Cysteines in ß-lactoglobulin affects its interfacial adsorption and protein film stabilization.

HYPOTHESIS: Disulfide bonds in proteins are strong chemical bonds forming the secondary and tertiary structure like in the dairy protein ß-lactoglobulin. We hypothesize that the partial or complete removal of disulfide bonds affects the structural rearrangement of proteins caused by intra- and intermolecular interactions that in turn define the interfacial activity of proteins at oil/water interfaces. The experimental and numerical investigations contribute to the mechanistic understanding of the structure-function relationship, especially for the interfacial adsorption behavior of proteins. EXPERIMENTAL AND NUMERICAL: Systematically, the 5 cysteines of ß-lactoglobulin were recombinantly exchanged by alanine. First, the protein structure of the variants in bulk was analyzed with Fourier-transform-infrared-spectroscopy and molecular dynamic simulations. Second, the structural changes after adsorption to the interface have been also analyzed by molecular dynamic simulations. The adsorption behavior was investigated by pendant drop analysis and the interfacial film properties by dilatational rheology. FINDINGS: The structural flexibility of ß-lactoglobulin with no cysteines encourages its unfolding at the interface, and accelerates the interfacial protein film formation that results in more visco-elastic films in comparison to the reference.

Epidermal Differentiation Genes of the Common Wall Lizard Encode Proteins with Extremely Biased Amino Acid Contents.

The epidermal differentiation complex (EDC) is a cluster of genes that code for protein components of cornified cells on the skin surface of amniotes. Squamates are the most species-rich clade of reptiles with skin adaptations to many different environments. As the genetic regulation of the skin epidermis and its evolution has been characterized for only a few species so far, we aimed to determine the organization of the EDC in a model species of squamates, the common wall lizard (Podarcis muralis). By comparative genomics, we identified EDC genes of the wall lizard and compared them with homologs in other amniotes. We found that the EDC of the wall lizard has undergone a major rearrangement leading to a unique order of three ancestral EDC segments. Several subfamilies of EDC genes, such as those encoding epidermal differentiation proteins containing PCCC motifs (EDPCCC) and loricrins, have expanded by gene duplications. Most of the EDPCCC proteins have cysteine contents higher than 50%, whereas glycine constitutes more than 50% of the amino acid residues of loricrin 1. The extremely biased amino acid compositions indicate unique structural properties of these EDC proteins. This study demonstrates that cornification proteins of the common wall lizard differ from homologous proteins of other reptiles, illustrating the evolutionary dynamics of diversifying evolution in squamates.

Functional Divergence in the Affinity and Stability of Non-Canonical Cysteines and Non-Canonical Disulfide Bonds: Insights from a VHH and VNAR Study.

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

A self-healing composite solid electrolyte with dynamic three-dimensional inorganic/organic hybrid network for flexible all-solid-state lithium metal batteries.

Composite solid electrolytes (CSEs), which combine the advantages of solid polymer electrolytes and inorganic solid electrolytes, are considered to be promising electrolytes for all-solid-state lithium metal batteries. However, the current CSEs suffer from defects such as poor inorganic/organic interface compatibility, lithium dendrite growth, and easy damage of electrolyte membrane, which hinder the practical application of CSEs. Herein, a CSE (PBHL@LLZTO@DDB) with polyurethane (PBHL) as the polymer matrix and Li6.4La3Zr1.4Ta0.6O12 (LLZTO) modified by silane coupling agent (DDB) as inorganic fillers (LLZTO@DDB) has been prepared. Disulfide bond exchange reactions between PBHL and LLZTO@DDB enable PBHL@LLZTO@DDB to form a dynamic three-dimensional (3D) inorganic/organic hybrid network, which promotes the uniform dispersion of LLZTO in PBHL@LLZTO@DDB, improves the Li+ conductivity (1.24 ± 0.08 × 10-4 S cm-1 at 30 â„ƒ), and broadens the electrochemical stability window (5.16 V vs. Li+/Li). Moreover, a combination of hydrogen bonds and disulfide bonds endows PBHL@LLZTO@DDB with excellent self-healing properties. As such, both all-solid-state symmetric and full cells exhibit excellent cycle performance at ambient temperature. More importantly, the healed PBHL@LLZTO@DDB can almost completely restore its original electrochemical properties, indicating its application potential in flexible electronic products.

An antireductant approach ameliorates misfolded proinsulin-induced hyperglycemia and glucose intolerance in male Akita mice.

Protein folding in the endoplasmic reticulum (ER) requires a high ratio of oxidized to reduced glutathione (GSSG/rGSH). Since the GSSG/rGSH depends on total glutathione (tGSH = GSSG + rGSH) levels, we hypothesized that limiting GSH biosynthesis will ameliorate protein misfolding by enhancing the ER oxidative milieu. As a proof-of-concept, we used DL-buthionine-(S,R)-sulfoximine (BSO) to inhibit GSH biosynthesis in Akita mice, which are prone to proinsulin misfolding. We conducted a 2-week intervention to investigate if BSO was safe and a 6-week intervention to find its effect on glucose intolerance. In both cohorts, male heterozygous Akita (AK) and wild-type (WT) mice were continuously administered 15 mM BSO. No adverse effects were observed on body weight, food intake, and water intake in either cohort. Unaltered levels of plasma aspartate and alanine aminotransferases, and cystatin-C, indicate that BSO was safe. BSO-induced decreases in tGSH were tissue-dependent with maximal effects in the kidneys, where it altered the expression of genes associated with GSH biosynthesis, redox status, and proteostasis. BSO treatment decreased random blood glucose levels to 80% and 67% of levels in untreated mice in short-term and long-term cohorts, respectively, and 6-h fasting blood glucose to 82% and 74% ï»¿of levels in untreated mice, respectively. BSO also improved glucose tolerance by 37% in AK mice in the long-term cohort, without affecting insulin tolerance. Neither glucose tolerance nor insulin tolerance were affected in WT. Data indicate that BSO might treat misfolded proinsulin-induced glucose intolerance. Future studies should investigate the effect of BSO on proinsulin misfolding and if it improves glucose intolerance in individuals with Mutant Insulin Diabetes of Youth.

Chitosan-Modified AgNPs Efficiently Inhibit Swine Coronavirus-Induced Host Cell Infections via Targeting the Spike Protein.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has filled a gap in our knowledge regarding the prevention of CoVs. Swine coronavirus (CoV) is a significant pathogen that causes huge economic losses to the global swine industry. Until now, anti-CoV prevention and control have been challenging due to the rapidly generated variants. Silver nanoparticles (AgNPs) with excellent antimicrobial activity have attracted great interest for biosafety prevention and control applications. In this study, we synthesized chitosan-modified AgNPs (Chi-AgNPs) with good biocompatibility to investigate their antiviral effects on swine CoVs. In vitro assays showed that Chi-AgNPs could significantly impaired viral entry. The direct interaction between Chi-AgNPs and CoVs can destroy the viral surface spike (S) protein secondary structure associated with viral membrane fusion, which is caused by the cleavage of disulfide bonds in the S protein. Moreover, the mechanism showed that Chi-AgNPs reduced the virus-induced apoptosis of Vero cells via the ROS/p53 signaling activation pathway. Our data suggest that Chi-AgNPs can serve as a preventive strategy for CoVs infection and provide a molecular basis for the viricidal effect of Chi-AgNPs on CoVs.

Unlocking Auxetic Behavior in Recyclable Thermosetting Foams Enabled by Dynamic Disulfide Cross-linking Strategy.

Auxetic foams with a negative Poisson's ratio (NPR) have attracted considerable attention in material engineering due to their outstanding performance in seismic and energy absorption. Nevertheless, thermoplastic auxetic foams are compromised by weak non-covalent crosslinking that diminishes the mechanical strength and durability of foams. Conversely, thermosetting foams with chemical crosslinking, although mechanically robust, face challenges in elaborating auxetic structure and in achieving recyclability. Herein, an alternative approach is proposed to tackle this dilemma by incorporating dynamic disulfide bonds into the polymer network for preparing a thermosetting polyurethane foam with covalent adaptable network. By leveraging the unidirectional multi-effect compression technique, the topological network reorganization of foam is induced, transforming the initial circular open-cell structure into a re-entrant cell structure. This structural transformation endows the foam with stable NPR capability, achieving a minimum Poisson's ratio value of -0.4 within 30% compressive strain. Benefiting from its reinforced network structure, the foam also demonstrates high compressive strength (6.47 MPa) and tensile strength (1.67 MPa). Furthermore, it is recyclable and can be recompressed into thermosetting films. This work offers a straightforward approach to making auxetic thermosetting foams with good mechanical and recyclable properties, which is interesting for the development of high-performance auxetic materials.

Enhancement of thermostability and expression level of Rasamsonia emersonii lipase in Pichia pastoris and its application in biodiesel production in a continuous flow reactor.

The acidic lipase from Rasamsonia emersonii named LIPR has great potential for biodiesel synthesis due to its strong methanol tolerance. Nonetheless, the limited thermostability of LIPR and low expression level in Escherichia coli remain major obstacles to its use in biodiesel synthesis. To enhance the thermostability, the mutant LIPR harboring mutations A126C-P238C for the formation of a new disulfide bond and amino acid substitution D214L was obtained through rational design. To our delight, the thermostability of LIPR mutant was greatly improved. Moreover, a comprehensive optimization strategy, such as employing the Mss signal peptide, co-expressing the molecular chaperone protein disulfide isomerase (PDI), knocking out the vacuolar sorting receptor gene VPS10-01, and overexpressing the dihydroxyacetone synthase gene DAS2, was adopted to obtain the combination-optimized mutant Pichia pastoris strain GS54. Furthermore, the biodiesel synthetic capability with the mutant GS54-LIPR was verified and the production yield was 52.2 % after 24 h in a shake flask. Subsequently, a continuous flow system was adopted to increase the biodiesel yield to 73.6 % within 3 h, demonstrating its efficacy in enhancing enzyme biocatalysis. The engineered GS54-LIPR mutant lipase is an efficient and reusable biocatalyst for the sustained production of biodiesel in a continuous flow reaction.

A spectrum of clinically-identified cysteine mutations in fibulin-3 (EFEMP1) highlight its disulfide bonding complexity and potential to induce stress response activation.

Fibulin-3 (FBLN3), also known as EFEMP1, is a secreted extracellular matrix (ECM) glycoprotein that contains forty cysteine residues. These cysteines, which are distributed across one atypical and five canonical calcium-binding epidermal growth factor (EGF) domains, are important for regulating FBLN3 structure, secretion, and presumably function. As evidence of this importance, a rare homozygous p.C55R mutation in FBLN3 negates its function, alters disulfide bonding, and causes marfanoid syndrome. Additional studies suggest that heterozygous premature stop codon mutations in FBLN3 may also cause similar, albeit less severe, connective tissue disorders. Interestingly, a series of twenty-four cysteine mutations in FBLN3 have been identified in the human population and published in the Clinical Variation (ClinVar) and gnomAD databases. We tested how seven of these cysteine mutants (five loss-of-cysteine variants: C42Y, C190R, C218R, C252F, and C365S, two gain-of-cysteine variants: R358C, Y369C) and two newly developed mutations (G57C and Y397C) altered FBLN3 secretion, disulfide bonding, MMP2 zymography, and stress response activation Surprisingly, we found a wide variety of biochemical behaviors: i) loss-of-cysteine variants correlated with an increased likelihood of disulfide dimer formation, ii) N-terminal mutations were less likely to disrupt secretion, and were less prone to aggregation, iii) in contrast to wild-type FBLN3, multiple, but not all variants failed to induce MMP2 levels in cell culture, and iv) C-terminal mutations (either loss or gain of cysteines) were more prone to significant secretion defects, intracellular accumulation/misfolding, and stress response activation. These results provide molecular and biochemical insight into FBLN3 folding, secretion, and function for many cysteine mutations found in the human population, some of which may increase the likelihood of subclinical connective tissue or other FBLN3-associated haploinsufficiency diseases.

Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

Microwave-induced heterogeneity in protein conformation and water mobility interferes with the distribution pattern and migration pathway of sodium ion in myofibrillar protein gel.

The aim of this study was to investigate the distribution pattern and migration pathway of sodium ion in the myofibrillar protein (MP) gel matrix during microwave heating. The results showed that the content of sodium ions in the outer layer of MP gel increased by 47.85% compared with that in the inner layer. In the inner layer of protein gel, the non-covalent disulfide bonds (mainly ε(γ-Glu)-Lys) increased (P < 0.05), which contributed to the formation of a better rigid structure of the protein. The free water content was significantly higher than that of the inner layer (P < 0.05), which was related to the higher mobility of sodium ions. The results of microstructure analysis showed that the outer layer of the MP gel formed a more porous network than the inner layer. This work is expected to give some insights into the development of promising salt-reduced meat products by microwave heating.

Design, Synthesis and Preliminary Bioactivity Evaluation of N-Acetylcysteine Derivatives as Antioxidative and Anti-Inflammatory Agents.

N-acetylcysteine (NAC) is a commonly used mucolytic agent and antidote for acetaminophen overdose. For pulmonary diseases, NAC exhibits antioxidative properties, regulates cytokine production, reduces apoptosis of lung epithelial cells, and facilitates the resolution of inflammation. However, the efficacy of NAC in clinical trials targeting different pathological conditions is constrained by its short half-life and low bioavailability. In the present study, a series of NAC derivatives were designed and synthesized to further enhance its pharmacological activity. Structure-activity relationship (SAR) studies were conducted to optimize the activating groups. In vitro evaluations revealed that compounds 4 r, 4 t, 4 w, and 4 x exhibited superior antioxidative and anti-inflammatory activities compared to the positive controls of NAC and fudosteine. The ADME prediction analysis indicated that these compounds exhibited a favorable pharmacological profile. In-vivo experiments with compound 4 r demonstrated that the high-dose group (80 mg/kg) exhibited improved therapeutic effects in reversing the HPY level in mice with pulmonary fibrosis compared to the NAC group (500 mg/kg), further proving its superior oral bioavailability and therapeutic effect compared to NAC.

Disulfide bonds as a molecular switch of enzyme-activatable anticancer drug precise release for fluorescence imaging and enhancing tumor therapy.

Enzyme-activatable drug delivery systems have been developed for cancer diagnosis and therapy. However, targeted intracellular drug delivery is a challenge for precisely tumor imaging and therapy due to the increased stability of copolymer nanoparticles (NPs) is accompanied by a notable decrease in enzyme degradation. Herein, disulfide bond was designed as an enzyme-activatable molecular switch of SS-P(G2)2/DOX NPs. The copolymer NPs consists of polyvinylpyrrolidone (PVP) with disulfide bonds in the center and enzyme-degradable peptide dendrites (Phe-Lys) to form dendritic-linear-dendritic triblock copolymers (TBCs). The amphiphilic TBCs could be split into two identical amphiphilic diblock copolymers (DBCs) by glutathione (GSH) in cancer cells specifically while maintaining the same hydrophilic-lipophilic equilibrium. This structural transformation significantly reduced the stability of copolymer NPs and enhanced sensitivity of DOX release by cathepsin B-activated. Subsequently, the released DOX acted as an indicator of fluorescence imaging and chemotherapy drug for cancer cells. The polymeric NPs achieved excellent drug-loaded stability and prolonged blood circulation in vivo, and realized fluorescence imaging and specific cancer cell killing capabilities by responding to the overexpression of GSH and cathepsin B in tumor cells. Furthermore, the copolymer NPs demonstrated excellent blood compatibility and biosafety. Therefore, a novel strategy based on one tumor marker acting as the switch for another tumor microenvironment responsive drug delivery system could be designed for tumor intracellular imaging and chemotherapy.

Protein vicinal thiols as intrinsic probes of brain redox states in health, aging, and ischemia.

The nature of brain redox metabolism in health, aging, and disease remains to be fully established. Reversible oxidations, to disulfide bonds, of closely spaced (vicinal) protein thiols underlie the catalytic maintenance of redox homeostasis by redoxin enzymes, including thioredoxin peroxidases (peroxiredoxins), and have been implicated in redox buffering and regulation. We propose that non-peroxidase proteins containing vicinal thiols that are responsive to physiological redox perturbations may serve as intrinsic probes of brain redox metabolism. Using redox phenylarsine oxide (PAO)-affinity chromatography, we report that PAO-binding vicinal thiols on creatine kinase B and alpha-enolase from healthy rat brains were preferentially oxidized compared to other selected proteins, including neuron-specific (gamma) enolase, under conditions designed to trap in vivo protein thiol redox states. Moreover, measures of the extents of oxidations of vicinal thiols on total protein, and on creatine kinase B and alpha-enolase, showed that vicinal thiol-linked redox states were stable over the lifespan of rats and revealed a transient reductive shift in these redox couples following decapitation-induced global ischemia. Finally, formation of disulfide-linked complexes between peroxiredoxin-2 and brain proteins was demonstrated on redox blots, supporting a link between protein vicinal thiol redox states and the peroxidase activities of peroxiredoxins. The implications of these findings with respect to underappreciated aspects of brain redox metabolism in health, aging, and ischemia are discussed.

Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

Advancing Peptide-Based Cancer Therapy with AI: In-Depth Analysis of State-of-the-Art AI Models.

Anticancer peptides (ACPs) play a vital role in selectively targeting and eliminating cancer cells. Evaluating and comparing predictions from various machine learning (ML) and deep learning (DL) techniques is challenging but crucial for anticancer drug research. We conducted a comprehensive analysis of 15 ML and 10 DL models, including the models released after 2022, and found that support vector machines (SVMs) with feature combination and selection significantly enhance overall performance. DL models, especially convolutional neural networks (CNNs) with light gradient boosting machine (LGBM) based feature selection approaches, demonstrate improved characterization. Assessment using a new test data set (ACP10) identifies ACPred, MLACP 2.0, AI4ACP, mACPred, and AntiCP2.0_AAC as successive optimal predictors, showcasing robust performance. Our review underscores current prediction tool limitations and advocates for an omnidirectional ACP prediction framework to propel ongoing research.

Chemical bonds and hair behaviour-A review.

When undertaking any review of the structure of the hair and its mechanical properties it becomes apparent that the overall behaviour of keratin fibres is commonly attributed to the presence of hydrogen, disulfide and ionic bonds. The action of physico-chemical agents used during various cosmetic treatments is viewed as the result of an interaction with these bonds. Thus, the breaking of bonds by chemical agents, or via mechanical or thermal stresses, affects the relative balance of disulfide and hydrogen bonds and the contribution of hydrophobic interactions, which are all important factors that may alter hair behaviour. Generally, these chemical bonds are considered as responding homogeneously to the environmental and cosmetic factors. This unitary image is challenged, however, by evaluating the results of chemical, nanomechanical, tensile and thermal measurements, which suggest that disulfide bonds may be grouped into several types, according to their location within the fibre and the way they respond to various agents. A compensatory effect of newly formed hydrogen bonds for broken disulfide bonds may also be seen, and additionally involves different types of hydrogen bonds. As a result, the picture of chemical bonding in hair appears to be far from a homogeneous one. In addition, it is apparent that further investigation is required for clarifying the action of ionic bonds and hydrophobic interactions within the hair fibre. The present review aims, thus, at offering a deeper background for understanding how the hair behaves under various conditions.

Comme l'indique l'étude de la littérature réalisée dans le cadre de cette revue, le comportement général des fibres kératiniques est généralement attribué à la présence de liaisons hydrogène, disulfure et ioniques. L'action des agents physico­chimiques utilisés au cours de divers traitements cosmétiques est alors considérée comme le résultat d'une interaction avec ces liaisons. Ainsi, la rupture des liaisons par des agents chimiques, ou par des contraintes mécaniques ou thermiques, affecte l'équilibre relatif des liaisons disulfure et hydrogène et la contribution des interactions hydrophobes, qui sont autant de facteurs importants susceptibles d'altérer le comportement du cheveu. En général, on considère que ces liaisons chimiques réagissent de manière homogène aux facteurs environnementaux et cosmétiques. Cette image unitaire est toutefois remise en question par l'évaluation des résultats des mesures chimiques, nanomécaniques, thermiques et de traction, qui suggèrent que les liaisons disulfures peuvent être regroupées en plusieurs types, en fonction de leur emplacement dans la fibre et de la manière dont elles réagissent aux différents agents. Un effet compensatoire des liaisons hydrogène nouvellement formées pour les liaisons disulfures rompues peut également être observé et implique en outre différents types de liaisons hydrogène. Par conséquent, l'image de la liaison chimique dans les cheveux est loin d'être homogène. En outre, il est évident que des recherches supplémentaires sont nécessaires pour clarifier l'action des liaisons ioniques et des interactions hydrophobes au sein de la fibre capillaire. La présente étude vise donc à offrir une base pour une compréhension plus approfondie du comportement du cheveu dans diverses conditions.

Cysteine, sodium metabisulfite, and glutathione enhance crosslinking between proteins during high moisture meat analog extrusion processing and may improve the fibrousness of the products.

BACKGROUND: High moisture meat analog (HMMA) products processed using extrusion have become increasingly popular in the last few years. Because the formation of disulfide bonds is believed to play a critical role in the texturization mechanism, this study aimed to understand how chemical compounds capable of reducing disulfide bonds, specifically cysteine, sodium metabisulfite, and glutathione, affect the texture and the chemical interactions between the proteins. METHOD: Wheat protein blended with cysteine, sodium metabisulfite, or glutathione at levels of 0, 0.5, 1.0, 2.5, 5.0, and 7.5 g kg-1 was extruded at three different temperatures (115, 140, and 165 °C) using a co-rotating twin-screw extruder. The feed rate (85 g min-1), the moisture content (600 g kg-1), and the screw speed (300 rpm) were kept constant. Unextruded and extruded material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polymeric protein fractionation, and sulfhydryl group/disulfide bond analysis. Extruded samples were further analyzed for their hardness and their anisotropic index. RESULTS: The inclusion of reductants significantly affected the structure of the obtained extrudates. Although reducing agents had a relatively small impact on the total amount of disulfide bonds, their action significantly enhanced crosslinking between the proteins. At select conditions, samples with high fibrousness were specifically obtained when cysteine or sodium metabisulfite was included at levels of 5.0 g kg-1. DISCUSSION: In the presence of reducing agents, it is believed that disulfide bonds are split earlier during the process without binding to them, giving the protein strands more time to unravel and align, leading to a better flow behavior and more fibrous products. © 2024 Society of Chemical Industry.

Functional metalloenzymes based on myoglobin and neuroglobin that exploit covalent interactions.

Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes.

Three-dimensional cross-linked network deep eutectic gel polymer electrolyte with the self-healing ability enable by hydrogen bonds and dynamic disulfide bonds.

Solid polymer electrolytes (SPEs) are effective solutions for the development of high-performance and flexible lithium metal batteries (LMBs). However, the key problems of SPEs including low ionic conductivity and inability to repair damage have hindered their industrialization process. In this work, a three-dimensional (3D) cross-linked network gel polymer electrolyte (CNGPE) is designed. The addition of deep eutectic solvent (DES) improves the ionic conductivity of CNGPE. The hydrogen bonds and dynamic disulfide bonds in the 3D cross-linked network endow CNGPE rapid self-healing ability at ambient temperature. In addition, the addition of lithium difluoro(oxalato)borate (LiDFOB) and lithium nitrate (LiNO3) helps to form a stable solid electrolyte interface (SEI). Due to the ingenious design, the Li/CNGPE/Li symmetrical cell exhibits excellent interface stability and no short circuit occurs for more than 800 h. The assembled LiFePO4/CNGPE/Li cell exhibits a discharge specific capacity of 126 mAh g-1 after 960 cycles at 0.5C. This work has shown that the self-healing gel polymer electrolyte containing DES provides an effective and feasible method for the development of high-performance LMBs.