Search Results (640)

Protein–polyelectrolyte nanostructures assembled via electrostatic interactions offer versatile applications in biomedicine, tissue engineering, and food science. However, several open questions remain regarding their intermolecular interactions and the influence of external conditions—such as temperature and pH—on their assembly, stability, and responsiveness. This study explores the formation and stability of networks between poly(acrylic acid) (PAA) and lysozyme (LYZ) at the nanoscale upon thermal treatment, using a combination of experimental and simulation measures. Experimental techniques of static and dynamic light scattering (SLS and DLS), Fourier transform infrared spectroscopy (FTIR), and circular dichroism (CD) are combined with all-atom molecular dynamics simulations. Model systems consisting of multiple PAA and LYZ molecules explore collective assembly and complexation in aqueous solution. Experimental results indicate that electrostatic complexation occurs between PAA and LYZ at pH values below LYZ’s isoelectric point. This leads to the formation of nanoparticles (NPs) with radii ranging from 100 to 200 nm, most pronounced at a PAA/LYZ mass ratio of 0.1. These complexes disassemble at pH 12, where both LYZ and PAA are negatively charged. However, when complexes are thermally treated (TT), they remain stable, which is consistent with earlier findings. Atomistic simulations demonstrate that thermal treatment induces partially reversible structural changes, revealing key microscopic features involved in the stabilization of the formed network. Although electrostatic interactions dominate under all pH and temperature conditions, thermally induced conformational changes reorganize the binding pattern, resulting in an increased number of contacts between LYZ and PAA upon thermal treatment. The altered hydration associated with conformational rearrangements emerges as a key contributor to the stability of the thermally treated complexes, particularly under conditions of strong electrostatic repulsion at pH 12. Moreover, enhanced polymer chain associations within the network are observed, which play a crucial role in complex stabilization. These insights contribute to the rational design of protein–polyelectrolyte materials, revealing the origins of association under thermally induced structural rearrangements. Full article

Pectin is a multifunctional polysaccharide whose structural attributes, including degree of esterification (DE), molecular weight (MW), and branching, directly affect its gelling, emulsifying, and bioactive properties. Conventional pectin extraction relies on acid- or alkali-based methods that degrade the pectin structure, generate chemical waste, and alter its physicochemical and functional properties. Although novel methods such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and enzyme-assisted extraction (EAE) are recognized as environmentally friendly alternatives, they frequently use acids or alkalis as solvents. This review focuses on pectin extraction methods that do not involve acidic or alkaline solvents such as chelating agents, super/subcritical water, and deep eutectic solvents (DESs) composed of neutral components. This review also discusses how these alternative extraction methods can preserve or modify the key structural features of pectin, thereby influencing its monosaccharide composition, molecular conformation, and interactions with other biopolymers. Furthermore, the influence of these structural variations on the rheological properties, gelling behaviors, and potential applications of pectin in the food, pharmaceutical, and biomedical fields are discussed. This review provides insights into alternative strategies for obtaining structurally intact and functionally diverse pectin by examining the relationship between the extraction conditions and pectin functionality. Full article

Protein S-nitrosylation is a selective post-translational modification in which a nitrosyl group is covalently attached to the reactive thiol group of cysteine, forming S-nitrosothiol. This modification plays a pivotal role in modulating physiological and pathological cardiovascular processes by altering protein conformation, activity, stability, and other post-translational modifications. It is instrumental in regulating vascular and myocardial systolic and diastolic functions, vascular endothelial cell and cardiomyocyte apoptosis, and cardiac action potential and repolarization. Aberrant S-nitrosylation levels are implicated in the pathogenesis of various cardiovascular diseases, including systemic hypertension, pulmonary arterial hypertension, atherosclerosis, heart failure, myocardial infarction, arrhythmia, and diabetic cardiomyopathy. Insufficient S-nitrosylation leads to impaired vasodilation and increased vascular resistance, while excessive S-nitrosylation contributes to cardiac hypertrophy and myocardial fibrosis, thereby accelerating ventricular remodeling. This paper reviews the S-nitrosylated proteins in the above-mentioned diseases and their impact on these conditions through various signaling pathways, with the aim of providing a theoretical foundation for the development of novel therapeutic strategies or drugs targeting S-nitrosylated proteins. Full article

Protein packing within crystal lattices plays a critical role in determining molecular flexibility; therefore, the observed conformation and flexibility of protein side chains can vary depending on the crystal space group. Protein crystal dehydration affects crystal lattice mosaicity, which can either reduce crystal quality or enhance X-ray diffraction intensity. It also often alters the crystal lattice, leading to space group transition. Accordingly, dehydration-induced space group transitions could theoretically offer an alternative when there are experimental limitations obstructing the obtainment of diverse crystal forms. However, this remains underexplored experimentally. Here, a dehydration-induced space group transition was explored to observe different conformations and flexibilities of the protein structure. Xylanase GH11 crystals from Thermoanaerobacterium saccharolyticum (TsaGH11) were air-dehydrated, and their structure at room temperature was determined. Upon dehydration, the space group of the TsaGH11 crystal changed from tetragonal to orthorhombic, affecting the protein–protein interfaces within the crystal lattice. The dehydrated crystal structure of TsaGH11 revealed multiple conformations of residues involved in substrate binding and recognition within the substrate-binding cleft. These diverse molecular conformations and flexibilities provide significant and previously unrevealed structural information for TsaGH11. This approach demonstrates the potential of dehydration-induced space group transitions to reveal diverse protein conformations, offering valuable insights into molecular properties and functions. Full article

The H1N1 swine influenza viruses CQ91 and CQ445, isolated from pigs in China, exhibited distinct virulence in mice despite sharing similar genomic constellations. CQ91 demonstrated higher pathogenicity (MLD₅₀: 5.4 log₁₀ EID₅₀) and replication efficiency in mice compared to CQ445 (MLD₅₀: 6.6 log₁₀ EID₅₀). Through reverse genetics, we found that the attenuation of CQ445 was due to a single substitution of glutamic acid (E) with lysine (K) at position 343 in the PB2 protein. Introducing the CQ445-PB2 (343K) into CQ91 significantly reduced viral replication and pathogenicity in mice, while replacing CQ445-PB2 with CQ91-PB2 (343E) restored virulence. In vitro studies showed that the K343E mutation impaired viral replication in MDCK and A549 cells and reduced polymerase activity in minigenome assays. Mechanistically, the amino acid at position 343 in the PB2 affects the transcription stage of the viral replication process. Structural modeling indicated that the charge reversal caused by E343K altered local electrostatic interactions without major conformational changes. Phylogenetic analysis revealed that PB2-343E is highly conserved (>99.9%) in human and swine H1/H3 influenza viruses, suggesting that PB2-343E confers an adaptive advantage. This study identifies PB2-343E as a critical determinant of influenza virus pathogenicity in mammals, highlighting its role in host adaptation. Full article

Background: The Nerve Growth Factor (NGF) is essential for neuronal survival and function and represents a key therapeutic target for pain and inflammation-related disorders, as well as for neurodegenerative diseases. Small-molecule antagonists of human NGF (hNGF) offer advantages over monoclonal antibodies, including oral availability and reduced immunogenicity. However, their development is often hindered by solubility challenges, necessitating the use of solvents like dimethyl sulfoxide (DMSO). This study investigates whether DMSO directly interacts with hNGF and affects its receptor-binding properties. Methods: Integrative/hybrid computational and experimental biophysical approaches were used to assess DMSO-NGF interaction by combining machine-learning tools and Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FT-IR) spectroscopy, Differential Scanning Fluorimetry (DSF) and Grating-Coupled Interferometry (GCI). These techniques evaluated binding affinity, conformational stability, and receptor-binding dynamics. Results: Our findings demonstrate that DMSO binds hNGF with low affinity in a specific yet non-disruptive manner. Importantly, DMSO does not induce significant conformational changes in hNGF nor affect its interactions with its receptors. Conclusions: These results highlight the importance of considering solvent–protein interactions in drug discovery, as these low-affinity yet specific interactions can affect experimental outcomes and potentially alter the small molecules binding to the target proteins. By characterizing DMSO-NGF interactions, this study provides valuable insights for the development of NGF-targeting small molecules, supporting their potential as effective alternatives to monoclonal antibodies for treating pain, inflammation, and neurodegenerative diseases. Full article

The misfolding and aggregation of proteins into amyloidogenic assemblies are key features of several metabolic and neurodegenerative diseases. Human insulin has long been known to form amyloid fibrils under various conditions, which affects its bioavailability and function. Clinically, insulin aggregation at recurrent injection sites poses a challenge for diabetic patients who rely on insulin therapy. Furthermore, decreased responsiveness to insulin in type 2 diabetic (T2D) patients may lead to its overproduction and accumulation as aggregates. Earlier reports have reported that various factors such as pH, temperature, agitation, and the presence of lipids or other proteins influence insulin aggregation. Our present study aims to elucidate the effects of non–micellar anionic DMPG (1,2–dimyristoyl–sn–glycero–3–phosphoglycerol) lipids on insulin aggregation. Distinct pathways of insulin aggregation and intermediate formation were observed in the presence of DMPG using a ThT fluorescence assay. The formation of soluble intermediates alongside large insulin fibrils was observed in insulin incubated with DMPG via TEM, DLS, and NMR as opposed to insulin aggregates generated without lipids. ¹³C magic angle spinning solid–state NMR and FTIR experiments indicated that lipids do not alter the conformation of insulin fibrils but do alter the time scale of motion of aromatic and aliphatic side chains. Furthermore, the soluble intermediates were found to be more cytotoxic than fibrils generated with or without lipids. Overall, our study elucidates the importance of anionic lipids in dictating the pathways and intermediates associated with insulin aggregation. These findings could be useful in determining various approaches to avoid toxicity and enhance the effectiveness of insulin in therapeutic applications. Full article

Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB) serves as a tethering factor that interacts with various proteins and recruits these proteins to the ER surface, exerting multiple functions, such as organelle membrane tethering, lipid transfer between organelles, regulation of calcium homeostasis, autophagy, and the unfolded protein response (UPR). Its interaction is often mediated by its MSP (major sperm) domain, which binds with FFAT (two phenylalanines in an acidic tract)-motif-containing proteins. However, pathogenic variations, such as P56S, P56H, and T46I, in the VAPB MSP domain lead to the familial form of amyotrophic lateral sclerosis (ALS8). Still, the underlying pathophysiology of ALS8 due to pathogenic variations in the VAPB MSP domain remains elusive. In this study, we conducted molecular dynamics (MD) simulations to understand the pathogenic-variant-derived changes in the structural dynamics of the VAPB MSP domain. We found that pathogenic variants altered the fluctuations and conformational dynamics of the VAPB protein. Analyzing the organizations of the secondary structure revealed that pathogenic variants changed the composition of secondary structure elements, especially increasing the proportion of α-helix while reducing β-sheet formation, which might affect the organelle tethering and other functions of VAPB, as well as VAPB homodimer and heterodimer formation. Taken together, these findings can be further investigated through in vivo and/or in vitro studies to not only clarify the pathophysiology of ALS8 resulting from VAPB MSP domain pathogenic variants but also develop novel therapeutics for the disease that restore the native structural organizations as well as fluctuations and motions. Full article

Reactive nitrogen species (RNS), particularly peroxynitrite (ONOO⁻), play a central role in post-translational modifications (PTMs) of proteins, including fibrinogen, a key component of the coagulation cascade. This review explores the structural and functional consequences of fibrinogen nitration, with a focus on its impact on clot formation, morphology, mechanical stability, and fibrinolysis. Nitration, primarily targeting tyrosine residues within functional domains of the Aα, Bβ, and γ chains, induces conformational changes, dityrosine crosslinking, and aggregation into high molecular weight species. These modifications result in altered fibrin polymerization, the formation of porous and disorganized clot networks, reduced mechanical resilience, and variable susceptibility to fibrinolysis. Moreover, nitrated fibrinogen may affect interactions with platelets and endothelial cells, although current evidence remains limited. Emerging clinical studies support its role as both a prothrombotic mediator and a potential biomarker of oxidative stress in cardiovascular and inflammatory diseases. Finally, we explore both pharmacological interventions, such as NOX inhibitors, and natural antioxidant strategies at counteracting fibrinogen nitration. Overall, fibrinogen nitration emerges as a critical molecular event linking oxidative stress to thrombotic risk. Full article

The increasing demand for sustainable protein sources has intensified interest in improving the processing efficiency of traditional proteins and developing novel alternatives, particularly those derived from plants and algae. Among various processing technologies, pH shifting has attracted attention due to its simplicity, low cost, and capacity to effectively alter protein structure and functionality. However, employing pH shifting alone requires extremely acidic or alkaline conditions, which can lead to protein denaturation and the generation of undesirable by-products. To address these limitations, this review explores the integration of pH shifting with physical processing techniques such as ultrasound, high-pressure processing, pulsed electric fields, and thermal treatments. Moreover, this review highlights the effects of these combined treatments on protein conformational transitions and the resulting improvements in functional properties such as solubility, emulsification, foaming capacity, and thermal stability. Importantly, they reduce reliance on extreme chemical conditions, providing greater sustainability in industrial applications, particularly in food product development where milder processing conditions help preserve nutritional quality and functional properties. In that sense, this combined treatment approach provides a promising and eco-efficient protein modification strategy, and bridges technological innovation with sustainable resource utilization. Full article

The kinetics of the depolymerization of natural rubber (NR) to hydroxyl-terminated natural rubber (HTNR) by hydrogen peroxide (H₂O₂) in the presence of a Fenton catalyst within an acidic milieu and under ultraviolet radiation has been rigorously examined utilizing infrared spectroscopy to determine the alterations in molar mass and the functional characteristics. The kinetic model was analyzed in accordance with the elementary reaction, encompassing the following mechanisms: the interaction between hydroxyl radicals and NR, producing radical NR and hydroxylated NR; the reaction wherein radical NR and hydroxyl radicals yield hydroxylated NR; and the subsequent reaction of hydroxylated NR with hydroxyl radicals producing lower radical NR, hydroxylated terminated NR, radical NR, and hydroxylated NR. The conversion of the NR polymer and the total hydroxyl content were discerned at the absorption bands of the CH₂-CH₂ and OH groups located at 850 cm⁻¹ and 3400 cm⁻¹, respectively. The absorption peak at 1850 cm⁻¹ attributed to CH₃ was employed as the reference group for calibration. The influence of the temperature on the depolymerization process conformed to the Arrhenius equation, characterized by activation energies of 750 K and 1200 K. The impact of the H₂O₂/Fenton ratio on the depolymerization process follows a power law with power coefficients of 1.97 and 1.82. Full article

Human immunodeficiency virus (HIV) continues to be a threat to public health. An emerging technique with promise in the context of fighting HIV type 1 (HIV-1) focuses on targeting ribosomal frameshifting. A crucial –1 programmed ribosomal frameshift (PRF) has been observed in several pathogenic viruses, including HIV-1. Altered folds of the HIV-1 RNA frameshift element (FSE) have been shown to alter frameshifting efficiency. Here, we use RNA-As-Graphs (RAG), a graph-theory based framework for representing and analyzing RNA secondary structures, to perform conformational analysis in motif space to propose how sequence length may influence folding patterns. This combined analysis, along with all-atom modeling and experimental testing of our designed mutants, has already proven valuable for the SARS-CoV-2 FSE. As a first step to launching the same computational/experimental approach for HIV-1, we compare prior experiments and perform SHAPE-guided 2D-fold predictions for the HIV-1 FSE embedded in increasing sequence contexts and predict structure-altering mutations. We find a highly stable upper stem and highly flexible lower stem for the core FSE, with a three-way junction connecting to other motifs at increasing lengths. In particular, we find little support for a pseudoknot or triplex interaction in the core FSE, although pseudoknots can form separately as a connective motif at longer sequences. We also identify sensitive residues in the upper stem and central loop that, when minimally mutated, alter the core stem loop folding. These insights into the FSE fold and structure-altering mutations can be further pursued by all-atom simulations and experimental testing to advance the mechanistic understanding and therapeutic strategies for HIV-1. Full article

Umami is a fundamental taste sensation, often described as a delicious and pleasant flavor perception. To enhance or complement the original flavor and meet the tastes of diverse regions, umami dipeptides have been extensively utilized in global food manufacturing. Currently, the application and purification techniques of dipeptides are relatively mature, while their umami mechanisms and molecular modification are both scarce. In this work, the 3D structure of the umami dipeptide target T1R1/T1R3 was first obtained through sequence alignment and homology modeling, then followed by the successful construction of a database containing 400 samples of dipeptides. Subsequently, the complex models of T1R1/T1R3, respectively, with DG (Asp-Gly) and EK (Glu-Lys) (i.e., T1R1_DG/T1R3, T1R1/T1R3_DG, T1R1_EK/T1R3, and T1R1/T1R3_EK) were obtained via molecular docking and virtual screening. Finally, based on comparative molecular dynamics (MD) simulation trajectories, the binding free energy was calculated to investigate receptor–ligand recognition and conformational changes, providing some implications for potential modifications of umami dipeptides. T1R1 tends to bind relatively small umami dipeptides, whereas T1R3 does the opposite, both of which favor the recognition of acidic and hydrophilic dipeptides. By comparing strategies such as hydroxyl introduction and chain length alteration, electrostatic effects may be more important than non-polar effects in molecular design. This work not only explores the recognition mechanism of umami dipeptides with the receptor T1R1/T1R3 showing certain theoretical significance, but also provides feasible suggestions for dipeptide screening and modification having certain application value. Full article

Lipopolysaccharide (LPS) endotoxin is a well-characterized microbe-associated molecular pattern (MAMP) that forms the outer membrane of both pathogenic and commensal Gram-negative bacteria. It plays a crucial role in triggering inflammatory disorders such as mastitis, acidosis, and septicemia. In heifers, an LPS challenge induces a dynamic stress response, marked by elevated cortisol levels, increased body temperature, and altered immune function. Research indicates that LPS administration leads to a significant rise in cortisol post-challenge. Building on this understanding, the present study aimed to estimate genetic parameters for serum cortisol response to LPS challenge in Holstein heifers and its linear associations with production, health, reproduction, and conformation traits. Additionally, a genome-wide association study (GWAS) was conducted to identify genetic regions associated with cortisol response. A total of 252 animals were evaluated for cortisol response, with correlations estimated between cortisol levels and 55 genomic breeding values for key traits. Genetic parameters and heritability for cortisol response were estimated using Residual Maximum Likelihood (REML) in the Blupf90+ v 2.57 software. Single-Step GWAS (ssGWAS) employing a 10-SNP window approach and 42,123 SNP markers was performed to identify genomic regions that explained at least 0.5% of additive genetic variance. Finally, candidate genes and QTLs located 50 kb up and downstream of those windows were identified. The cortisol response showed significant but weak linear associations with cystic ovaries, body maintenance requirements, lactation persistency, milk yield, and protein yield (p-value ≤ 0.05) and showed suggestive weak linear associations with udder texture, clinical ketosis, heel horn erosion, and milking speed (p-value ≤ 0.15). Cortisol response showed significant additive genetic variance, along with moderate heritability of 0.26 (±0.19). A total of 34 windows explained at least 0.5% of additive genetic variance, and 75 QTLs and 11 candidate genes, comprising the genes CCL20, DAW1, CSMD2, HMGB4, B3GAT2, PARD3, bta-mir-2285aw, CFH, CDH2, ENSBTAG00000052242, and ENSBTAG00000050498, were identified. The functional enrichment analysis allowed us to infer two instances where these gene products could interfere with cortisol production: the first instance is related to the complement system, and the second one is related to the EMT (Epithelium–Mesenchymal Transition) and pituitary gland formation. Among the QTLs, 13 were enriched in the dataset, corresponding to traits related to milk (potassium content), the exterior (udder traits, teat placement, foot angle, rear leg placement, and feet and leg conformation), production (length of productive life, net merit, and type), and reproduction (stillbirth and calving ease). In summary, the cortisol response to LPS challenge in Holstein heifers seems to be moderately heritable and has weak but significant linear associations with important production and health traits. Several candidate genes identified could perform important roles, in at least two ways, for cortisol production, and QTLs were identified close to regions of the genome that explained a significant amount of additive genetic variance for cortisol response. Therefore, further investigations are warranted to validate these findings with a larger dataset. Full article

Minced lamb remains one of the most produced meat products in the meat industry, across both the food service and retail sectors. Tea polyphenols (TPs), renowned for their diverse biological activities, are increasingly being employed as natural food additives in research and development. Tea polyphenols at concentrations of 0.00% (CG), 0.01% (TP1), 0.10% (TP2), and 0.30% (TP3) were added to lamb which had undergone a series of freeze–thaw cycles. The presence of tea polyphenols led to a significant decrease in the number of disulfide bonds, resulting in a slower oxidation rate. In addition, the surface hydrophobicity and juice loss of the minced lamb supplemented with tea polyphenols were 91.23 ± 0.22 and 20.00 ± 0.46, respectively, representing a reduction of 1.5% and 7.59% compared to the group without the addition of tea polyphenols. However, the addition of high-dose tea polyphenols also led to a reduction in emulsification stability, alterations in protein conformation, and changes in water migration. Furthermore, the incorporation of a minimal quantity of tea polyphenols (0.01%) resulted in enhanced emulsification stability, water retention, textural properties, and microstructures in minced lamb. This suggests that tea polyphenols have the potential to improve the quality of minced lamb following freezing and thawing processes. Full article