Unfolded Structure Research Articles

Potential effects of pH changes in the micro-environment during desalination on infant powder: The perspective of pH shift affecting whey protein molecular structure and function

In order to simulate the effect of pH environment change caused by different desalting methods on whey protein (WP) in actual production, the WP was treated under different acidic pH (2.0–6.0) and pH shift (pH recovery). The molecular structure, properties, and functions of the proteins were further analyzed. The results showed that the spatial structure of WP was unfolded under acidic conditions, α-helix and β-sheet were transformed into random coils, hydrophobic residues and free sulfhydryl groups were exposed. The particle size of WP was increased, and the emulsification, foaming and thermal stability were improved significantly. Especially after pH2 treatment, WP has the largest expansion degree, emulsification and foaming properties increased to 232.96 m2/g and 158.83 %. After pH recovery, the partially unfolded structure refolded, reducing the surface hydrophobicity, forming a smaller particle size of soluble protein particles, solubility has increased to 83.95 % compared to the natural. However, the emulsification, foamability, and thermal stability after pH shift are slightly reduced compared to acidic conditions, making it easier to form a gel after heating.

Support-free printing origami-based 3D negative Poisson's ratio-structured piezoresistive sensor for motion monitoring

A convenient strategy for preparing meta-structures with a negative Poisson's ratio (NPR), which enhance mechanoelectrical responsiveness and are typically based on inner concave hollow cells, is 3D printing without support structures. However, this process requires the material to have high melt strength, often linked to high viscosity and low printability. In this work, we introduced 0D carbon black (CB) into a thermoplastic polyurethane (TPU)/1D carbon nanotubes (CNT) composite to create a dimension-hybrid filler system, enhancing strength with minimal viscosity increase. The results show that the bending modulus and Young's modulus of CB/CNT(1:3)/TPU composites are increased by 1.6 times and 1.8 times that of TPU, while its viscosity (2697.98 Pa⋅s) is lower than the allowed value of printer. Then, the 3D-printed suspension bridge of TPU is 42.7 % less saggy, significantly benefiting to preparing meta-structures. Based on a support-free 3D printable enclosed hollow structure, the TPU / CNT / CB composite material achieves an adjustable NPR between −0.59 and −0.20. Compared to unfolded structure, this NPR characteristic further improves the piezoelectric output voltage by 9.00 times, enhances the compressive modulus by 3.15 times, and improves the piezoresistive sensitivity by 122.0 %. Sensors based on this 3D-printed material showcase high performance stability and reproducibility, demonstrating their potential in wearable equipment.

Advanced sampling simulations of coupled folding and binding of phage P22 N-peptide to boxB RNA

Protein-RNA interactions are crucially important for numerous cellular processes and often involve coupled folding and binding of peptide segments upon association. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes such a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics free energy simulations were used to calculate the absolute binding free energy of the N-peptide of bacteriophage P22 in complex with the boxB RNA hairpin motif at different salt concentrations and using two different water force field models. We obtained good agreement with experiment also at different salt concentrations for the TIP4P-D water model that has a stabilizing effect on unfolded protein structures. It allowed us to estimate the free energy contribution resulting from restricting the molecules’ spatial and conformational freedom upon binding, which makes a large opposing contribution to binding. In a second set of umbrella sampling simulations to dissociate/associate the complex along a separation coordinate, we analyzed the onset of preorientation of the N-peptide and onset of structure formation relative to the RNA and its dependence on the salt concentration. Peptide orientation and conformational transitions are significantly coupled to the first contact formation between peptide and RNA. The initial contacts are mostly formed between peptide residues and the boxB hairpin loop nucleotides. A complete transition to an α-helical bound peptide conformation occurs only at a late stage of the binding process a few angstroms before the complexed state has been reached. However, the N-peptide orients also at distances beyond the contact distance such that the sizable positive charge points toward the RNA’s center-of-mass. Our result may have important implications for understanding protein- and peptide-RNA complex formation frequently involving coupled folding and association processes.

Application of high-pressure homogenization-assisted pH-shift to enhance techno-functional and interfacial properties of lentil protein isolate

High-pressure homogenization (HPH) is a promising physical non-thermal approach to improve protein techno-functionality. This study aims to examine the effects of HPH on the lentil proteins through the perspective of the interfacial adsorption mechanism. The impact of HPH treatment on lentil protein isolate (LPI) at varying pressure levels (0–150 MPa) was determined using several analytical techniques, including SDS-PAGE, FTIR, solubility, and techno-functional properties (foaming and emulsifying properties), alongside analyses of interfacial tension and interfacial shear rheology at the o/w and a/w interfaces for two pH values (2.0 and 4.5). Results reveal that HPH treatment up to 100 MPa effectively unfolds lentil proteins by disrupting disulfide-bonded subunits into lower molecular weight fractions and unfolding highly-ordered secondary structures into random coils. LPI's capacity to produce emulsions and foams was found to be enhanced concurrently with these physicochemical changes, particularly at pressures up to 50 MPa. The findings aligned with the interfacial tension and shear rheology analyses, which show that proteins can form interfacial viscoelastic films on both o/w and a/w interfaces. Furthermore, the interfacial behavior of LPI and the effect of HPH on the interfacial behavior were found to be pH-dependent. The lower interfacial tension and the higher interfacial viscoelastic moduli (G′ and G″) were recorded at 50 MPa and 0 MPa at pH 2.0 and 4.5, respectively. These results stated that the effects of the HPH on the technofunctionality of LPI can be further enlightened by investigating the interfacial adsorption kinetics.

Unlocking the unfolded structure of ubiquitin: Combining time-resolved x-ray solution scattering and molecular dynamics to generate unfolded ensembles.

The unfolding dynamics of ubiquitin were studied using a combination of x-ray solution scattering (XSS) and molecular dynamics (MD) simulations. The kinetic analysis of the XSS ubiquitin signals showed that the protein unfolds through a two-state process, independent of the presence of destabilizing salts. In order to characterize the ensemble of unfolded states in atomic detail, the experimental XSS results were used as a constraint in the MD simulations through the incorporation of x-ray scattering derived potential to drive the folded ubiquitin structure toward sampling unfolded states consistent with the XSS signals. We detail how biased MD simulations provide insight into unfolded states that are otherwise difficult to resolve and underscore how experimental XSS data can be combined with MD to efficiently sample structures away from the native state. Our results indicate that ubiquitin samples unfolded in states with a high degree of loss in secondary structure yet without a collapse to a molten globule or fully solvated extended chain. Finally, we propose how using biased-MD can significantly decrease the computational time and resources required to sample experimentally relevant nonequilibrium states.

Peptide production through enhanced solid-state fermentation of soybean meal with Bacillus subtilis SBM_1: Fermentation process and product characteristics

This study aimed to assess the impact of additional inoculation of Bacillus subtilis SBM_1 on solid-state fermentation (SSF) of soybean meal (SBM). The peptide yield of fermented SBM increased to 220.93 mg/g under the optimal SSF conditions, marking a 47.74 % rise compared to natural SSF. Throughout enhanced SSF, the physicochemical properties of metabolites were analyzed. The results showed that the pH and total bacterial counts increased in the initial 8 h, along with elevated levels of peptide, soluble protein, and free amino acid (FAA). Protease activities is maintained at a relatively high level (103.37–241.93 U/g), leading to the substantial degradation of trypsin inhibitor (TI) and macromolecular protein. Following enhanced SSF, the TI activity of the fermented SBM decreased by 76.11 %, and small peptides with molecular weight (MW) < 3000 Da dominated the fermented products (71.64 %). Additionally, the enhanced SSF process demonstrated an improvement in nutrient content in the fermented products. Structural analysis of the fermented SBM showed that macromolecular soybean globulin and allergenic proteins were degraded, with the unfolded and loosened protein spatial structures and the loosely packed and dispersed surface microstructure. Biosafety analysis revealed no detectable microbial contaminants, including mold, salmonella, and coliforms, in compliance with national standards.

Disassembling the components of virtual clothing presentation: exploring their impact on consumers' perception and purchase intention

PurposeVirtual clothing presentation has a visual 3D effort and allows consumers to interact. Currently, most scholars make virtual clothing presentations as a whole or focus on one element to study its impact on consumers’ purchase intentions. This study disassembles the components of virtual clothing presentation in Taobao and explores how each component (virtual clothing, virtual model, presentation space, presentation technology and system quality) affects consumers’ purchase intention, which can provide clothing companies recommendations for developing it and help improve consumers’ online shopping experience.Design/methodology/approachThis study created the parts of a virtual clothing presentation and took advantage of Taobao’s 3D interactive module to simulate a shopping scenario. Participants experienced a 3D interactive virtual clothing presentation in which they could change clothing colors, show actual clothing sizes, try different model poses, switch presentation backgrounds and unfold clothing structure through interactive buttons. Then they were randomly assigned to view two kinds of images and videos of experiment clothing (real images and videos vs virtual ones), respectively. Hypotheses were tested using SEM and applying SmartPLS 4.FindingsThe results demonstrated that five components of virtual clothing presentation have different degrees of influence on consumers’ purchase intention through different consumer perceptions (perceived usefulness, ease of use and enjoyment). It is necessary to consider the design of the virtual clothing presentation in those five dimensions for different needs. In addition, it is feasible to replace images and animated videos of real clothing with virtual ones in Taobao.Originality/valueDifferent types of virtual clothing presentations can lead to various results in terms of diverse impacts. This study disassembles and researches each component of a virtual clothing presentation. Overall, this study could provide guidance and suggestions for clothing companies to develop and design or invest in virtual clothing presentations.

Uncovering the freezing energy release of salted grass carp (Ctenopharyngodon idella) flesh: Effects of water state and protein structure on the thermal properties

The effect of NaCl content on the protein structures, water status and thermal properties of grass carp flesh, along with its relationship with energy required for freezing was investigated to improve the quality of frozen flesh and to reduce energy consumption for freezing. Adding salt prompted the shifting of the secondary structure of α-helix to β-sheet, β-turn and random coil. The interaction between water molecules and hydrophilic groups in the unfolded protein structure increased nonfreezing water content, which decreased specific heat capacity, thermal conductivity of sample during freezing. The lowest energy required (235.69 kJ/kg) for freezing was found in 7% NaCl salted sample, indicating a 38.39% reduction compared to the unsalted sample. Few pores between muscle fibers were observed in the 3% and 5% NaCl salted sample. Therefore, an optimal salt concentration improved quality of frozen flesh and reduced the energy required for freezing, promoting energy-efficient freezing of aquatic products.

Modification of myofibrillar protein structural characteristics: Effect of ultrasound-assisted first-stage thermal treatment on unwashed Silver Carp surimi gel

The hardness properties of unwashed surimi gel are considered as the qualities of gelation defect. This research investigated the effect of ultrasound-assisted first-stage thermal treatment (UATT) on the physicochemical properties of unwashed Silver Carp surimi gel, and the enhancement mechanism. UATT could reduce protein particle size, which significantly reduced from 142.22 μm to 106.70 μm after 30 min of UATT compared with the nature protein. This phenomenon can promote the protein crosslinking, resulting in the hardness of surimi gel increased by 15.08 %. Partially unfolded structure of myofibrillar protein and exposures of tryptophan to water, lead to the increase in the zeta potential absolute value, driven by UATT. The reduced SH group level and the conformational conversion of proteins from random coiling to α-helix and β-sheet, which was in support of intermolecular interaction and gel network construction. The results are valuable for processing protein gels and other food products.

Conjugates of glycosylated antarctic krill proteins and curcumin: Maintaining the storage quality of salmon fillets coupling with photodynamic inactivation

Antarctic krill proteins (AKP) were successfully glycosylated by inulin driven by ultrasound (300 W, 20 min) and pH shift (11.0) to prepare the conjugates of glycosylated AKP and curcumin. The preservation effects of the conjugates-mediated photodynamic inactivation (PDI) on the storage quality of salmon fillets were investigated. Results showed that the glycosylated AKP (AKP-pH11-U20) exhibited a significantly increased solubility, and a relatively small and loose morphology characterized by SEM, an unfolded and disorganized structure by FTIR and intrinsic fluorescence analysis, and a decreased surface hydrophobicity by bromophenol blue staining, and all these changes contributed to its reduced surface tension. On this basis, the glycosylated AKP efficiently loaded curcumin (323.4 μg/mg) to form the conjugates, and the conjugates-mediated PDI greatly inhibited 99.8% bacterial cells and the migration of water, retarded the change of color and pH, suppressed the lipid oxidation and degradation during 4 °C storage for 7 days, compared to the conventional curcumin-mediated PDI. This study expands the application of new marine protein resources in preserving the quality of aquatic products.

Enhancing the emulsion properties and bioavailability of loaded astaxanthin by selecting the reaction sequence of ternary conjugate emulsifiers in nanoemulsions

This study investigated the effects of the conjugate reaction sequences of whey protein concentrate (WPC), epigallocatechin gallate (EGCG) and dextran (DEX) on the structure and emulsion properties of conjugates and the bioaccessibility of astaxanthin (AST). Two types of ternary covalent complexes were synthesised using WPC, EGCG and DEX, which were regarded as emulsifiers of AST nanoemulsions. Results indicated that the WPC-DEX-EGCG conjugate (referred to as ‘con’) exhibits a darker SDS-PAGE dispersion band and higher contents of α-helix (6%), β-angle (24%) and random coil (32%), resulting in a greater degree of unfolding structure and fluorescence quenching. These findings suggested WPC-DEX-EGCG con had the potential to exhibit better emulsification properties than WPC-EGCG-DEX con. AST encapsulation efficiency (76.22%) and bioavailability (31.89%) also demonstrated the superior performance of the WPC-DEX-EGCG con emulsifier in nanoemulsion delivery systems. These findings indicate that altering reaction sequences changes protein conformation, enhancing the emulsification properties and bioavailability of AST.

Structural, emulsifying and curcumin-embedded properties of arachin nanoparticles induced by ultrasound and heat

Different types of arachin (one of the major globulins in peanut proteins) nanoparticles were prepared by ultrasound (US) and heat treatments, respectively. The results showed that short-term US treatment induced large nanoparticles (named US200 and US500, ∼267 nm) that maintained a compact globular structure; long-term US treatment induced small nanoparticles (named US500L, ∼197 nm) with a moderately unfolded structure; heat treatment induced medium-sized nanoparticles with an unfolded structure. The large nanoparticles had significantly lower surface hydrophobicity, β-sheet content, and λmax but higher enthalpy of denaturation (ΔH) than other nanoparticles. US500L and US500 exhibited the highest emulsifying activity and stability indices, respectively. As for curcumin-embedded properties, both US and heat treatments enhanced the curcumin encapsulation efficiency (from 88.53 to 94.08–97.88%) and load amount (from 1.77 to 1.88–1.96 μg/mg) of arachin. However, only curcumin in large nanoparticles was effectively protected from damage by heat and light. In simulated digestion tests, curcumin in US500L and heat-induced nanoparticles showed higher stability and bioavailability, which was mainly attributed to the slower digestion of these nanocomplexes. We have established a simple method for the preparation of arachin nanoparticles by US and the nanoparticles have potential applications in the food industry due to their favorable bifunctional properties.

Soy protein isolate-catechin complexes conjugated by pre-heating treatment for enhancing emulsifying properties: Molecular structures and binding mechanisms

This study aimed to investigate the impact of different pre-heating temperatures (ranging from 40 °C to 80 °C) on the interactions between soy protein isolate (SPI) and catechin to effectively control catechin encapsulation efficiency and optimize the emulsifying properties of soy protein isolate. Results showed that optimal heat treatment at 70 °C improved catechin encapsulation efficiency up to 93.71 ± 0.14 %, along with the highest solubility, enhanced emulsification activity index and improved thermal stability of the protein. Multiple spectroscopic techniques revealed that increasing pretreatment temperature (from 40 °C to 70 °C) altered the secondary structures of SPI, resulting in a more stable unfolded structure for the composite system with a significant increase in α-helical structures and a decrease in random coil and β-sheet structures. Moreover, optimal heat treatment also leads to an augmentation of free sulfhydryl groups within complex as well as exposure of more internal chromophore amino acids on molecular surface. Size-exclusion high-performance liquid chromatography and SDS-PAGE analysis indicated that the band intensity of newly formed high-molecular-weight soluble macromolecules (>180 kDa) increased as the pre-heating temperature rose. Furthermore, fluorescence spectroscopy and molecular docking analysis suggest that hydrophobic and covalent interactions were involved in complex formation, which intensified with increasing temperature.

Small angle X-ray scattering analysis of thermophilic cytochrome P450 CYP119 and the effects of the N-terminal histidine tag

Combining size exclusion chromatography-small angle X-ray scattering (SEC-SAXS) and molecular dynamics (MD) analysis is a promising approach to investigate protein behavior in solution, particularly for understanding conformational changes due to substrate binding in cytochrome P450s (CYPs). This study investigates conformational changes in CYP119, a thermophilic CYP from Sulfolobus acidocaldarius that exhibits structural flexibility similar to mammalian CYPs. Although the crystal structure of ligand-free (open state) and ligand-bound (closed state) forms of CYP119 is known, the overall structure of the enzyme in solution has not been explored until now. It was found that theoretical scattering profiles from the crystal structures of CYP119 did not align with the SAXS data, but conformers from MD simulations, particularly starting from the open state (46 % of all frames), agreed well. Interestingly, a small percentage of closed-state conformers also fit the data (9 %), suggesting ligand-free CYP119 samples ligand-bound conformations. Ab initio SAXS models for N-His tagged CYP119 revealed a tail-like unfolded structure impacting protein flexibility, which was confirmed by in silico modeling. SEC-SAXS analysis of N-His CYP119 indicated pentameric structures in addition to monomers in solution, affecting the stability and activity of the enzyme. This study adds insights into the conformational dynamics of CYP119 in solution.

Osmolyte-specific counteraction of ethanol-induced aggregation and structure-function distortion of catalase

Catalase, an industrially important enzyme, was found to unfold and aggregate in presence of ethanol. Aggregation of catalase was observed in 20% and higher ethanol concentrations as evident by typical light scattering aggregation profile with complete absence of lag phase, enhanced Thioflavin T fluorescence, increased 8-anilinonaphthalene-1-sulfonic acid binding depicting enhanced hydrophobic exposure, unfolded tertiary structure with an altered Fourier transform infrared and soret spectra. To prevent ethanol-induced aggregation of catalase, osmolytes were screened for their ability to counteract the ethanol effects on catalase. Among all the osmolytes tested, only three i.e., betaine, trehalose and sucrose counteracted the ethanol-induced aggregation of catalase by about 30–50%. Results infer that addition of osmolytes to ethanol-treated catalase persuaded changes in solvent polarity and protein microenvironment leading to overall counteraction of ethanol-induced structural changes in catalase responsible for its aggregation. Fluorescence microscopy also confirmed the counteraction of ethanol-induced aggregation by osmolytes as evident from the decreased presence of aggregates in osmolyte-incubated ethanol-treated catalase. Overall, the osmolyte-induced counteraction of ethanol effects on catalase is biologically significant as it provides significant insights regarding the prevention of organic solvent induced aggregation of industrially important enzymes, being otherwise vulnerable to the solvent-induced distortion of their structure-function integrity.

Role of water in the pressure-induced unfolding of Bovine Pancreatic Trypsin Inhibitor (BPTI) protein: A molecular dynamics study

The mechanism of protein denaturation under pressure is still an active area of research. Using extensive molecular dynamics simulations, for a partially unfolded structures of BPTI protein, we have studied the role of water in the unfolding of proteins at pressures of 0.001, 5, and 10 kbar at 298K. Our results show that high enough pressure leads to insertion of water into the protein’s interior, causing structural perturbation to proteins, facilitated by a decrease in the intact time of intra-protein hydrogen bond acceptor–donor distances or an increase in the residence times of shell waters or by a combination of both.

Molecular Deformation Is a Key Factor in Screening Aggregation Inhibitor for Intrinsically Disordered Protein Tau.

Direct inhibitor of tau aggregation has been extensively studied as potential therapeutic agents for Alzheimer's disease. However, the natively unfolded structure of tau complicates the structure-based ligand design, and the relatively large surface areas that mediate tau-tau interactions in aggregation limit the potential for identifying high-affinity ligand binding sites. Herein, a group of isatin-pyrrolidinylpyridine derivative isomers (IPP1-IPP4) were designed and synthesized. They are like different forms of molecular "transformers". These isatin isomers exhibit different inhibitory effects on tau self-aggregation or even possess a depolymerizing effect. Our results revealed for the first time that the direct inhibitor of tau protein aggregation is not only determined by the previously reported conjugated structure, substituent, hydrogen bond donor, etc. but also depends more importantly on the molecular shape. In combination with molecular docking and molecular dynamics simulations, a new inhibition mechanism was proposed: like a "molecular clip", IPP1 could noncovalently bind and fix a tau polypeptide chain at a multipoint to prevent the transition from the "natively unfolded conformation" to the "aggregation competent conformation" before nucleation. At the cellular and animal levels, the effectiveness of the inhibitor of the IPP1 has been confirmed, providing an innovative design strategy as well as a lead compound for Alzheimer's disease drug development.

First-principles study on the electronic structure of n-type magnetic semiconductor Ba(Zn 1−x Co x )2As2

We perform systematic first-principles calculations on the electronic structure of n-type magnetic semiconductor Ba(Zn 1−x Co x )2As2 with the facilitation of HSE06 hybrid functional. Supercells are used to consider the doping of Co atoms, and the first-principles band structures are unfolded for clarity. Based on the calculation results, magnetic states are preferred by individual Co atoms doped in Ba(Zn 1−x Co x )2As2 at diluted limit, and carriers are originated mainly from situations where only one Co atom exists in the nearest neighbor Zn sites out of certain doped Co atoms. The origination of carriers can be explained by the density of states and the unfolded band structure, where it is found that the scattering effects from single Co atom is small but quite large when more Co atoms are located at adjacent Zn sites. The large scattering effects of two adjacent Co atoms will alter the band structures near the Fermi-level. Carriers in Ba(Zn 1−x Co x )2As2 mainly originate from the As-4p orbitals, with partial contributions from the Co-3d orbitals. Our work provides new insights into the origin of the n-type carriers in magnetic semiconductors and will inspire the development of new magnetic semiconducting systems.

Mesoscopic irreversible thermodynamics of aging kinetics of alpha polypeptides [DNA] under various constraints: Special reference to the simple spring mechanics

The mesoscopic irreversible thermodynamic treatment of α-polypeptides and the helical polynucleotides (DNA) furnishes two sets of analytical expressions, which allow us not only to analyze the reversible force–extension experiments performed by atomic force microscopy (AFM) but also to predict the irreversible “aging” kinetics of the single-stranded and double-stranded polynucleotides (ssDNA and dsDNA) helical conformations exposed to aqueous solutions and applied static stress systems under the various constraints. The present physicochemical cage model emphasizes the fact that the global Helmholtz free energy of the helical conformation acts not only under the stored “intrinsic” unusual torsional and bending elastic energies inherited by the unfolded helical structure of the amino-acid (peptides) or the nucleic-acid (nucleotide) backbone but also reveals the importance of the interfacial Helmholtz free energy density associated with the interaction of the side-wall branches within the surrounding aqueous solutions. The analytical expression obtained for the unfolding force vs extension (FE) shows a strong non-linear elasticity behavior under the twist angle constraint when the interfacial Helmholtz energy term is incorporating into the scenario. This behavior is in excellent quantitative agreement with the AFM test results obtained by Idiris et al. (2000) on the poly-L-glutamic acid [Glu(n)-Cys] exposed to aqueous solutions, which show that acidity increases the degrees of helicity.

Tailoring the Techno-Functional Properties of Fava Bean Protein Isolates: A Comparative Evaluation of Ultrasonication and Pulsed Electric Field Treatments

The fava bean protein isolate (FBPI) holds promise as a sustainable plant-based protein ingredient. However, native FBPIs exhibit limited functionality, including unsuitable emulsifying activities and a low solubility at a neutral pH, restricting their applications. This study is focused on the effect of ultrasonication (US) and pulsed electric fields (PEF) on modulating the techno-functional properties of FBPIs. Native FBPIs were treated with US at amplitudes of 60–90% for 30 min in 0.5 s on-and-off cycles and with PEF at an electric field intensity of 1.5 kV/cm with 1000–4000 pulses of 20 μs pulse widths. US caused a reduction in the size and charge of the FBPIs more prominently than the PEF. Protein characterization by means of SDS-PAGE illustrated that US and PEF caused severe-to-moderate changes in the molecular weight of the FBPIs. In addition, a spectroscopic analysis using Fourier-transform infrared (FTIR) and circular dichroism (CD) revealed that US and the PEF induced conformational changes through partial unfolding and secondary structure remodeling from an α-helix to a β-sheet. Crystallographic and calorimetric determinations indicated decreased crystallinity and lowered thermal transition temperatures of the US- and PEF-modified FBPIs. Overall, non-thermal processing provided an effective strategy for upgrading FBPIs’ functionality, with implications for developing competitive plant-based protein alternatives.