Role Of Disulfide Bonds Research Articles

The role of disulfide bonds in the formation of the spatial structure of the human epidermal growth factor

The epidermal growth factor (EGF) is a peptide of the EGF-like growth factor family with a common conserved EGF-like domain formed by three intramolecular disulfide bonds. This article describes changes in the spatial structure of EGF and its mutant form with the D46G substitution in its C-terminal fragment observed upon disulfide bonds reduction in the corresponding synthetic peptides in a 0.01 M phosphate buffer (pH = 7.4). The structure was analyzed using circular dichroism spectroscopy, spectrofluorimetry, native polyacrylamide gel electrophoresis, and centrifugal ultrafiltration. It was shown that disulfide bonds reduction changes the geometry of the EGF-like domain towards an increase in the content of the beta-structure, while these peptides remain in dimeric form. According to the molecular modeling results, this can lead to the elongation of the main beta-hairpin of the EGF-like domain, to the elongation of the intermolecular beta-structure, or to the formation of a new beta-structure between the N- and C-terminal fragments of each molecule, which will change the intermolecular interface in dimeric form. Disulfide bonds reduction prevents EGF dimer dissociation to monomers. Under physiological conditions, this can lead to the inability of EGF to form binding sites for EGFR (epidermal growth factor receptor) and to cause its activation.

Electrochemical analysis on the role of disulfide bonds in polysulfide-co-polyoxide as cathode active binder for enhanced storage capacity of lithium-metal batteries

The present article is aimed at elucidation on the role of disulfide bonds in enhancement of energy storage capacity of lithium metal battery using polysulfide-co-polyoxide as cathode active binder in lithium metal batteries. Upon photocuring via ‘thiol-ene’ reaction, the resulting thioplast-co-trimethylolpropane ethoxylate triacrylate (TMPETA) co-network is flexible containing disulfide and ether oxygen moieties. Ionic conductivity of the polymer electrolyte membrane containing Thioplast-co-TMPETA network/plasticizer/lithium salt reaches the superionic conductor level of 10−3 S cm−1 at room temperature. To further improve the electrochemical performance, Thioplast-co-TMPETA co-networks were used as a binder, in lieu of polyvinylidene fluoride (PVDF), for lithium iron phosphate (LFP) cathode. Of particular interest is the achievement of a high overall specific capacity of ∼265 mAh g−1 initially which stabilizes at ∼185 mAh g−1 after 35 cycles. These values certainly exceeded the theoretical value of 170 mAh g−1 for neat LFP cathode. This capacity enhancement may be attributed to the disulfide bonds of polysulfide constituent undergoing SS bond scission followed by electron acceptor-donor interactions between sulfide radicals and Li cations. Moreover, the polysulfide-co-polyoxide binder also works as a cathode active material with a specific capacity of 122–103 mAh g−1 in the potential range of 1.0–3.0 V showing reasonable capacity retention of ∼85% and Coulombic efficiency of ∼97%.

Role of Disulfide Bonds and Sulfhydryl Blocked by N-Ethylmaleimide on the Properties of Different Protein-Stabilized Emulsions.

To investigate the role of sulfhydryl groups and disulfide bonds in different protein-stabilized emulsions, N-ethylmaleimide (NEM) was used as a sulfhydryl-blocking agent added in the emulsion. The addition of NEM to block the sulfhydryl groups resulted in a reduction in disulfide bond formation, which enabled the internal structure of the protein molecule to be destroyed, and then decreased the restriction of protein membrane on the oil droplets. Furthermore, with the NEM content increasing in the emulsion, a reduction in the protein emulsifying activity and emulsion stability also occurred. At the same time, the intermolecular interaction of the protein on the oil droplet interface membrane was destroyed, and the emulsion droplet size increased with the NEM content in the emulsion. Although NEM blocking sulfhydryl groups from forming disulfide bonds has similar effects on three types of protein emulsion, the degree of myofibrillar protein (MP), egg-white protein isolate (EPI), and soybean protein isolate (SPI) used as emulsifiers had a subtle difference.

Removal of disulfide from acid stress chaperone HdeA does not wholly eliminate structure or function at low pH

HdeA is an acid-stress chaperone that operates in the periplasm of various strains of pathogenic gram-negative bacteria. Its primary function is to prevent irreversible aggregation of other periplasmic proteins when the bacteria enter the acidic environment of the stomach after contaminated food is ingested; its role is therefore to help the bacteria survive long enough to enter and colonize the intestines. The mechanism of operation of HdeA is unusual in that this helical homodimer is inactive when folded at neutral pH but becomes activated at low pH after the dimer dissociates and partially unfolds. Studies with chemical reducing agents previously suggested that the intramolecular disulfide bond is important for maintaining residual structure in HdeA at low pH and may be responsible for positioning exposed hydrophobic residues together for the purpose of binding unfolded client proteins. In order to explore its role in HdeA structure and chaperone function we performed a conservative cysteine to serine mutation of the disulfide. We found that, although residual structure is greatly diminished at pH 2 without the disulfide, it is not completely lost; conversely, the mutant is almost completely random coil at pH 6. Aggregation assays showed that mutated HdeA, although less successful as a chaperone than wild type, still maintains a surprising level of function. These studies highlight that we still have much to learn about the factors that stabilize residual structure at low pH and the role of disulfide bonds.

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics.

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

A disulfide constrains the ToxR periplasmic domain structure, altering its interactions with ToxS and bile-salts

ToxR is a transmembrane transcription factor that, together with its integral membrane periplasmic binding partner ToxS, is conserved across the Vibrionaceae family. In some pathogenic Vibrios, including V. parahaemolyticus and V. cholerae, ToxR is required for bile resistance and virulence, and ToxR is fully activated and protected from degradation by ToxS. ToxS achieves this in part by ensuring formation of an intra-chain disulfide bond in the C-terminal periplasmic domain of ToxR (dbToxRp). In this study, biochemical analysis showed dbToxRp to have a higher affinity for the ToxS periplasmic domain than the non-disulfide bonded conformation. Analysis of our dbToxRp crystal structure showed this is due to disulfide bond stabilization. Furthermore, dbToxRp is structurally homologous to the V. parahaemolyticus VtrA periplasmic domain. These results highlight the critical structural role of disulfide bond in ToxR and along with VtrA define a domain fold involved in environmental sensing conserved across the Vibrionaceae family.

Templateless Synthesis of Organosilica Nanotoroids. The Effect of Precursor Concentration

The features of (γ-mercaptopropyl)trimethoxysilane (MPTMS) hydrolytic condensation in alkaline aqueous solutions have been studied. The interrelation between the process time, MPTMS concentration, and structure-related morphological characteristics of the obtained organosilica (silsesquioxane) particles has been determined. It has been shown that toroidal particles are mainly formed at low precursor concentrations (≤2.5 mM) in a solution. The formation kinetics of such nanotoroids has been analyzed, and preliminary data have been obtained on the mechanism of this process. These data have been discussed taking into account the information available from the literature on the structure of silsesquioxanes and the role of disulfide bonds in the formation of anisotropic particles of these compounds.

Sulfur dioxide induced aggregation of wine thaumatin-like proteins: Role of disulfide bonds

Aggregation of heat unstable wine proteins is responsible for the economically and technologically detrimental problem called wine protein haze. This is caused by the aggregation of thermally unfolded proteins that can precipitate in bottled wine. To study the influence of SO2 in this phenomenon, wine proteins were isolated and thaumatins were identified has the most prone to aggregate in the presence of this compound. Isolated wine thaumatins aggregation was followed by dynamic light scattering (DLS), circular dichroism (CD), fluorescence spectroscopy and size exclusion chromatography (SEC). Our experimental results demonstrate that protein thermal unfolding after exposure of the protein to 70 °C does not present differences whether SO2 is present or not. Conversely, when the protein solution is cooled to 15 °C (after heat stress) significant analytical changes can be observed between samples with and without SO2. A remarkable change of circular dichroism spectra in the region 220–230 nm is observed (which can be related to S-S torsion angles), as well as an increase in tryptophan fluorescence intensity (absence of fluorescence quenching by S-S bonds). Formation of covalently-linked dimeric and tetrameric protein species were also detected by SEC. The ability to dissolve the aggregates with 8 M urea seems to indicate that hydrophobic interactions are prevalent in the formed aggregates. Also, the reduction of these aggregates with tris (2-carboxyethyl) phosphine (TCEP) to only monomeric species reveals the presence of intermolecular S-S bonds.

The Role of Intermolecular Disulfide Bonds in Stabilizing the Structure of Peroxiredoxins

The comparative characterization of thermal stability of human peroxiredoxins 1–6 (Prx1–Prx6) has been performed by physicochemical and biochemical methods and the role of disulfide bonds in stabilizing their structure has been shown. Prx1 and Prx2 among the tested peroxiredoxins exhibit the highest peroxidase activity and thermal stability. Prx1 and Prx2 are more than 2 times more active on average with H2O2 and tert-butyl hydroperoxide as substrates compared to other peroxiredoxins and retain at least 50% activity after 30 min heating at a temperature of 64°C, which is more than 10°C higher compared to Prx3–Prx6. The reduction of the disulfide bonds between Prx1 and Prx2 leads to a decrease of their thermal stability, comparable to the thermal stability of Prx3–Prx6, which confirms the important role of the intermolecular S–S bonds in stabilizing the structure of these proteins.

Role of Disulfide Bonds on DNA Packaging Forces in Bull Sperm Chromatin

Short arginine-rich proteins called protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin.

Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions: The Roles of Disulfide Bonds and Effects of Denaturant.

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.

Role of Disulfide Bonds in Stabilizing the Conformation of Selected Enzymes—An Approach Based on Divergence Entropy Applied to the Structure of Hydrophobic Core in Proteins

One of the factors responsible for tertiary structural stabilization in proteins is the presence of the hydrophobic core—a result of hydrophobic interactions within the protein body. In some proteins (especially extracellular ones) additional stabilization is provided by covalent bonds between selected Cys residues, commonly referred to as disulfide bonds. The mutual interplay of both factors and their respective contributions to stabilization are the focus of this work. The assessment of the effects of disulfide bonds isinterpreted by Fuzzy Oil Drop (FOD) model in which individual polypeptide chain fragments (including fragments which participate in SS bonds) can be evaluated in the context of their influence upon tertiary structural stabilization by comparing their corresponding theoretical and idealized hydrophobicity density distributions. The proteins were identified with both factors reinforcing each other, as well as proteins where they seem to counteract each other. The analysis presents a number of enzymes, including ribonuclease, lysozyme, disulfide isomerase and phospholipase.

The role of disulfide bonds in structure and activity of chlorotoxin.

Chlorotoxin is a small scorpion peptide that inhibits glioma cell migration. We investigated the importance of a major component of chlorotoxin's chemical structure - four disulfide bonds - to its tertiary structure and biological function. Five disulfide bond analogs of chlorotoxin were synthesized, with l-α-aminobutyric acid residues replacing each or all of the disulfide bonds. Chemical oxidation and circular dichroism experiments revealed that Cys III-VII and Cys V-VIII were essential for native structure formation. Cys I-IV and Cys II-VI were important for stability of enzymatic proteolysis but not for the inhibition of human umbilical vein endothelial cell migration. The disulfide bonds of chlorotoxin are important for its structure and stability and have a minor role in its activity against cell migration.

Folding of peptides and proteins: role of disulfide bonds, recent developments.

Disulfide-containing proteins are ideal models for studies of protein folding as the folding intermediates can be observed, trapped, and separated by HPLC during the folding reaction. However, regulating or analyzing the structures of folding intermediates of peptides and proteins continues to be a difficult problem. Recently, the development of several techniques in peptide chemistry and biotechnology has resulted in the availability of some powerful tools for studying protein folding in the context of the structural analysis of native, mutant proteins, and folding intermediates. In this review, recent developments in the field of disulfide-coupled peptide and protein folding are discussed, from the viewpoint of chemical and biotechnological methods, such as analytical methods for the detection of disulfide pairings, chemical methods for disulfide bond formation between the defined Cys residues, and applications of diselenide bonds for the regulation of disulfide-coupled peptide and protein folding.

Insights into Stabilization of a Viral Protein Cage in Templating Complex Nanoarchitectures: Roles of Disulfide Bonds

As a typical protein nanostructure, virus-based nanoparticle (VNP) of simian virus 40 (SV40), which is composed of pentamers of the major capsid protein of SV40 (VP1), has been successfully employed in guiding the assembly of different nanoparticles (NPs) into predesigned nanostructures with considerable stability. However, the stabilization mechanism of SV40 VNP remains unclear. Here, the importance of inter-pentamer disulfide bonds between cysteines in the stabilization of quantum dot (QD)-containing VNPs (VNP-QDs) is comprehensively investigated by constructing a series of VP1 mutants of cysteine to serine. Although the presence of a QD core can greatly enhance the assembly and stability of SV40 VNPs, disulfide bonds are vital to stability of VNP-QDs. Cysteine at position 9 (C9) and C104 contribute most of the disulfide bonds and play essential roles in determining the stability of SV40 VNPs as templates to guide assembly of complex nanoarchitectures. These results provide insightful clues to understanding the robustness of SV40 VNPs in organizing suprastructures of inorganic NPs. It is expected that these findings will help guide the future design and construction of protein-based functional nanostructures.

Deciphering structural and functional roles of disulfide bonds in decorsin

Decorsin, an antagonist of integrin glycoprotein IIb/IIIa, contains Arg-Gly-Asp (RGD) sequence and three disulfide bridges. The function of RGD sequence has already been well defined, but the roles of conserved disulfide bonds in antihemostatic proteins still remain unclear. Herein we use the fusion expression and characterization of mutant decorsin to study the functions of disulfide bonds in protein structure, stability and biological activity. The purified protein shows an apparent inhibition of activity to platelet aggregation induced by ADP with IC50 of 500 nM. The removal of cys7-cys15 (from cysteine to serine) at the N-terminal causes a thirty-fold decrease of the inhibition activity with IC50 of 15 μM, whereas the mutation of cys22-cys38 at the C-terminal completely impairs the biological activity of decorsin. The overall secondary and tertiary structures of decorsin are disrupted inevitably without disulfide bonds. Using a domain insertion mutation, the retaining of RGD loop and the adjacent disulfide bond produces a week antihemostatic activity of decorsin. This reveals that the overall structure of decorsin stabilized by the three conserved disulfide bridges is cooperative for antihemostatic function. Our study on the effect of disulfide bonds together with RGD-sequence on the protein function is helpful for structure-based drug design of antithrombotic research.

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing conditions these values decrease by 4°C and 8°C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90°C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.

Molecular Dynamics Simulations on Pars Intercerebralis Major Peptide-C (PMP-C) Reveal the Role of Glycosylation and Disulfide Bonds in its Enhanced Structural Stability and Function

Fucosylation of Thr 9 in pars intercerebralis major peptide-C (PMP-C) enhances its structural stability and functional ability as a serine protease inhibitor. In order to understand the role of disulfide bonds and glycosylation on the structure and function of PMP-C, we have carried out multiple explicit solvent molecular dynamics (MD) simulations on fucosylated and non-fucosylated forms of PMP-C, both in the presence and absence of the disulfide bonds. Our simulations revealed that there were no significant structural changes in the native disulfide bonded forms of PMP-C due to fucosylation. On the other hand, the non-fucosylated form of PMP-C without disulfide bonds showed larger deviations from the starting structure than the fucosylated form. However, the structural deviations were restricted to the terminal regions while core β-sheet retained its hydrogen bonded structure even in absence of disulfide bonds as well as fucosylation. Interestingly, fucosylation of disulfide bonded native PMP-C led to a decreased thermal flexibility in the residue stretch 29–32 which is known to interact with the active site of the target proteases. Our analysis revealed that disulfide bonds covalently connect the residue stretch 29–32 to the central β-sheet of PMP-C and using a novel network of side chain interactions and disulfide bonds fucosylation at Thr 9 is altering the flexibility of the stretch 29–32 located at a distal site. Thus, our simulations explain for the first time, how presence of disulfide bonds between conserved cysteines and fucosylation enhance the function of PMP-C as a protease inhibitor.

Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop

Heterologous expression of factor VIII (FVIII) is about two to three orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels. Disulfide bonds are vital to the proper folding, secretion and stability of most secretory proteins. In an effort to explore additional targeted bioengineering approaches, the role of disulfide bonds in FVIII secretion and function was probed in this study. Single and paired cysteine mutants were generated by substituting with serine or glycine residues and analyzed by transient transfection into COS-1 and CHO cells. Seven of the eight disulfide bonds in FVIII were found to be indispensable for proper secretion and function. However, elimination of the disulfide bond formed by C1899 and C1903 within the conserved A3 domain improved the secretion of FVIII. The addition of the C1899G/C1903G mutations to a previously described FVIII variant, 226/N6, with high secretion efficiency increased its secretion by 2.2-fold. Finally, the addition of the A1-domain mutation, F309S, in conjunction with the disulfide mutation had an additive effect, resulting in a net improvement in secretion of between 35 and 45-fold higher than wild-type FVIII in CHO cells. Such combined targeted bioengineering strategies may facilitate more efficient production of recombinant FVIII and contribute toward low-cost factor replacement therapy for hemophilia A.

When reduction leads to expansion part 2: on‐going undergraduate laboratory investigations on the roles of disulfide bonds within E. coli alkaline phosphatase

When reduction leads to expansion part 2: on‐going undergraduate laboratory investigations on the roles of disulfide bonds within E. coli alkaline phosphatase