fragmentation. SlinkS is able to provide the accurate monoisotopic precursor masses of the two disulfide-cleaved peptides and the sequence of each linked peptide by matching the remaining EThcD product ions against a linear peptide database. The workflow was validated using a protein mixture containing six proteins rich in natural disulfide bridges. Using this pepsin-based workflow, we were able to efficiently and confidently identify a total of 31 unique Cys-Cys bonds (out of 43 disulfide bridges present), with no disulfide reshuffling products detected. Pepsin digestion not only outperformed trypsin digestion in terms of the number of detected authentic Cys-Cys bonds, but, more important, prevented the formation of artificially reshuffled disulfide bridges due to protein digestion under neutral pH. Our new workflow therefore provides a precise and generic approach for disulfide bridge mapping, which can be used to study protein folding, structure, and stability.

Influence of overalkylation in enzymatic digestion on the qualitative and quantitative analysis of proteins

Reduction and alkylation with iodoacetamide (IAA) of the disulfide bridges in proteins are important procedures used in protein digestion. But the alkylation with IAA takes place not only on cysteine residues but also on other amino acid residues. Here we conducted a systematic study of all alkylated peptides under the usual protein digestion conditions. It showed that the potentials for alkylation reaction of different amino acid residues were cysteine > N-terminal amino acid > aspartic acid > glutamic acid > histidine > asparagine > lysine > tyrosine. Furthermore, we found that the alkylation reaction happened either exclusively or cooperatively among different amino acid residues in the same peptide. Based on the qualitative results on overalkylation at several peptides, the targeted multiple reaction monitoring (MRM) technique was used to evaluate the effect of overalkylation on quantitative protein analysis. The results showed that overalkylation has large effect on the qualitative and quantitative analysis of proteins and should be avoided in enzymatic digestion.

Automatic Determination of Disulfide Bridges in Proteins

Precise determination of disulfide linkages between cysteine (Cys) residues in proteins is essential in the determination of protein structure. Therefore, a reliable automated method for the identification of disulfide bridges can serve as an important tool in the analysis of the tertiary structure of proteins of interest. Here, we describe the current and past methods used to identify disulfide bridges in proteins, with a focus on mass spectrometry (MS)-based methods and a particular emphasis on nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS)-based methods. We also show the development of an easy method based on the separation of disulfide-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing and nonreducing conditions and selective in-gel digestion of proteins using reducing and nonreducing conditions, followed by analysis of the resulting peptide mixture by nanoACQUITY UPLC coupled to a quadrupole time-of-flight (QTOF) Micro mass spectrometer (nanoLC-MS/MS). Data-dependent analysis (DDA) nanoLC-MS/MS and information-dependent analysis (IDA) nanoLC-MS/MS were used for random and targeted identification of disulfide-linked peptides. Finally, an example of electrospray-MS (ESI-MS) and ESI-MS/MS-based determination of disulfide-linked peptides is shown.

Fibril Formation of Bovine α-Lactalbumin Is Inhibited by Glutathione

The current research is aimed at exploring the inhibitory effect of glutathione on fibril formation of an important four disulfide bond-containing whey protein, α-lactalbumin. Through numerous spectroscopic techniques and transmission electron microscopy, we found that the inhibition of amyloid formation of α-lactalbumin was dependent on the glutathione concentration and fibrillation was significantly attenuated in the presence of 5 mM glutathione. Moreover, the data from the measurements using 4,4′-dithiodipyridine reagent revealed that the treatment of α-lactalbumin with glutathione led to the exposure of sulfhydryl groups. Also, the observed inhibition of α-lactalbumin by glutathione was correlated with the reduction of disulfide bridges of protein. The results presented here suggest that the addition of food compatible reducing agent/dietary supplement such as glutathione would be useful for preventing the formation of milk protein fibrillar aggregates. The presence of these resulting aggregates can then, in turn, be used to modulate the key properties of food products such as protein beverage and yogurt.

Lipogenesis Is Decreased by Grape Seed Proanthocyanidins According to Liver Proteomics of Rats Fed a High Fat Diet

Bioactive proanthocyanidins have been reported to have several beneficial effects on health in relation to metabolic syndrome, type 2 diabetes, and cardiovascular disease. We studied the effect of grape seed proanthocyanidin extract (GSPE) in rats fed a high fat diet (HFD). This is the first study of the effects of flavonoids on the liver proteome of rats suffering from metabolic syndrome. Three groups of rats were fed over a period of 13 weeks either a chow diet (control), an HFD, or a high fat diet supplemented for the last 10 days with GSPE (HFD + GSPE). The liver proteome was fractionated, using a Triton X-114-based two-phase separation, into soluble and membrane protein fractions so that total proteome coverage was considerably improved. The data from isobaric tag for relative and absolute quantitation (iTRAQ)-based nano-LC-MS/MS analysis revealed 90 proteins with a significant (p < 0.05) minimal expression difference of 20% due to metabolic syndrome (HFD versus control) and 75 proteins due to GSPE treatment (HFD + GSPE versus HFD). The same animals have previously been studied (Quesada, H., del Bas, J. M., Pajuelo, D., Díaz, S., Fernandez-Larrea, J., Pinent, M., Arola, L., Salvadó, M. J., and Bladé, C. (2009) Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver. Int. J. Obes. 33, 1007-1012), and GSPE was shown to correct dyslipidemia observed in HFD-fed rats probably through the repression of hepatic lipogenesis. Our data corroborate those findings with an extensive list of proteins describing the induction of hepatic glycogenesis, glycolysis, and fatty acid and triglyceride synthesis in HFD, whereas the opposite pattern was observed to a large extent in GSPE-treated animals. GSPE was shown to have a wider effect than previously thought, and putative targets of GSPE involved in the reversal of the symptoms of metabolic syndrome were revealed. Some of these novel candidate proteins such as GFPT1, CD36, PLAA (phospholipase A(2)-activating protein), METTL7B, SLC30A1, several G signaling proteins, and the sulfide-metabolizing ETHE1 and SQRDL (sulfide-quinone reductase-like) might be considered as drug targets for the treatment of metabolic syndrome.

MetalDetector: a web server for predicting metal-binding sites and disulfide bridges in proteins from sequence

The web server MetalDetector classifies histidine residues in proteins into one of two states (free or metal bound) and cysteines into one of three states (free, metal bound or disulfide bridged). A decision tree integrates predictions from two previously developed methods (DISULFIND and Metal Ligand Predictor). Cross-validated performance assessment indicates that our server predicts disulfide bonding state at 88.6% precision and 85.1% recall, while it identifies cysteines and histidines in transition metal-binding sites at 79.9% precision and 76.8% recall, and at 60.8% precision and 40.7% recall, respectively. Freely available at http://metaldetector.dsi.unifi.it. Details and data can be found at http://metaldetector.dsi.unifi.it/help.php.

Site-Specific Pegylation of G-CSF by Reversible Denaturation

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.

Esterase activity of bovine serum albumin up to 160 °C: A new benchmark for biocatalysis

Albumins are highly conserved proteins that carry out a multitude of physiological functions and exhibit a broad range of catalytic activities. Moreover, amino acid sequence comparisons of the albumin multigene family indicate that albumins diverged from a common ancestor. Here we report that bovine serum albumin (BSA) can catalyze ester hydrolysis at high temperatures (as high as 160 °C) well beyond the temperature limits reported for enzymatic catalysis, including for enzymes from known hyperthermophiles. Furthermore, BSA exhibited a ∼133-fold increase in its turnover number ( k cat) toward p-nitrophenyl palmitate from 70 to 150 °C. When BSA was incubated for 1 h at 150 °C in the presence of 25 mM SDS, it retained complete esterase activity, indicating that a catalytically competent orientation of amino acid residues exists in the denatured or partially unfolded protein. However, esterase activity diminished to ∼50% upon disruption of the protein's disulfide bridges and disappeared completely when BSA was digested by proteases. These results point to a new standard of robustness for biocatalytic activity at high temperatures. Catalytic activity and promiscuity at very high temperatures could have been advantageous to enzymes in primitive organisms evolving in hot environments, making BSA an intriguing model for early enzymes.