Search Results (192)

This study used soybean meal as the substrate and systematically optimized its enzymatic hydrolysis through single-factor experiments and response surface methodology. A predictive model based on a Box–Behnken design was developed to improve protein hydrolysis efficiency and increase the yield of functional products. The optimal conditions were 1.45% enzyme addition, a reaction time of 62 h, a temperature of 36.5 °C, and a moisture content of 35%. Under these conditions, the small-peptide content increased 16.33-fold. Structural analyses showed that enzymatic treatment markedly disrupted the compact surface of soybean meal, converting it into a loose, porous matrix. In addition, enzymolysis altered the protein secondary structure from ordered α-helices and folded conformations to more disordered, flexible forms, thereby improving the molecular-weight distribution. Composition analyses showed an 114.2% increase in total free amino acids, including essential amino acids. Moreover, DPPH radical-scavenging activity increased from 18.37% to 57.99%. Overall, this study optimized the enzymatic hydrolysis conditions for soybean meal and provides valuable insights for the development of high-value protein-peptide products. Full article

Photodynamic therapy is a cancer treatment that relies on a photosensitizer (PS) to generate reactive oxygen species upon light activation, thereby destroying cancer cells. The photophysical properties of porphyrins make them effective PSs, while nanoparticles (NPs) enhance their delivery and stability. The bioavailability and targeting efficiency of NPs-PS complexes may be improved through transport via human serum albumin (HSA). This study investigates the HSA binding affinity with 5,10,15,20-(Tetra-4-carboxyphenyl)porphyrin (TCPP) and with TiO₂-TCPP complexes. The interactions were analyzed using UV-Vis absorption, laser-induced fluorescence (LIF), and FTIR spectroscopy. Molecular docking was performed and provided consistent binding constant values for the TCPP–HSA complex with UV-Vis absorption measurements. LIF data revealed a slightly lower affinity when compare free porphyrin with TiO₂-TCPP, possibly due to competitive binding between TiO₂ and HSA. Docking simulations indicated that TCPP favorably interacts with amino acid residues located in subdomains IB and IIIA of HSA, supporting a preferential binding near Sudlow site I. FTIR measurements revealed conformational changes in HSA for both its interactions with TCPP and TiO₂-TCPP, including alterations in α-helical content and reorganization of the hydrogen bonding network within the polypeptide backbone. Full article

Background/Objectives: Non-syndromic tooth agenesis (NSTA) is a congenital condition that causes the absence of one or more teeth without accompanying systemic abnormalities, which significantly affects quality of life. Genetic factors, including mutations in several specific genes, contribute to the pathogenesis of NSTA. This study investigates a novel EVC2 mutation in a patient with NSTA and explores its potential pathogenic mechanism, with the aim of enriching the spectrum of pathogenic genes. Methods: Whole-exome sequencing (WES) was performed on peripheral blood samples from a patient diagnosed with NSTA. Bioinformatics analysis was utilized to identify the mutation and assess its potential impact on protein structure and function. Molecular dynamics simulations were conducted to analyze structural alterations in the EVC2 protein. The binding affinity between EVC2, EVC, and Smoothened (SMO) was to determine the effect of mutation on protein–protein interaction. Protein localization and expression were analyzed using immunofluorescence and Western blotting. Reverse transcription quantitative PCR (RT-qPCR) was employed to evaluate downstream signaling pathway alterations. Results: A novel EVC2 mutation (c.1657_1660delinsA, p.Glu553_leu554delinsMet) was identified in the proband, and the mutation was maternally inherited. Molecular dynamics simulations revealed that the mutation resulted in a decrease in α-helical content and significant conformational changes in the protein structure. This led to reduced binding affinity between EVC2 and its ligands EVC and SMO, destabilizing the structural integrity of the protein complex. Despite these structural changes, EVC2 protein localization and expression were unaffected. Furthermore, a downregulation of GLI1 and SHH expression was observed, indicating impaired Hedgehog (Hh) signaling. The downregulation of the Hh signaling pathway impairs the tooth development process and may lead to the occurrence of tooth agenesis. Conclusions: A novel EVC2 mutation was identified in a patient with NSTA. Based on molecular dynamics simulations, it is hypothesized that this EVC2 variant could contribute to the pathogenesis of NSTA by impairing the EVC2-EVC-SMO complex formation, which may lead to downregulation of downstream GLI1 and SHH. These findings provide new insights into the molecular mechanisms underlying EVC2-mediated NSTA, suggesting that disruption of Hh signaling may represent a critical pathogenic mechanism. Full article

This work explores the impact of ergosterol (ERG) addition (0%, 0.5%, 1.0%, 1.5%, and 2.0%) on the physicochemical properties, conformational changes, and digestive characteristics of soy protein isolate (SPI) and wheat gluten (WG) processed by high-moisture extrusion. The results demonstrated that the incorporation of ERG significantly reduced the apparent viscosity and dynamic moduli of the feedstock system, enhancing melt fluidity and consequently reducing extrusion torque, die pressure, and specific mechanical energy. An appropriate amount of ERG (1.0%) effectively facilitated the development of a distinct fibrous morphology, increased the fibrous degree, lightened the color, and softened the texture. However, excessive addition weakened the fibrous structure due to excessively high fluidity. ERG influenced protein aggregation behavior through hydrophobic interactions, reduced thermal stability, and induced a transition in secondary structure from β-turns to α-helices. The in vitro digestibility initially decreased and then increased, with the lowest value observed at 1.0% ERG. This study indicates that ERG can elevate the performance and value of extruded products by modulating protein structure and rheological behavior, providing a theoretical basis for its application in plant-based meat analogue products. Full article

Elastin is the principal protein found in the elastic fibers of vertebrate tissues, and the water within these fibers plays a crucial role in preserving the structure and function of this hydrophobic protein. Soluble elastin was successfully obtained by repeatedly treating insoluble elastin, extracted from pig aorta, with oxalic acid. Solid-state NMR analysis was performed on the soluble elastin, focusing on conformation-dependent chemical shifts of alanine residues. This analysis revealed that cross-linked alanine residues exhibited both α-helix and random coil structures in the dry state. In contrast, the hydrated state favored random coil structures, with some distorted helices possibly present, indicating that the cross-linked configuration is relatively unstable. Similar conformational changes were observed in insoluble elastin, mirroring those found in the soluble form. Additionally, when the soluble elastin was re-cross-linked using 1,12-dodecanedicarboxylic acid and 4-hydroxyphenyl dimethylsulfonium methylsulfate, it retained a mixture of α-helix and random coil structures in the dry state. Remarkably, in the hydrated state, α-helix structures were more prominently preserved alongside random coils. These structural changes corresponded with increased stiffness of molecular chains in the hydrophobic regions compared to their state prior to re-cross-linking, even under hydrated conditions. Full article

Background/Objectives: Intrinsically disordered protein regions (IDRs) are difficult to study due to their flexible nature and transient interactions. Computational folding using AlphaFold may offer one way to explore potential folding of these regions under various conditions. Human DNA topoisomerase IIα (TOP2A) is an essential enzyme involved in regulating DNA topology during replication and cell division. TOP2A has an IDR at the C-terminal domain (CTD) that has been shown to be important for regulating TOP2A function, but little is known about potential conformations that it may undertake. Methods: Utilizing the AlphaFold 3 (AF3) model by way of AlphaFold Server, TOP2A was folded as a dimer first without and then with 29 literature-supported post-translational modifications (PTMs) and DNA to observe whether there is predicted folding. Results: TOP2A CTD does not fold in the absence of PTMs. With the addition of PTMs, however, the CTD is predicted to fold into a globular bundle of loops and α-helices. While DNA alone did not induce folding, in the presence of PTMs, DNA ligands increased helicity of the folded CTD and caused it to interact at different core domain interfaces. In addition, DNA is predicted to enable folding of the TOP2A CTD in the presence of fewer PTMs when compared to the absence of DNA. Conclusions: AF3 predicts the folding of TOP2A CTD in the presence of specific PTMs, and this folding appears to shift to allow binding to DNA in functionally relevant regions. These studies provide predicted folding patterns that can be tested by biochemical approaches. AF3 may support the development of testable hypotheses regarding IDRs and enables researchers to model protein-DNA interactions. Full article

Cold denaturation of gluten proteins during prolonged frozen storage or repeated freeze–thaw cycles can severely affect the quality of frozen cereal products. While both processes have been studied individually, their combined effects and underlying mechanisms remain unclear. This study systematically evaluated the hydration properties and conformational changes in gluten proteins stored at −73 °C and −23 °C, with or without freeze–thaw cycling. Compared to continuous storage, freeze–thaw cycles reduced water-holding capacity by 9.1–12.2% and increased oil-holding capacity by 5.3–10.3%, indicating aggravated structural damage. Ultra-low temperature storage (−73 °C) suppressed ice crystal growth, preserved hydration, and limited hydrophobic residue exposure. Spectroscopic analyses revealed a temperature-dependent shift from α-helices to β-sheets and β-turns, which was accelerated by freeze–thaw cycles. Enhanced hydrophobic interactions and tryptophan exposure further indicated destabilization. Molecular dynamics simulations showed that increased hydrogen bonding between proteins and water contributed to unfolding at low temperatures, while temperature fluctuations intensified denaturation through repeated hydrogen bond breakage and reformation. These results underscore the critical role of thermal instability in cold denaturation and offer mechanistic insights for improving cryoprotection strategies in frozen food systems. Full article

Cancer cells can adapt to their surrounding microenvironment by upregulating glucose-regulated protein 78 kDa (GRP78) and vacuolar-type ATPase (V-ATPase) proteins to increase their proliferation and resilience to anticancer therapy. Therefore, targeting these proteins can obstruct cancer progression. A comprehensive computational study was conducted to investigate the inhibitory potential of four proton pump inhibitors (PPIs), dexlasnoprazole (DEX), esomeprazole (ESO), pantoprazole (PAN), and rabeprazole (RAB), against GRP78 and V-ATPase. Molecular docking revealed high-affinity scores for PPIs against both proteins. Moreover, molecular dynamics showed favorable root mean square deviation values for GRP78 and V-ATPase complexes, whereas root mean square fluctuations were high at the substrate-binding subdomains of GRP78 complexes and the α-helices of V-ATPase. Meanwhile, the radius of gyration and the surface-accessible surface area of the complexes were not significantly affected by ligand binding. Trajectory projections of the first two principal components showed similar motions of GRP78 structures and the fluctuating nature of V-ATPase structures, while the free-energy landscape revealed the thermodynamically favored GRP78-RAB and V-ATPase-DEX conformations. Furthermore, the binding free energy was −16.59 and −18.97 kcal/mol for GRP78-RAB and V-ATPase-DEX, respectively, indicating their stability. According to our findings, RAB and DEX are promising candidates for GRP78 and V-ATPase inhibition experiments, respectively. Full article

Background/Objectives: Cancer is one of the leading causes of death worldwide, and skin cancer is especially prevalent and lethal in Brazil. Despite advancements in treatment, there is still a need for new anticancer agents that are effective, selective, and less toxic. This study aimed to evaluate the cytotoxic and therapeutic potential of the peptide PEPAD. Methods: The cytotoxicity of PEPAD was assessed by MTT assay in murine melanoma (B16F10-Nex2), human melanoma (SK-MEL-28), breast (MCF-7), and cervical (HeLa) cancer cell lines. Selectivity was evaluated in healthy cells (RAW 264.7 and FN1). Morphological changes were analyzed by microscopy. Cell migration was assessed using scratch assays. Apoptotic features were evaluated using MitoTracker Deep Red, NucBlue, CaspACETM labeling, and flow cytometry. Immunogenic cell death was investigated by calreticulin and HMGB1 release. Molecular dynamics simulations explored peptide structure and interaction with lipid membranes. Results: PEPAD showed IC₅₀ values of 7.4 µM and 18 µM in B16F10-Nex2 and SK-MEL-28 cells, respectively, and >60 µM in MCF-7 and HeLa cells. Low toxicity was observed in healthy cells (IC₅₀ > 56 µM), indicating high selectivity. Apoptotic morphology and reduced cell migration were observed. Flow cytometry and fluorescence probes confirmed apoptosis and mitochondrial swelling. Calreticulin and HMGB1 release indicated immunogenic cell death. Simulations showed that PEPAD maintains a stable α-helical conformation and interacts with membranes. Conclusions: These findings highlight PEPAD’s selective cytotoxicity and its potential as an anticancer agent with apoptotic and immunogenic properties, making it a promising candidate for therapeutic development. Full article

Antimicrobial peptides (AMPs) are a primary defense against pathogens. Here, we examined the interaction of two BP100 analogs, R²R⁵-BP100 (where Arg substitutes Lys 2 and 5) and R²R⁵-BP100-A-NH-C₁₆ (where an Ala and a C₁₆ hydrocarbon chain are added to the R²R⁵-BP100 C-terminus), with membrane models. Large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs) were prepared with the major lipids in Gram-positive (GP) and Gram-negative (GN) bacteria, as well as red blood cells (RBCs). Fluorescence data, dynamic light scattering (DLS), and zeta potential measurements revealed that upon achieving electroneutrality through peptide binding, vesicle aggregation occurred. Circular dichroism (CD) spectra corroborated these observations, and upon vesicle binding, the peptides acquired α-helical conformation. The peptide concentration, producing a 50% release of carboxyfluorescein (C₅₀) from LUVs, was similar for GP-LUVs. With GN and RBC-LUVs, C₅₀ decreased in the following order: BP100 > R²R⁵-BP100 > R²R⁵BP100-A-NH-C₁₆. Optical microscopy of GP-, GN-, and RBC-GUVs revealed the rupture or bursting of the two former membranes, consistent with a carpet mechanism of action. Using GUVs, we confirmed RBC aggregation by BP100 and R²R⁵-BP100. We determined the minimal inhibitory concentrations (MICs) of peptides for a GN bacterium (Escherichia coli (E. coli)) and two GP bacteria (two strains of Staphylococcus aureus (S. aureus) and one strain of Bacillus subtilis (B. subtilis)). The MICs for S. aureus were strain-dependent. These results demonstrate that Lys/Arg replacement can improve the parent peptide’s antimicrobial activity while increasing hydrophobicity renders the peptide less effective and more hemolytic. Full article

This study investigates the structural and functional enhancement of corn zein–chitosan composites via mild alkaline treatment to develop biodegradable protein-polysaccharide materials for diverse applications. Films with varying zein-to-chitosan ratios were fabricated and characterized using Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Both untreated and sodium hydroxide (NaOH)-treated films were evaluated to assess changes in physicochemical properties. FTIR analysis revealed that NaOH treatment promoted deprotonation of chitosan’s amine groups, partial removal of ionic residues, and increased deacetylation, collectively enhancing hydrogen bonding and resulting in a denser molecular network. Simultaneously, partial unfolding of zein’s α-helical structures improved conformational flexibility and strengthened interactions with chitosan. These molecular-level changes led to improved thermal stability, reduced degradation, and the development of porous microstructures. Controlled NaOH treatment thus provides an effective strategy to tailor the physicochemical properties of zein–chitosan composite films, supporting their potential in sustainable food packaging, wound healing, and drug delivery applications. Full article

Aggregation of tau protein is a hallmark feature of tauopathies such as Alzheimer’s disease. The microtubule-binding domain of tau plays a crucial role in the tau aggregation process. In this study, we investigated the dual effects of membrane interactions of tau_298–317, a fragment peptide from the microtubule-binding domain, on peptide-induced membrane disruption and membrane-mediated peptide self-assembly. Our results show that neither wild-type tau_298–317 nor its P301L or Ser305-phosphorylated mutants aggregate in the presence of zwitterionic POPC vesicles or cause lipid vesicle leakage, indicating weak peptide–membrane interactions. In contrast, tau_298–317 strongly interacts with negatively charged POPG liposomes, leading to a rapid transition of the peptide conformation from random coils to α-helical intermediate conformation upon membrane adsorption, which may further promote peptide self-association to form oligomers and β-sheet-rich fibrillar structures. Tau_298–317-induced rapid POPG membrane leakage indicates a synergistic process of the peptide self-assembly at the membrane interface and the aggregation-induced membrane disruption. Notably, phosphorylation at Ser305 disrupts favorable electrostatic interactions between the peptide and POPG membrane surface, thus preventing peptide aggregation and membrane leakage. In contrast, the P301L mutation significantly enhances membrane-mediated peptide aggregation and peptide-induced membrane disruption, likely due to alleviation of local conformational constraints and enhancement of local hydrophobicity, which facilitates fast conformational conversion to β-sheet structures. These findings provide mechanistic insights into the molecular mechanisms underlying membrane-mediated aggregation of crucial regions of tau and peptide-induced membrane damage, indicating potential strategies to prevent tau aggregation and membrane rupture by targeting critical electrostatic interactions between membranes and key local regions of tau. Full article

The increasing circulation of multi-drug-resistant pathogens, coupled with the sluggish development of new antibiotics, is weakening our capacity to combat human infections, resulting in elevated death tolls. To address this worldwide crisis, antimicrobial peptides (AMPs) are viewed as promising substitutes or adjuvants for combating bacterial infections caused by multidrug-resistant organisms. Here, the antimicrobial activity and structural characterization of a novel 13-amino acid cationic peptide named RKW (RKWILKWLRTWKK-NH2), designed based on known AMPs sequences and the identification of a key tryptophan-rich structural motif, were described. RKW displayed a broad-spectrum and potent antimicrobial and antibiofilm activity against Gram-positive and Gram-negative pathogens, including ESKAPE bacteria and fungi with minimal inhibitory concentrations (MBC) ranging from 5 µM to 20 μM. Structural results by fluorescence and Circular Dichroism (CD) spectroscopy revealed that the peptide was folded into a regular α-helical conformation in a membrane-like environment, remaining stable in a wide range of pH and temperature for at least 48 h of incubation. Furthermore, RKW showed low toxicity in vitro against mammalian fibroblast cells, indicating its potential as a promising candidate for the development of new antimicrobial or antiseptic strategies. Full article

This review investigates the novel idea that proteins catalyze chemical reactions through conformational changes driven by energy derived from their collisions with water molecules. Recent studies have suggested that proteins in solution undergo constant deformation due to collisions with water molecules, generating potential energy that can be harnessed for catalytic functions. We detail the existing evidence supporting this idea, including how structures in proteins such as α-helices and β-sheets facilitate energy conversion, how conformational changes can affect the ways in which substrates attach, and how reactions occur. Combining information from computer-based methods—such as molecular dynamics simulations and machine learning models (e.g., AlphaFold)—we suggest a more complete model for understanding how proteins function beyond simply looking at their fixed shapes. This emerging view has implications for drug design, enzyme engineering, and our fundamental understanding of biological catalysis. Full article

Iron deficiency is a global health issue, making the development of novel iron supplements to enhance iron absorption critically important. In this study, low molecular weight donkey-hide gelatin peptides (LMW DHGP) were enzymatically hydrolyzed from donkey-hide gelatin. Experimental results demonstrated that the iron chelating capacity of LMW DHGP reached 249.98 μg/mg. Key amino acids (Asn, Gly, Cys, Lys) may participate in chelation. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis showed rough, porous amorphous structures of LMW DHGP-iron complexes. The results of circular dichroism spectroscopy (CD) indicated that the self-assembly of LMW DHGP-iron complexes appears to be primarily mediated by peptide α-helical structural conformations. Fourier transform infrared (FTIR) spectroscopy further indicated that the interaction between LWM DHGP and Fe²⁺ likely occurs through carboxyl and amino functional groups. In vitro digestion stability studies demonstrated that LMW DHGP-iron complexes exhibited superior iron ion solubility compared to FeSO₄ in simulated gastrointestinal conditions. PGPAG-iron complexes exhibited the highest antioxidant activity, with scavenging rates of 71.64% (DPPH radical) and 88.79% (ABTS radical). These findings collectively suggest that LMW DHGP-iron complexes possess significant potential as a novel iron supplement in food applications, which provides valuable theoretical insights for the development of innovative iron supplementation strategies. Full article