Stable Disulfide Bonds Research Articles

Exploring the effect of natural deep eutectic solvents on zein: Structural and functional properties.

This study evaluated the effects of chemical modification, including ethanol, acetic acid, and natural deep eutectic solvents (NADES), on the secondary and tertiary structures, hydrophobicity, free amine content, protein-protein interactions, and functional properties of zein. The NADES used included choline chloride: oxalic acid, choline chloride: urea, choline chloride: glycerol, and glucose: citric acid. The results reveal that the NADES system significantly altered zein's structures, as evidenced by Fourier transform infrared spectroscopy, fluorescence, and Ultraviolet-Visible Spectroscopy analysis. Circular dichroism spectroscopy analysis indicated significant conformational change in modified zein, with decreased α-helix and increased random coil content. Notably, the NADES system leads to greater disruption of hydrogen bonds and facilitates the exposure of hydrophobic regions compared to water, ethanol, and acetic acid systems. This resulted in enhanced solubility, surface hydrophobicity, and free amine content in zein, indicating a more significant change in protein structure. In contrast, water and acetic acid solvents maintained more stable disulfide bonds within zein, which correlates with lower solubility and less unfolding. The NADES system promoted interactions between zein and its solvent components, improving emulsifying properties. Water, ethanol, and acetic acid systems had higher solubility in urea, thiourea, and dithiothreitol than the NADES system, revealing disruption of both covalent and noncovalent bonds in zein modified by NADES. Overall, this study highlights the superior ability of the NADES system to modify zein's structure and functionality compared to conventional solvents, suggesting its potential for enhancing protein applications in the industrial production of foods.

Oxaliplatin-loaded amphiphilic hyaluronic acid nanohydrogel formed via interfacial reactions enhances the therapeutic effect of targeted tumor

Hyaluronic acid (HA) nanogels have attracted widespread attention, aiming to improve cancer treatment paradigms and overcome the limitations of free-form drugs. However highly hydrophilic nature of HA nanogels limits their potential application where amphiphilic interactions are required for the delivery of hydrophobic drugs. In this study, we developed amphiphilic structure oxaliplatin (OXA) loaded oligo-hyaluronic acid (oHA)-PEG-Octane nanogel using stable disulfide bonds with ultrasonic re-emulsion method. 1,8-Mercaptooctane present in the organic phase underwent crosslinking with sulfhydryl groups conjugated on oHA and PEG in the inner aqueous phase through interfacial reactions, resulting in the formation of an amphiphilic structure of the nanogels. The nanogels demonstrated uniform size, stability, excellent biocompatibility, and achieved a high drug-loading capacity of 10.8 %. OXA NGs targeted tumor cells through CD44-mediated endocytosis, disassembled under the influence of high glutathione (GSH) levels within the tumor microenvironment (TME) and released OXA inside the reductive cytosol to trigger immunogenic cell death (ICD) of tumor cells. In vivo experiments showed OXA NGs could inhibit tumor growth. Furthermore, the amphiphilic hyaluronic acid nanohydrogel may have clinical potential due to its ability to accommodate various therapeutic agents; therefore, OXA NGs are a potential candidate for clinical translation.

Investigating the molecular mechanism of high-molecular-weight glutenin subunit affects gluten aggregation during dough mixing: Experimental characterizations and computational simulations

This study investigated the impact of high-molecular-weight glutenin subunits (HMW-GS) on gluten aggregation and dough rheology at different mixing stages, using wheat lines with deletions at the Glu-B1 locus. Dough rheology was analyzed across varying mixing levels, while the multiscale structure and composition of gluten were systematically characterized. Additionally, molecular dynamics simulations under increased pressure (10 bar) provided detailed insights into the structural dynamics of different HMW-GSs. The results showed that optimum mixing promoted gluten aggregation, enhancing viscoelasticity, while over-mixing led to disaggregation. HMW-GS deletions, particularly of Bx7, significantly hindered gluten aggregation under optimum mixing, limiting stable disulfide bonds, intermolecular β-sheet formation, and hydrophobic interactions essential for tertiary structure. Conversely, HMW-GS deletions facilitated disaggregation during over-mixing, with Bx7 deletion having a stronger impact. Molecular dynamics simulations further illustrated Bx7's role, showing its more hydrophobic and flexible structure compared to By8, supporting the experimental observation that Bx7 deletion affects gluten network integrity more markedly. These findings underscore the critical role of HMW-GS in modulating gluten aggregation, providing a molecular basis for targeted HMW-GS manipulation in wheat breeding to enhance dough functionality and improve processing stability across various mixing conditions.

Thioredoxin-1 protein interactions in neuronal survival and neurodegeneration

Neuronal cell death remains the principal pathophysiologic hallmark of neurodegenerative diseases and the main challenge for treatment strategies. Thioredoxin1 (Trx1) is a major cytoplasmic thiol oxidoreductase protein involved in redox signaling, hence a crucial player in maintaining neuronal health. Trx1 levels are notably reduced in neurodegenerative diseases including Alzheimer's and Parkinson's diseases, however, the impact of this decrease on neuronal physiology remains largely unexplored. This is mainly due to the nature of Trx1 redox regulatory role which is afforded by a rapid electron transfer to its oxidized protein substrates. During this reaction, Trx1 forms a transient bond with the oxidized disulfide bond in the substrate. This is a highly fast reaction which makes the identification of Trx1 substrates a technically challenging task. In this project, we utilized a transgenic mouse model expressing a Flag-tagged mutant form of Trx1 that can form stable disulfide bonds with its substrates, hence allowing identification of the Trx1 target proteins. Autophagy is a vital housekeeping process in neurons that is critical for degradation of damaged proteins under oxidative stress conditions and is interrupted in neurodegenerative diseases. Given Trx1's suggested involvement in autophagy, we aimed to identify potential Trx1 substrates following pharmacologic induction of autophagy in primary cortical neurons. Treatment with rapamycin, an autophagy inducer, significantly reduced neurite outgrowth and caused cytoskeletal alterations. Using immunoprecipitation and mass spectrometry, we have identified 77 Trx1 target proteins associated with a wide range of cellular functions including cytoskeletal organization and neurodegenerative diseases. Focusing on neuronal cytoskeleton organization, we identified a novel interaction between Trx1 and RhoB which was confirmed in genetic models of Trx1 downregulation in primary neuronal cultures and HT22 mouse immortalized hippocampal neurons. The applicability of these findings was also tested against the publicly available proteomic data from Alzheimer's patients. Our study uncovers a novel role for Trx1 in regulating neuronal cytoskeleton organization and provides a mechanistic explanation for its multifaceted role in the physiology and pathology of the nervous system, offering new insights into the molecular mechanisms underlying neurodegeneration.

Magnetic field improves the functional properties of frozen gluten by inhibiting structural deteriorations of glutenin and gliadin

The structural and functional properties of glutenin and gliadin after magnetic field (MF)-assisted freezing-thawing cycles treatment was compared to clarify the protective mechanism of MF on frozen dough. Compared with conventional frozen glutenin and gliadin, MF inhibited the freezing-induced structural unfolding of glutenin and the rearrangement of gliadin, whereby the surface hydrophobicity individually increased and decreased by 23.5% and 47.5%. Raman spectra and atomic force microscope analyses showed that MF-assisted freezing treatment provided both the glutenin and gliadin with a higher aggregation, as manifest with the increased stable disulfide bond conformation (g-g-g), average molecular chain height and width. Small angle X-ray scattering indicated that the conformation and fractal dimensions of MF-assisted frozen glutenin and gliadin were close to those of fresh groups. MF individually enhanced the water retention capacity and emulsifying performances of gliadin and glutenin, which synergistically contributed to an improved foaming performances in a reconstituted gluten model system. This study established a substantial relationship between the individual component structural changes and whole gluten functional properties, which is benefit to the application of MF in frozen market.

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.

Systematic computer-aided disulfide design as a general strategy to stabilize prefusion class I fusion proteins.

Numerous enveloped viruses, such as coronaviruses, influenza, and respiratory syncytial virus (RSV), utilize class I fusion proteins for cell entry. During this process, the proteins transition from a prefusion to a postfusion state, undergoing substantial and irreversible conformational changes. The prefusion conformation has repeatedly shown significant potential in vaccine development. However, the instability of this state poses challenges for its practical application in vaccines. While non-native disulfides have been effective in maintaining the prefusion structure, identifying stabilizing disulfide bonds remains an intricate task. Here, we present a general computational approach to systematically identify prefusion-stabilizing disulfides. Our method assesses the geometric constraints of disulfide bonds and introduces a ranking system to estimate their potential in stabilizing the prefusion conformation. We hypothesized that disulfides restricting the initial stages of the conformational switch could offer higher stability to the prefusion state than those preventing unfolding at a later stage. The implementation of our algorithm on the RSV F protein led to the discovery of prefusion-stabilizing disulfides that supported our hypothesis. Furthermore, the evaluation of our top design as a vaccine candidate in a cotton rat model demonstrated robust protection against RSV infection, highlighting the potential of our approach for vaccine development.

Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a β-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the β-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual β-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

Heterogeneity in Disulfide Bond Reduction in IgG1 Antibodies Is Governed by Solvent Accessibility of the Cysteines.

We studied unpaired cysteine levels and disulfide bond susceptibility in four different γ-immunoglobulin antibodies using liquid chromatography-mass spectrometry. Our choice of differential alkylating agents ensures that the differential peaks are non-overlapping, thus allowing us to accurately quantify free cysteine levels. For each cysteine residue, we observed no more than 5% to be unpaired, and the free cysteine levels across antibodies were slightly higher in those containing lambda light chains. Interchain and hinge residues were highly susceptible to reducing stresses and showed a 100-1000-fold higher rate of reduction compared to intrachain cysteines. Estimations of the solvent-accessible surface for individual cysteines in IgG1, using an implicit all-atom molecular dynamics simulation, show that interchain and hinge cysteines have >1000-fold higher solvent accessibility compared to intrachain cysteines. Further analyses show that solvent accessibility and the rate of reduction are linearly correlated. Our work clearly establishes the fact that a cysteine's accessibility to the surrounding solvent is one of the primary determinants of its disulfide bond stability.

Evidence that protein thiols are not primary targets of intracellular reactive oxygen species in growing Escherichia coli.

The oxidizability of cysteine residues is exploited in redox chemistry and as a source of stabilizing disulfide bonds, but it also raises the possibility that these side chains will be oxidized when they should not be. It has often been suggested that intracellular oxidative stress from hydrogen peroxide or superoxide may result in the oxidation of the cysteine residues of cytoplasmic proteins. That view seemed to be supported by the discovery that one cellular response to hydrogen peroxide is the induction of glutaredoxin 1 and thioredoxin 2. In this study we used model compounds as well as alkaline phosphatase to test this idea. Our results indicate that molecular oxygen, superoxide, and hydrogen peroxide are very poor oxidants of N-acetylcysteine and of the protein thiols of alkaline phosphatase in vitro. Copper could accelerate thiol oxidation, but iron did not. When alkaline phosphatase was engineered to remain in the cytoplasm of live cells, unnaturally high concentrations of hydrogen peroxide were required to oxidize it to its active, disulfide-dependent form, and toxic levels of superoxide had no effect. At the same time, far lower concentrations of these oxidants were sufficient to poison key metalloenzymes. The elimination of glutaredoxin 1 and thioredoxin 2 did not change these results, raising the question of why E. coli induces them during peroxide stress. In fact, when catalase/peroxidase mutants were chronically stressed with hydrogen peroxide, the absence of glutaredoxin 1 and thioredoxin 2 did not impair growth at all, even in a minimal medium over many generations. We conclude that physiological levels of reduced oxygen species are not potent oxidants of typical protein thiols. Glutaredoxin and thioredoxin must either have an alternative purpose or else play a role under culture conditions that differ from the ones we tested.

Spectral Features Differentiate Aging-Induced Changes in Parchment-A Combined Approach of UV/VIS, µ-ATR/FTIR and µ-Raman Spectroscopy with Multivariate Data Analysis.

From the moment of production, artworks are constantly exposed to changing environmental factors potentially inducing degradation. Therefore, detailed knowledge of natural degradation phenomena is essential for proper damage assessment and preservation. With special focus on written cultural heritage, we present a study on the degradation of sheep parchment employing accelerated aging with light (295-3000 nm) for one month, 30/50/80% relative humidity (RH) and 50 ppm sulfur dioxide with 30/50/80%RH for one week. UV/VIS spectroscopy detected changes in the sample surface appearance, showing browning after light-aging and increased brightness after SO2-aging. Band deconvolution of ATR/FTIR and Raman spectra and factor analysis of mixed data (FAMD) revealed characteristic changes of the main parchment components. Spectral features for degradation-induced structural changes of collagen and lipids turned out to be different for the employed aging parameters. All aging conditions induced denaturation (of different degrees) indicated by changes in the secondary structure of collagen. Light treatment resulted in the most pronounced changes for collagen fibrils in addition to backbone cleavage and side chain oxidations. Additional increased disorder for lipids was observed. Despite shorter exposure times, SO2-aging led to a weakening of protein structures induced by transitions of stabilizing disulfide bonds and side chain oxidations.

Mechanism of active acetylcholinesterase inhibition by organic sulfanes in garlic: Non-covalent binding and covalent modifications

Numerous secondary metabolites in medicinal food homology plants such as Allium inhibit the activity of acetylcholinesterase (AChE), but the current understanding of the inhibition mechanism is limited. In this study, we employed ultrafiltration, spectroscopic, molecular docking, and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) techniques to investigate the inhibition mechanism of AChE by garlic organic sulfanes, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). The results of UV-spectrophotometry and ultrafiltration experiments showed the inhibition of AChE activity by DAS and DADS was reversible (competitive inhibition), but inhibition by DATS was irreversible. Molecular fluorescence and molecular docking indicated DAS and DADS changed the positions of key amino acids inside the catalytic cavity through hydrophobic interactions with AChE. By using MALDI-TOF-MS/MS, we found DATS irreversibly inhibited AChE activity by opening disulfide-bond switching of disulfide bond 1 (Cys-69 and Cys-96) and disulfide bond 2 (Cys-257 and Cys-272) in AChE, as well as by covalently modifying Cys-272 in disulfide bond 2 to generate AChE–SSA derivatives (strengthened switch). This study provides a basis for further exploration of natural AChE inhibitors using organic active substances in garlic and presents a hypothesis of U-shaped spring force arm effect based on the disulfide bond-switching reaction of DATS that can be used to evaluate the stability of disulfide bonds in proteins.

The intestinal MUC2 mucin C-terminus is stabilized by an extra disulfide bond in comparison to von Willebrand factor and other gel-forming mucins

The MUC2 mucin polymer is the main building unit of the intestinal mucus layers separating intestinal microbiota from the host epithelium. The MUC2 mucin is a large glycoprotein with a C-terminal domain similar to the MUC5AC and MUC5B mucins and the von Willebrand factor (VWF). A structural model of the C-terminal part of MUC2, MUC2-C, was generated by combining Cryo-electron microscopy, AlphaFold prediction, information of its glycosylation, and small angle X-ray scattering information. The globular VWD4 assembly in the N-terminal of MUC2-C is followed by 3.5 linear VWC domains that form an extended flexible structure before the C-terminal cystine-knot. All gel-forming mucins and VWF form tail-tail disulfide-bonded dimers in their C-terminal cystine-knot domain, but interestingly the MUC2 mucin has an extra stabilizing disulfide bond on the N-terminal side of the VWD4 domain, likely essential for a stable intestinal mucus barrier.

Aggregation mechanism and branched 3D morphologies of pathological human light chain proteins under reducing conditions

Here, we examined the aggregation mechanism and structures of the pathological human multiple myeloma light chain aggregates (hLC) after disrupting stabilizing disulfide bonds by various reducing agents. The aggregation kinetics were measured in the presence of three commonly used disulfide reducers (TCEP, DTT and glutathione), and the resulting aggregates were visualized by the combination of light and confocal/super-resolution STED microscopy. We find that aggregation kinetics can be described by two apparent macroscopic rate constants of the Finke-Watzky model related to the nucleation and the growth process. Surprisingly, the growth rate constants decreased at higher protein concentrations, which we interpret as the involvement of an aggregation active monomer particle that is successively depleted at high concentrations due to shifts in a monomer/dimer equilibrium. Seeding experiments demonstrated the specificity of the aggregates; only certain seeds accelerated the aggregation, while others eventually slowed down the aggregation. Three-dimensional visualization of the overall structures of the final aggregates at submicrometer resolution showed variable, reducer-specific branched morphologies with non-trivial fractal dimensions. Thus, the disruption of the stabilizing disulfide bonds in hLC leads to specific large, branched aggregates formed by the monomer-addition mechanism.

New In Vivo Approach to Broaden the Thioredoxin Family Interactome in Chloroplasts.

Post-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.

Mechanical Stability of Ribonuclease A Heavily Depends on the Redox Environment.

Disulfide bonds are covalent bonds that connect nonlocal fragments of proteins, and they are unique post-translational modifications of proteins. They require the oxidizing environment to be stable, which occurs for example during oxidative stress; however, in a cell the reductive environment is maintained, lowering their stability. Despite many years of research on disulfide bonds, their role in the protein life cycle is not fully understood and seems to strictly depend on a system or process in which they are involved. In this article, coarse-grained UNited RESidue (UNRES), and all-atom Assisted Model Building with Energy Refinement (AMBER) force fields were applied to run a series of steered molecular dynamics (SMD) simulations of one of the most studied, but still not fully understood, proteins—ribonuclease A (RNase A). SMD simulations were performed to study the mechanical stability of RNase A in different oxidative–reductive environments. As disulfide bonds (and any other covalent bonds) cannot break/form in any classical all-atom force field, we applied additional restraints between sulfur atoms of reduced cysteines which were able to mimic the breaking of the disulfide bonds. On the other hand, the coarse-grained UNRES force field enables us to study the breaking/formation of the disulfide bonds and control the reducing/oxidizing environment owing to the presence of the designed distance/orientation-dependent potential. This study reveals that disulfide bonds have a strong influence on the mechanical stability of RNase A only in a highly oxidative environment. However, the local stability of the secondary structure seems to play a major factor in the overall stability of the protein. Both our thermal unfolding and mechanical stretching studies show that the most stable disulfide bond is Cys65–Cys72. The breaking of disulfide bonds Cys26–Cys84 and Cys58–Cys110 is associated with large force peaks. They are structural bridges, which are mostly responsible for stabilizing the RNase A conformation, while the presence of the remaining two bonds (Cys65–Cys72 and Cys40–Cys95) is most likely connected with the enzymatic activity rather than the structural stability of RNase A in the cytoplasm. Our results prove that disulfide bonds are indeed stabilizing fragments of the proteins, but their role is strongly redox environment-dependent.

Engineering Antiviral Agents via Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 µM as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-µ-molar range.

A highly stable covalent adaptable network through π-π conjugated confinement effect

Covalent adaptable networks, which can shuffle chemical bonds through exchange reactions of dynamic covalent bonds in permanent crosslinking networks, could address the pollution issue caused by thermosets. However, the covalent adaptable networks’ stability would be deteriorated under external stimulus because of intrinsic dynamic feature of dynamic bonds, which will seriously restrict the stable application of covalent adaptable networks in emerging fields such as self-adaptive materials and devices. In this work, we fabricate a confined dynamic crosslinking polyurethane (CDPU) by constructing π-π conjugation to improve the stability of dynamic disulfide bonds while retaining dynamic activity. The resultant CDPU remains stable even at 160 °C (above its topology freezing transition temperature: 129.2 °C) under single heat stimulus. Whereas this material can be activated under the co-stimulation of 140 °C and 10 MPa stress. Therefore, the CDPU shows improved thermostability and mechanical robustness compared with traditional covalent adaptable networks without confinement effect on dynamic bonds. This strategy provides an effective way to improve the stability of covalent adaptable networks so that they can be applied in emerging fields such as soft robotics, electronic skins, etc. • The dynamic thermosetting polyurethane with confinement effect were fabricated. • The dynamic thermosetting polyurethane with confinement effect exhibit improved stability. • The dynamic bonds with confinement effect can proceed exchange reaction under stimuli of heat and extra force.

Monitoring Protein Global and Local Parameters in Unfolding and Binding Studies: The Extended Applicability of the Diffusion Coefficient─Molecular Size Empirical Relations.

Protein unfolding and denaturation are main issues in biochemical and pharmaceutical research. Using a global parameter, the translational diffusion coefficient D, folded, unfolded, and intrinsically disordered proteins of a given molar mass M can be distinguished based on their distinct hydrodynamic properties. For broader applications, we provide generalized, PFG-NMR-based empirical D-M relations validated at different temperatures and ready to use with the corresponding corrections in different media. We demonstrate that these relations enable a more accurate molecular mass determination and show fewer potential errors than those of the common methods based on small-molecular diffusion standards. We monitor unfolding of three model proteins using 8 M urea and dimethyl sulfoxide (DMSO)-water mixtures as denaturing agents, highlighting the effect of disulfide bonds. Denaturation in 8 M urea is pH-dependent; in addition, for proteins with highly stable disulfide bonds, a reducing agent (TCEP) is required to achieve complete unfolding. Regarding the effect of local parameters, we show that at low DMSO concentrations─common conditions in pharmaceutical binding studies─the PFG-NMR-derived global parameters are not significantly affected. Still, the atomic environments can change, and the bound solvent molecule can inhibit the binding of a partner molecule. Using proteins with natural isotopic abundance, this effect can be proven by fast 1H-15N 2D correlation spectra. Our results enable fast and easy estimation of protein molecular mass and the degree of folding in various media; moreover, the effect of the cosolvent on the atomic-level structure can be traced without the need of isotope labeling.

Genome-Wide Identification of TLP Gene Family and Their Roles in Carya cathayensis Sarg in Response to Botryosphaeria dothidea.

Hickory (Carya cathayensis) is a critical tree species of the genus Carya from the Juglandaceae family that contains nutrient-rich nuts. Due to large-scale soil degradation, the pests and diseases of hickory are becoming more and more serious. Thaumatin-like proteins (TLPs) are vital proteins involved in the complex defense process of plant pathogens. In this study, 40 CcTLP genes were identified genome-widely and phylogenetically grouped into three subfamilies. The sequence of CcTLPs had a conservative pattern, such as eight stable disulfide bonds, REDDD, and G-X-[GF]-X-C-X-T-[GA]-D-C-X(1,2)-G-X-(2,3)-C structure. In total, 57 cis-elements related to stress-responsive, light-responsive, phytohormone-responsive, and plant-responsive were discovered. Under salicylate (SA), methyl jasmonate (MeJA), and ethephon (ETH) treatments, the expressions of CcTLP28, CcTLP29, CcTLP30, CcTLP31, CcTLP32, CcTLP33, CcTLP37, CcTLP38, and CcTLP39 had different patterns. This is an indication that most of the TLP genes were upregulated by SA and downregulated by MeJA. Notably, seven TLP genes were significantly upregulated under the Botryosphaeria dothidea inoculation, especially CcTLP31, with an over 20-fold change. Nine genes were shown by subcellular localization analysis to be located at the plasma membrane and cytoplasm. The knowledge of the disease-resistant function of the CcTLP family in hickory is promoted by these results. A foundation reference for the molecular breeding of this plant in the future is provided by our findings.