Search Results (29)

Soybean protein isolate (SPI) gel has been demonstrated to exhibit suboptimal stability and a coarse texture. Selective enzymatic hydrolysis modification has been demonstrated to effectively enhance the functional properties and structural stability of the protein. The objective of this study was to modify SPI using alkaline protease and papain. The impact of selective enzymatic hydrolysis on SPI was examined through the analysis of hydrolysis degree (DH), particle size, and protein purity. A systematic exploration was conducted in order to investigate the structural and quality characteristics of SPI gel. Indicators such as secondary structure changes, texture characteristics, water-holding capacity (WHC), rheology, and microstructure were analyzed. The findings indicate that when the DH of the SPI solution is 1%, its particle size is reduced relative to that when DH is 0.5%. The SDS-PAGE results indicated that alkaline protease could hydrolyze most of the 7S and 11S components in SPI into shorter peptides, while papain retained more of the 7S and 11S components and generated peptides with larger molecular weights. Fourier-transform infrared (FT-IR) spectral analysis indicated that following the process of enzymatic modification, the contents of α-helix and β-sheet in the secondary structure of SPI increased, while the contents of β-turns and random coils decreased. In the context of gel performance, it has been demonstrated that papain-modified SPI, attributable to its elevated content of macromolecular peptides, manifests superior WHC, hardness, springiness, cohesiveness, chewiness, storage modulus (G), and microstructure in comparison to alkaline protease-modified gel. Concurrently, the gel performance of papain modified SPI is significantly superior to that of unmodified SPI gel. This research provides a significant theoretical foundation and practical reference for promoting the efficient application of SPI in the domain of food processing. Full article

Cold environments challenge the structural and functional integrity of membrane proteins, requiring specialized adaptations to maintain activity under low thermal energy. Here, we investigate the molecular basis of cold tolerance in the peptide transporter PepT1 from the Antarctic icefish (Chionodraco hamatus, ChPepT1) using molecular dynamics simulations, binding free energy calculations (MM/GBSA), and dynamic network analysis. We compare ChPepT1 to its human ortholog (hPepT1), a non-cold-adapted variant, to reveal key features enabling psychrophilic function. Our simulations show that ChPepT1 displays enhanced global flexibility, particularly in domains adjacent to the substrate-binding site and the C-terminal domain (CTD). While hPepT1 loses substrate binding affinity as temperature increases, ChPepT1 maintains stable peptide interactions across a broad thermal range. This thermodynamic buffering results from temperature-sensitive rearrangement of hydrogen bond networks and more dynamic lipid interactions. Importantly, we identify a temperature-responsive segment (TRS, residues 660–670) within the proximal CTD that undergoes an α-helix to coil transition, modulating long-range coupling with transmembrane helices. Dynamic cross-correlation analyses further suggest that ChPepT1, unlike hPepT1, reorganizes its interdomain communication in response to temperature shifts. Our findings suggest that cold tolerance in ChPepT1 arises from a combination of structural flexibility, resilient substrate binding, and temperature-sensitive interdomain dynamics. These results provide new mechanistic insight into thermal adaptation in membrane transporters and offer a framework for engineering proteins with enhanced functionality in extreme environments. Full article

Acute kidney injury (AKI) resulting from ischemia/reperfusion (I/R) poses a significant clinical challenge due to its high mortality and complex pathophysiology. Here, the protective actions of Coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) in carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced adenosine triphosphate depletion and recovery (ATP-D/R) injury in human kidney-2 (HK2) cells are examined. During ATP-D/R, expression levels of CHCHD2 were significantly reduced. The overexpression of CHCHD2 substantially reduced the levels of ROS, lipid peroxidation, apoptosis, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL), whereas the knockdown of CHCHD2 exacerbated cellular injury. Mechanistic studies further demonstrated that overexpression of CHCHD2 restored Nrf2 expression under ATP-D/R conditions, facilitated its nuclear translocation, and upregulated the downstream antioxidant enzyme HO-1. In contrast, the knockdown of Nrf2 reduced the cytoprotective actions of CHCHD2. These findings indicate that CHCHD2 reduces cellular damage by enhancing antioxidant defenses and reducing apoptosis through activating the Nrf2 axis, underscoring its potential as a therapeutic target for AKI. Full article

Anthocyanin synthetase (ANS), a key enzyme in the final step of the anthocyanin synthesis pathway, catalyzes the conversion of leucoanthocyanidins to anthocyanins. In this study, an ANS structural protein (TRINITY_DN18024_c0_g1) was found to be associated with anthocyanin accumulation in Acer pseudosieboldianum leaves, named ApsANS. Real-time quantitative fluorescence PCR analysis revealed that the expression of ApsANS was significantly higher in red-leaved (variant) than green-leaved (wild-type) strains, which was consistent with the transcriptome data. The UPLC results showed that the cyanidin metabolites may be the key substance influencing the final color formation of Acer pseudosieboldianum. The ApsANS gene was cloned and analyzed through bioinformatics analysis. ApsANS has a total length of 1371 bp, and it encodes 360 amino acids. Analysis of the structural domain of the ApsANS protein revealed that ApsANS contains a PcbC functional domain. Protein secondary structure predictions indicate that α-helix, irregularly coiled, and extended chains are the major building blocks. Subcellular localization predicted that ApsANS might be localized in the nucleus. The phylogenetic tree revealed that ApsANS is relatively closely related to ApANS in Acer palmatum. The prediction of miRNA showed that the ApsANS gene is regulated by miR6200. This study provides a theoretical reference for further analyzing the regulatory mechanism of leaf color formation in Acer pseudosieboldianum. Full article

The bacterial flagellar motor consists of a rotor and stator units and is driven by ion flow through the stator. The activation of the ion flow is coupled with the anchoring of the stator units to the peptidoglycan layer by the stator B-subunit around the rotor. Gram-negative bacteria, such as Salmonella and Vibrio, change the conformation of the N-terminal helix of the periplasmic domain of the B-subunit to anchor the stator units. However, a recent high-speed atomic force microscopic study has suggested that the periplasmic domain of MotS, the stator B-subunit of the sodium (Na⁺)-driven stator of Bacillus subtilis, a gram-positive bacterium, unfolds at low external Na⁺ concentrations and folds at high Na⁺ concentrations to anchor the stator units. Here, we report the crystal structures of MotS_68–242, a periplasmic fragment of MotS, from B. subtilis at high and low Na⁺ concentrations. We also performed far-UV CD spectroscopic analysis of the wild-type MotS_68–242 and MotS_78–242 proteins and mutant variants of MotS_68–242 under high and low Na⁺ concentrations and found that the N-terminal disordered region of MotS_68–242 shows a Na⁺-dependent coil–helix transition. We propose a mechanism of the Na⁺-dependent structural transition of Bs-MotS to anchor the stator units. Full article

Microtubules, polymerized from α-tubulin (TUA) and β-tubulin (TUB) monomers, play a pivotal role in shaping plant morphogenesis according to developmental and environmental cues. Salix psammophila C. Wang & C. Y. Yang is an important shrub plant in sand-fixing afforestation in arid regions, with three significantly distinct plant types shaped under various environments, namely, upright, intermediate, and scattered types. However, how tubulin genes respond to the developmental and environmental signs in S. psammophila has been far less studied. Here, based on RNA-seq, Sanger sequencing, and real-time PCR (RT-PCR) data, we analyzed the phylogeny of tubulins and their expression profiles in S. psammophila among the three plant types. Furthermore, we analyzed the genetic structure and expression pattern of SpsTUB10 in S. psammophila under various abiotic stress treatments. In total, we identified 26 SpsTubulin genes in S. psammophila. The homologous alignment and phylogenetic analysis revealed that these SpsTubulin genes can be classified into two groups, corresponding to the TUA and TUB genes. The expression profiles of these SpsTubulin genes in various organs showed that most SpsTubulin genes were mainly expressed in the root. SpsTUB10 is a member of the TUB IIa group, consisting of two intros and three exons. The SpsTUB10 protein contains a typical GTPase domain and a C-terminal domain, with α-helix and random coil dominant in the secondary and tertiary structures. The RT-PCR results of SpsTUB10 showed an extremely significant difference in expression levels among the root and stem-developing organs between the upright and scattered types, and the transcript level of SpsTUB10 had a significantly negative correlation with the crown-height ratio. Under different treatments, we found that cold, osmotic stress, and short daylight could significantly increase SpsTUB10 expression levels compared to those in the controls, thereby supporting the positive role of SpsTUB10 in stress-induced responses. These results will provide evidence for the SpsTubulin genes’ response to the developmental and environmental cues in S. psammophila. Full article

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an $\alpha$ helical switch towards a $\beta$ hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin–membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water–ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two $\alpha$ helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed. Full article

Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) is a nuclear-encoded mitochondrial protein which is primarily mutated in the spectrum of familial and sporadic amyotrophic lateral sclerosis (ALS)–frontotemporal dementia (FTD). Endogenous CHCHD10 levels decline in the brains of ALS–FTD patients, and the CHCHD10^S59L mutation in Drosophila induces dominant toxicity together with PTEN-induced kinase 1 (PINK1), a protein critical for the induction of mitophagy. However, whether and how CHCHD10 variants regulate mitophagy flux in the mammalian brain is unknown. Here, we demonstrate through in vivo and in vitro models, as well as human FTD brain tissue, that ALS/FTD-linked CHCHD10 mutations (R15L and S59L) impair mitophagy flux and mitochondrial Parkin recruitment, whereas wild-type CHCHD10 (CHCHD10^WT) normally enhances these measures. Specifically, we show that CHCHD10^R15L and CHCHD10^S59L mutations reduce PINK1 levels by increasing PARL activity, whereas CHCHD10^WT produces the opposite results through its stronger interaction with PARL, suppressing its activity. Importantly, we also demonstrate that FTD brains with TAR DNA-binding protein-43 (TDP-43) pathology demonstrate disruption of the PARL–PINK1 pathway and that experimentally impairing mitophagy promotes TDP-43 aggregation. Thus, we provide herein new insights into the regulation of mitophagy and TDP-43 aggregation in the mammalian brain through the CHCHD10–PARL–PINK1 pathway. Full article

4-Coumarate: coenzyme A ligase (4CL; EC 6.2.1.12) is an important enzyme in the phenylpropanoid metabolic pathway that controls the biosynthesis of lignin and flavonoids. In this study, to identify the function of the Ag4CL3 gene of celery, the Ag4CL3 gene was cloned from celery cv. “Nanxuan Liuhe Ziqin”. Sequence analysis results showed that the Ag4CL3 gene contained an open reading frame (ORF) with a length of 1688 bp, and 555 amino acids were encoded. The Ag4CL3 protein was highly conserved among different plant species. Phylogenetic analysis demonstrated that the 4CL proteins from celery and carrot belonged to the same clade. The Ag4CL3 protein was mainly composed of 31.89% α-helixes, 18.02% extended strands, 6.67% β-turns, and 43.42% random coils, and the signal peptide was unfound. A total of 62 phosphorylation sites and a class-I superfamily of adenylate-forming domains were found. As the growth time increased, the plant height and stem thickness also increased, and the petiole lignin content increased and became lignified gradually. The relative expression levels of the Ag4CL3 gene in “Nanxuan Liuhe Ziqin” petioles were higher than those in other tissues, with the highest level occurring 70 d after sowing. The lignin contents in the transgenic Arabidopsis thaliana lines hosting the Ag4CL3 gene were higher than those in the WT. In this study, the overexpression of Ag4CL3 led to the significant upregulation of lignin biosynthesis gene expression in transgenic A. thaliana plants, except for AtPAL, AtCCR, and AtLAC. This study speculates that Ag4CL3 genes are related to lignin synthesis in A. graveolens. Full article

Electrode-pair towed antennas are widely utilized for marine electromagnetic detection, underwater communication, and other purposes. However, the motion-induced noise created by antenna vibrations due to environmental turbulence affects extremely low frequency and super low frequency (ELF/SLF) communications. In this article, we presented a method for detecting the motion-induced noise of electrode-pair towed antennas using helix coil sensors. The equivalent resistance, inductance, capacitance, and conductance (RLCG) transmission model and parameters were derived based on the mirror method of the twisted structure with a shielding layer inside. A water-flow cycling experimental platform was constructed to evaluate two types of antenna sections. Electrode-pair, accelerators, and helix coils sensors signals were sampled and analyzed in time and frequency domain. The comparison results demonstrated that the helix coil sensors achieved a high correlation with the electrode-pair towed antenna in various vibration speeds ranging from 0.7 to 0.9. Full article

Ca²⁺/calmodulin (Ca²⁺/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca²⁺/CaM is not fully understood. Ca²⁺/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca²⁺/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca²⁺/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca²⁺/CaM with dissociation constants (K_d(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca²⁺-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca²⁺-dependent association at resting [Ca²⁺] of 50–100 nM is indicated in these complexes as one of the CaM Ca²⁺ binding sites displays high affinity with K_d of 70 and 30 nM for Ca²⁺, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca²⁺/CaM in a dose dependent manner, further cementing Ca²⁺/CaM as a regulator of gap junction function. The motion of a stretched CaM–CL2 complex compacting upon Ca²⁺ binding may bring about the Ca²⁺/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane. Full article

In this project, we combine two areas of research, experimental characterization and molecular docking studies of the interaction of positively charged oligopeptides with crucial blood plasma proteins. The investigated peptides are rich in NH₂ groups of amino acid side chains from Dap, Orn, Lys, and Arg residues, which are relevant in protein interaction. The peptides are 9- and 11-mer with the following sequences: (Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt), (Lys-Dab-Ala-Gly-Orn-Pro-His-Lys-Arg), and (Lys-Dab-Dab-Gly-Orn-Pro-Phe(2-F)-Lys-Arg). The net charge of the compound strongly depends on the pH environment and it is an important aspect of protein binding. The studied oligopeptides exhibit therapeutic properties: anti-inflammatory activity and the capacity to diminish reactive oxygen species (ROS). Therefore, the mechanism of potential binding with blood plasma components is the next challenge. The binding interaction has been investigated under pseudo-physiological conditions with the main blood plasma proteins: albumin (BSA), α1-acid glycoprotein (AAG), and γ-globulin fraction (GGF). The biomolecular quenching constant (k_q) and binding constant (K_b) were obtained by fluorescence spectroscopy at various temperatures. Simultaneously, the changes in the secondary structure of proteins were monitored by circular dichroism (CD) and infrared spectroscopy (IR) by quantity analysis. Moreover, molecular docking studies were conducted to estimate the binding affinity, the binding domain, and the chemical nature of these interactions. The results show that the investigated oligopeptides could be mainly transported by albumin, and the binding domain I is the most favored cavity. The BSA and GGF are able to form stable complexes with the studied compounds as opposed to AAG. The binding reactions are spontaneous processes. The highest binding constants were determined for Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt peptide, in which the values of the binding constants K_b to BSA and GGF were 10.1 × 10⁴ dm³mol⁻¹ and 3.39 × 10³ dm³mol⁻¹, respectively. The positively charged surface of peptides participated in salt bridge interaction with proteins; however, hydrogen bonds were also formed. The secondary structure of BSA and GGF after contact with peptides was changed. A reduction in the α-helix structure was observed with an increase in the β-sheet and β-turn and random coil structures. Full article

Insulin degrading enzyme (IDE) has been detected in the cerebrospinal fluid media and plays a role in encapsulating and degrading the amyloid β (Aβ) monomer, thus regulating the levels of Aβ monomers. The current work illustrates a first study by which IDE encapsulates polymorphic early-stage Aβ oligomers. The main goal of this study was to investigate the molecular mechanisms of IDE activity on the encapsulated early-stage Aβ dimers: fibril-like and random coil/α-helix dimers. Our work led to several findings. First, when the fibril-like Aβ dimer interacts with IDE-C domain, IDE does not impede the contact between the monomers, but plays a role as a ‘dead-end’ chaperone protein. Second, when the fibril-like Aβ dimer interacts with the IDE-N domain, IDE successfully impedes the contacts between monomers. Third, the inhibitory activity of IDE on random coil/α-helix dimers depends on the stability of the dimer. IDE could impede the contacts between monomers in relatively unstable random coil/α-helix dimers, but gets hard to impede in stable dimers. However, IDE encapsulates stable dimers and could serve as a ‘dead-end’ chaperone. Our results examine the molecular interactions between IDE and the dimers, and between the monomers within the dimers. Hence, this study provides insights into the inhibition mechanisms of the primary nucleation of Aβ aggregation and the basic knowledge for rational design to inhibit Aβ aggregation. Full article

The crystal protein Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis, has been demonstrated to have excellent anthelmintic activity. While a previous structure of the three-domain core region of Cry5B(112–698) had been reported, this structure lacked a key N-terminal extension critical to function. Here we report the structure of Cry5B(27–698) containing this N-terminal extension. This new structure adopts a distinct quaternary structure compared to the previous Cry5B(112–698) structure, and also exhibits a change in the conformation of residues 112–140 involved in linking the N-terminal extension to the three-domain core by forming a random coil and an extended α-helix. A role for the N-terminal extension is suggested based on a computational model of the tetramer with the conformation of residues 112–140 in its alternate α-helix conformation. Finally, based on the Cry5B(27–698) structure, site-directed mutagenesis studies were performed on Tyr495, which revealed that having an aromatic group or bulky group at this residue 495 is important for Cry5B toxicity. Full article

The less virulent human (h) coronaviruses (CoVs) 229E, NL63, OC43, and HKU1 cause mild, self-limiting respiratory tract infections, while the more virulent SARS-CoV-1, MERS-CoV, and SARS-CoV-2 have caused severe outbreaks. The CoV envelope (E) protein, an important contributor to the pathogenesis of severe hCoV infections, may provide insight into this disparate severity of the disease. We, therefore, generated full-length E protein models for SARS-CoV-1 and -2, MERS-CoV, HCoV-229E, and HCoV-NL63 and docked C-terminal peptides of each model to the PDZ domain of the human PALS1 protein. The PDZ-binding motif (PBM) of the SARS-CoV-1 and -2 and MERS-CoV models adopted a more flexible, extended coil, while the HCoV-229E and HCoV-NL63 models adopted a less flexible alpha helix. All the E peptides docked to PALS1 occupied the same binding site and the more virulent hCoV E peptides generally interacted more stably with PALS1 than the less virulent ones. We hypothesize that the increased flexibility of the PBM in the more virulent hCoVs facilitates more stable binding to various host proteins, thereby contributing to more severe disease. This is the first paper to model full-length 3D structures for both the more virulent and less virulent hCoV E proteins, providing novel insights for possible drug and/or vaccine development. Full article