Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state.

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

Insight into the mechanism of the aggregation behavior of wheat protein modulated by l-lysine under microwave irradiation.

This study explored the mechanism of l-lysine intervention in wheat gluten protein (WG) gel formation under a microwave (MW) field. The results showed that the MW treatment had higher ζ-potential values at the same heating rate. After adding l-lysine, the solution conductivity and dielectric loss were significantly increased. Moreover, the WG gel strength enhanced 4.40% under the MW treatment. The Fourier spectra showed that the α-helix content was decreased 13.78% with the addition of lysine. The ultraviolet absorption spectra and fluorescence spectra indicated that MW irradiation impacted the interactions between WG molecules more effectively than the water bath heating, promoting the denaturation and unfolding of the protein structure. In addition, scanning electron microscopy analysis showed that the incorporation of lysine promoted an ordered network structure formation of the protein, which enhanced the gel properties. This indicated that the zwitterion of l-lysine played a regulatory role in the aggregation of proteins in the MW field.

Delicate Analysis of Interacting Proteins and Their Assemblies by Flow Field-Flow Fractionation Techniques.

We study the efficiency of several asymmetrical flow field-flow fractionation (AF4) techniques to investigate self-associating wheat gluten proteins. We compare the use of a denaturing buffer including sodium dodecyl sulfate (SDS) and a mild chaotropic solvent, water/ethanol, as the eluent, on a model gluten sample. Through a thorough analysis of the data obtained from coupled light scattering detectors and with the identification of molecular composition of the eluted protein, we evidence coelution events in several conditions. We show that the focus step used in conventional AF4 with the SDS buffer leads to the formation of aggregates that coelute with monomeric proteins. By contrast, a frit-inlet device enables the fractionation of individual wheat proteins in the SDS buffer. Interestingly conventional AF4, using water/ethanol as eluent, is an effective method for fractionating gluten proteins and their complex dynamic assemblies, which involve weak forces and are composed of both monomeric and polymeric proteins.

Development of environmentally friendly glyoxal-based adhesives with outstanding water repellency utilizing wheat gluten protein.

To reduce the release of volatile organic compounds (VOCs) from formaldehyde-based adhesives at the source, the use of low-toxicity and biodegradable glyoxal instead of formaldehyde for the preparation of novel urea-glyoxal resins is a simple and promising strategy. The limited water resistance and adhesive strength of the new urea-glyoxal resins (UG) restrict their extensive application. This study prepared a high-performance, water-resistant WP-UG wood adhesive by combining UG prepolymer with wheat gluten protein (WP). FTIR, XRD, and XPS confirmed the existence of a chemical reaction between the two components, and thermal analysis showed that WP-UG plywood had better thermal stability. Evaluation of the gluing properties revealed that the dry and wet strengths of WP-UG adhesive bonded plywood reached 1.39 and 0.87 MPa, respectively, which were significantly higher than those of UG resin by 35 % and 314 %. The bond strength increased from 0 to 0.89 MPa after immersion in water at 63 °C for 3 h. The results indicated that the introduction of WP promoted the formation of a more complex and tightly packed crosslinking network and developed a glyoxal-based adhesive with high bond strength and water resistance. This study provides a new green pathway for novel urea-formaldehyde binders to replace harmful formaldehyde-based binders, which helps to increase their potential application value in the wood industry.

Wheat gluten structure and (non-)covalent network formation during deep-fat frying.

The aim of this work was to investigate wheat gluten protein network structure throughout the deep-frying process and evaluate its contribution to frying-induced micro- and macrostructure development. Gluten polymerization, gluten-water interactions, and molecular mobility were assessed as a function of the deep-frying time (0 - 180 s) for gluten-water model systems of differing hydration levels (40 - 60 % moisture content). Results showed that gluten protein extractability decreased considerably upon deep frying (5 s) mainly due to glutenin polymerization by disulfide covalent cross-linking. Stronger gliadin and glutenin protein-protein interactions were attributed to the formation of covalent linkages and evaporation of water interacting with protein chains. Longer deep-frying (> 60 s) resulted in progressively lower protein extractabilities, mainly due to the loss in gliadin protein extractability, which was associated with gliadin co-polymerization with glutenin by thiol-disulfide exchange reactions. The mobility of gluten polymers was substantially reduced during deep-frying (based on the lower T2 relaxation time of the proton fraction representing the non-exchanging protons of gluten) and gluten proteins gradually transitioned from the rubbery to the glassy state (based on the increased area of said protons). The sample volume during deep-frying was strongly correlated to the reduced protein extractability (r = -0.792, p < 0.001) and T2 relaxation time of non-exchanging protons of gluten proteins (r = -0.866, p < 0.001) thus demonstrating that the extent of gluten structural expansion as a result of deep-frying is dictated both by the polymerization of proteins and the reduction in their molecular mobility.

Effects of different polysaccharide colloids on the structure and physicochemical properties of peanut protein and wheat gluten composite system under extrusion.

The structure and physicochemical properties of the complex system of peanut protein and gluten with different concentrations (0 %, 0.5 %, 1 %, and 2 %) of carboxymethyl cellulose (CMC) or sodium alginate (SA) under high-moisture extrusion were studied. The water absorption index and low-field nuclear magnetic resonance showed that adding 0.5 % SA could significantly improve the water uniformity of peanut protein extrudates, while the increase in water absorption was not significant. The texture properties showed that adding CMC or SA increased the hardness, vertical shearing force, and parallel shearing force of the system. Furthermore, adding 0.5 % SA increased approximately 33 % and 75.2 % of the tensile distance and strength of the system, respectively. The secondary structure showed that CMC or SA decreased the proportion of α-helix, ß-turn, and random coil, while increased ß-sheet proportion. The results of hydrophobicity, unextractable protein, and endogenous fluorescence revealed that CMC and SA reduced the surface hydrophobicity of the system and caused fluorescence quenching in the system. Additionally, it was found that CMC generally increased the free sulfhydryl group content, while SA exhibited the opposite effect.

Parameter calibration of discrete element model for gluten densification molding.

The densified powder material is convenient for storage and transportation, with broad market application prospects. In this study, the discrete element model parameters required for simulating gluten densification were calibrated using the Hertz-Mindlin with JKR contact model. Initially, physical testing techniques were utilized to assess the size distribution, density, and angle of repose (AoR) of gluten particles. Following this, the Plackett-Burman test, the steepest ascent test, and the Box-Behnken test were conducted, and the significant factors were obtained: The coefficient of rolling friction (P-P) was 1.038, the coefficient of static friction (P-P) was 0.071, and the surface energy (P-P) was 0.047. Finally, the AoR and densification simulations were performed under the optimal parameter combination, along with validation tests. The results showed that the relative error between the simulated and tested AoR was 0.52%. The compression ratio and compression force curves of simulated and actual were similar.

Rheological, thermal, and structural properties of heat-induced gluten gel: Effects of starch with varying degrees of debranching.

This study evaluated the effects of starch with varying degree of debranching on the rheological, thermal, and structural properties of heat-induced gluten gel. As the duration of starch debranching treatment increased from 0 to 8 h, the viscoelasticity of the gel containing debranched starch (DBS) improved. Compared with the gluten gel (G), the gel strength of the G + DBS (8 h) sample increased by 65.2 %. The degradation temperature of gluten was minimally affected by DBS, while the weight loss rate increased by 4.4 %. Furthermore, the α-helical structure of gluten decreased, concomitant with an increase in ß-sheet content. Notably, DBS treated for 8 h exhibited more hydrogen bonds with the tyrosine of gluten and triggered disulfide bridge conformation to transition from g-g-g to t-g-g, thereby reducing the stability of the molecular conformation of gluten proteins, as evidenced by the decreased height and width of the molecular chains observed in atomic force microscopy images. Overall, the composite gel structure induced by DBS exhibited a more continuous and homogeneous owing to the improved compatibility between DBS and gluten proteins, favoring the formation of a robust gel. These findings provide valuable insights for utilizing DBS to enhance gluten gel properties.

Effect of starch degradation induced by extruded pregelatinization treatment on the quality of gluten-free brown rice bread.

There is considerable interest in preparing high-quality gluten-free bread. The effect of the molecular structure of extruded pregelatinization starch on the dough's rheological properties and the brown rice bread's quality was investigated. Extruded rice starch (ERS) was prepared with various added moisture contents of 20 % (ERS20), 30 % (ERS30), and 40 % (ERS40), respectively. ERS had smaller molecular weight and more short branched chains as the moisture content decreased. The dough elasticity and deformation resistance were improved with the ERS supplementation and in the order of ERS40 > ERS30 > ERS20 at the same level. Fortification with ERS improved the gluten-free brown rice bread quality. Compared to the control group, breadcrumbs supplemented with ERS20 at the 10 % level showed an increase in cell density from 17.87 cm-2 to 28.32 cm-2, a decrease in mean cell size from 1.22 mm2 to 0.81 mm2, and no significant change in cell area fraction. In addition, the specific volume increased from 1.50 cm3/g to 2.04 cm3/g, the hardness decreased from 14.34 N to 6.28 N, and the springiness increased from 0.56 to 0.74. The addition of extruded pregelatinization starches with smaller molecular weights and higher proportions of short chains is promising for preparing high-quality gluten-free bread.

Fabrication, characterization, and in vitro digestion of gelatin/gluten oleogels from thermally crosslinked electrospun short fiber aerogel templates.

In this work, the electrospun short fiber-based oleogels (ESFO) were formed by thermal crosslinking. Gelatin and gluten nanofibers were obtained via electrospinning, then homogenized and transformed into short fiber dispersions. Through freeze-drying, electrospun short fiber-based aerogel (ESF-A) templates were obtained for oil adsorption. All ESF-A exhibited the micromorphology of loose fibrous pore structure and prominent changes of characteristic peaks in the thermal and infrared analyses. Moreover, the highly crosslinked templates owned excellent hydrophobicity and mechanical performances (elastic modulus: 0.25 kPa, yield strength: 14.56 kPa, compressive strength: 52.54 kPa, and the final compression recovery: 91.27%). Meanwhile, the oil adsorption/oil holding capacity could reach 76.56 g/g and 80.04%, respectively. Through thermal crosslinking, ESF-O presented good and controllable rheological/in vitro digestion properties, which were further confirmed by PCA analysis. According to different application conditions, ESF-O properties could be adjusted by different degrees of fiber addition or thermal crosslinking.

Dynamic behaviors of protein and water associated with fresh noodle quality during processing based on different HMW-GSs at Glu-D1.

In this study, dynamic behaviors of proteins and water during fresh noodles processing associated with the quality of fresh noodles were systematically investigated by using wheat near-isogenic lines carrying high-molecular-weight glutenin subunits (HMW-GS) 2 + 12, 3 + 12 or 5 + 10 at the Glu-D1 locus. The results showed that subunits 5 + 10 tend to form a complex gluten network and had a poorly hydrated ability, that prevent the intrusion of external water during cooking; subunits 3 + 12 formed a moderate strength gluten network that generated a medium ability to resist the hydrated and mechanical treatment, which explained the highest water absorption and less cooking loss of cooked noodles; while subunits 2 + 12 formed fragile protein aggregates that had a poor ability to resist mechanical. The findings demonstrated that subunits 3 + 12 provided a suitable gluten network which was crucial for intrusion and hydration of external water thus formed a uniform gluten network and excellent fresh noodle quality.

Valorization of Red Beetroot (Beta vulgaris L.) Pomace Combined with Golden Linseed (Lini semen) for the Development of Vegetable Crispbreads as Gluten-Free Snacks Rich in Bioactive Compounds.

This work aimed to develop gluten-free snacks such as crispbread based on beetroot pomace (Beta vulgaris L.) and golden linseed (Lini semen). Beetroot is attracting more and more consumer attention because of its nutritional and health properties. The use of beet pomace contributes to waste management. Linseed, known as a superfood with many health-promoting properties, was used to produce crispbreads as an alternative to cereals, which are allergens. Beetroot pomace and whole or ground linseed were used in different proportions to produce crispbread snacks. Chemical and physical analyses were performed including water activity, dry matter, betalains, and polyphenols content, as well as Fourier transform infrared spectroscopy (FTIR). A sensory evaluation and microstructure observations were also performed. The obtained snacks were characterized by low water activity (0.290-0.395) and a high dry matter content (93.43-97.53%), which ensures their microbiological stability and enables longer storage. Beetroot pomace provided betalains-red (14.59-51.44 mg betanin/100 g d.m.) and yellow dyes (50.02-171.12 mg betanin/100 g d.m.)-while using linseed enriched the product with polyphenols (730-948 mg chlorogenic acid/100 g d.m.). FTIR analysis showed the presence of functional groups such as the following: -OH, -C-O, -COOH, and -NH. The most desired overall consumer acceptability was achieved for snacks containing 50% beetroot pomace and 50% linseed seeds. The obtained results confirmed that beetroot pomace combined with linseed can be used in the production of vegetable crispbread snacks.

Effects of zein extractions on the structural properties of SPI-zein composite gels: Implications for gluten-free plant-based products.

The growing global interest in physical and environmental health has led to the development of plant-based products. Although soy protein and wheat gluten are commonly utilized, concerns regarding gluten-related health issues have driven exploration into alternative proteins. Zein has emerged as a promising option. This research investigated the impact of extraction methods on zein characteristics and the structures of SPI-zein composite gels. Different extraction methods yielded zein with protein contents ranging from 48.12 % to 64.34 %. Ethanol-extracted Z1 and Z3, obtained at different pH conditions, exhibited zeta potential of -3.25 and 5.43 mV, respectively. They displayed similar characteristics to commercial zein and interacted comparably in composite gels. Conversely, alkaline-extracted Z2 had a zeta potential of -2.37 mV and formed distinct gels when combined with SPI. These results indicated that extraction methods influence zein behaviour in composite gels, offering possibilities for tailored formulations and expanding zein's applications, particularly in gluten-free plant-based products.

Amyloid-like Aggregation of Wheat Gluten and Its Components during Cooking: Mechanisms and Structural Characterization.

Amyloid-like aggregation widely occurs during the processing and production of natural proteins, with evidence indicating its presence following the thermal processing of wheat gluten. However, significant gaps remain in understanding the underlying fibrillation mechanisms and structural polymorphisms. In this study, the amyloid-like aggregation behavior of wheat gluten and its components (glutenin and gliadin) during cooking was systematically analyzed through physicochemical assessment and structural characterization. The presence of amyloid-like fibrils (AFs) was confirmed using X-ray diffraction and Congo red staining, while Thioflavin T fluorescence revealed different patterns and rates of AFs growth among wheat gluten, glutenin, and gliadin. AFs in gliadin exhibited linear growth curves, while those in gluten and glutenin showed S-shaped curves, with the shortest lag phase and fastest growth rate (t1/2 = 2.11 min) observed in glutenin. Molecular weight analyses revealed AFs primarily in the 10-15 kDa range, shifting to higher weights over time. Glutenin-derived AFs had the smallest ζ-potential value (-19.5 mV) and the most significant size increase post cooking (approximately 400 nm). AFs in gluten involve interchain reorganization, hydrophobic interactions, and conformational transitions, leading to additional cross ß-sheets. Atomic force microscopy depicted varying fibril structures during cooking, notably longer, taller, and stiffer AFs from glutenin.

Heat-moisture treatment can modulate all-purpose wheat flour for short dough biscuit making: Evidences and mechanism.

In view of the merits of all-purpose wheat flour (APWF) to soft wheat flour (SWF) in cost and protein supply, the feasibility of heat-moisture treatment (HMT, 19% moisture for 1 h at 60, 80 and 100 °C, respectively) to modify APWF as a substitute SWF in making short dough biscuits was explored. For underlying mechanisms, on the one hand, HMT reduced the hydration capacity of damaged starch particles by coating them with denatured proteins. On the other hand, HMT at 80 °C and 100 °C significantly denatured gluten proteins to form protein aggregates, highly weakening the gluten network in dough. These two aspects jointly conferred APWF dough with higher deformability and therefore significantly improved the qualities of biscuits. Moreover, the qualities of biscuits from APWF upon HMT-100 °C were largely comparable to that from SWF, even higher values were concluded in spread ratio, volume, specific volume and consumer acceptance.

Blueberry anthocyanin based active intelligent wheat gluten protein films: Preparation, characterization, and applications for shrimp freshness monitoring.

The aim of this study was to prepare active intelligent gluten protein films using wheat gluten protein (WG) and apple pectin (AP) as film-forming matrices, and blueberry anthocyanin extract (BAE) as a natural indicator. SEM and FT-IR analyses demonstrated the successful immobilization of BAE in the film matrix by hydrogen bonding interactions and its compatibility with WG and AP. The resultant WG-AP/BAE indicator films demonstrated notable antioxidant activity, color stability, barrier qualities, pH and ammonia response sensitivity, and mechanical properties. Among them, WG-AP/BAE5 exhibited the best mechanical properties (TS: 0.83 MPa and EB: 242.23%) as well as the lowest WVP (3.92 × 10-8 g.m/m2.Pa.s), and displayed high sensitivity to volatile ammonia. In addition, WG-AP/BAE5 showed a color shift from purplish red to green to yellowish green, demonstrating the monitoring of shrimp freshness in real time. Consequently, this study offers a firm scientific foundation for the development of active intelligent gluten protein films and their use in food freshness assessments.

Effect of magnetic fields on the structure, properties, baking performance of frozen wheat dough at different frozen stage.

As an emerging physical technology, magnetic fields have been used to improve the quality of frozen and refrigerated foods. This study compared the effect of applying a static magnetic field (2 mT) at different stages of freezing and storage on the quality of frozen dough. Results suggested that the magnetic field significantly impacted frozen dough quality. It not only prevented the formation of ice crystals during the pre-freezing stage but also inhibited ice crystal growth during the following frozen storage. This effect helped to maintain the integrity of gluten proteins and their adhesion to starch granules by preventing the breakage of disulfide bonds and the depolymerization of gluten macromolecules. It was also observed that yeast inactivation and glutathione release were reduced, resulting in improved air retention and air production capacity of the dough. This, in turn, led to a more appealing volume and texture quality of the finished bread.

Enhanced B-type starch granules proportion modulates starch-gluten interactions during the thermal processing of reconstituted doughs.

This work investigated structure-properties changes of reconstituted wheat A/B starch doughs under different ratios during dynamic thermal processing. Results indicated that a change in spatial conformation and aggregation structure of the starch-gluten system was induced with heating (30 °C-86 °C). Moderately increased B starch ratio can effectively fill the gluten network and improve starch-protein interactions, which promotes the free sulfhydryl group oxidation and results in the formation of more glutenin macropolymer; this contributes to a higher degree of cross-linking and stability to the gluten network matrix. This improvement is enhanced as the heating temperature is increased. Notably, the B starch ratio requires to be controlled within a suitable range (≤ 75%) to avoid aggregation and accumulation on the gluten matrix triggered by its excess. This work may provide insights and optimization for clarifying the on-demand regulation strategy of A/B starch in dough processing.

Improvement on wheat bread quality by in situ produced dextran-A comprehensive review from the viewpoint of starch and gluten.

Deterioration of bread quality, characterized by the staling of bread crumb, the softening of bread crust and the loss of aroma, has caused a huge food waste and economic loss, which is a bottleneck restriction to the development of the breadmaking industry. Various bread improvers have been widely used to alleviate the issue. However, it is noteworthy that the sourdough technology has emerged as a pivotal factor in this regard. In sourdough, the metabolic breakdown of carbohydrates, proteins, and lipids leads to the production of exopolysaccharides, organic acids, aroma compounds, or prebiotics, which contributes to the preeminent ability of sourdough to enhance bread attributes. Moreover, sourdough exhibits a "green-label" feature, which satisfies the consumers' increasing demand for additive-free food products. In the past two decades, there has been a significant focus on sourdough with in situ produced dextran due to its exceptional performance. In this review, the behaviors of bread crucial compositions (i.e., starch and gluten) during dough mixing, proofing, baking and bread storing, as well as alterations induced by the acidic environment and the presence of dextran are systemically summarized. From the viewpoint of starch and gluten, results obtained confirm the synergistic amelioration on bread quality by the coadministration of acidity and dextran, and also highlight the central role of acidification. This review contributes to establishing a theoretical foundation for more effectively enhancing the quality of wheat breads through the application of in situ produced dextran.

Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties.

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://MSV000093372@massive.ucsd.edu).