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Food Chemistry: X Jun 2024Knowledge regarding the denaturation process and control methods for depolymerized sol-state myofibrillar proteins (MPs) during freezing remains scant. This study...
Effects of different protein cross-linking degrees on physicochemical and subsequent thermal gelling properties of silver carp myofibrillar proteins sol subjected to freeze-thaw cycles.
Knowledge regarding the denaturation process and control methods for depolymerized sol-state myofibrillar proteins (MPs) during freezing remains scant. This study investigated the effects of protein cross-linking treatment before freezing on physicochemical and subsequent gelation properties of MPs sol subjected to freeze-thaw (F-T) cycles. Results indicated that after five F-T cycles, cross-linked MPs sols showed increased high molecular weight polymers and bound water (T and T) mobility, suggesting enhanced protein-protein interactions at the expense of protein-water interactions. Upon heating after F-T cycles, gels formed from cross-linked sols exhibited significantly higher hardness, springiness, and cooking loss ( < 0.05), alongside more contracted gel networks. Correlation analysis revealed that the formation and properties of thermal gel after freezing closely relate to changes in molecular conformation and chemical bonds of cross-linked MPs sol during freezing. This study provides new insights into regulating the freezing stability and post-thawed thermal processing properties of sol-based surimi products.
PubMed: 38764785
DOI: 10.1016/j.fochx.2024.101448 -
Food Science of Animal Resources Mar 2024This study aimed to determine the effects of humectants on moisture content, water activity, tenderness, color, microbiological analysis, protein denaturation, and...
This study aimed to determine the effects of humectants on moisture content, water activity, tenderness, color, microbiological analysis, protein denaturation, and oxidation of jerky. A thorough search for papers published in scientific journals that examined the impacts of humectants on jerky was carried out using Web of Science, Google Scholar, PubMed, and Science Direct. Only 14 studies matched inclusion requirements. They were used in the meta-analysis to synthesise quantitative findings. In the current investigation, jerky produced with beef, poultry, goat, or pork was used. The standardised mean difference (SMD) between treatments with humectants and controls was examined to investigate the effects of humectants using random-effects models. Heterogeneity was investigated using meta-regression. A subgroup analysis was carried out for significant factors. Results revealed that the addition of humectants had no significant impact on water activity, pH, fat, ash, CIE L*, or CIE a* (p>0.05). However, humectant addition significantly increased moisture (SMD=1.28, p<0.05), CIE b* (SMD=1.67, p<0.05), and overall acceptability (SMD=1.73, p<0.05). It significantly decreased metmyoglobin (SMD=-0.96, p<0.05), shear force (SMD=-0.84, p<0.05), and protein (SMD=-1.61, p<0.05). However, it was difficult to get a firm conclusion about how humectants affected the myofibrillar fragmentation index, total plate count, and 2-thiobarbituric acid-reactive substances because there were fewer than ten studies. To sum up, the proper use of humectants in jerky demands careful attention to both type and quantity, needing a delicate balancing act with other contributing factors.
PubMed: 38764511
DOI: 10.5851/kosfa.2024.e3 -
Biochemistry Jun 2024Global substitution of leucine for analogues containing CHF instead of methyl groups delivers proteins with multiple sites for monitoring by F nuclear magnetic resonance...
Global substitution of leucine for analogues containing CHF instead of methyl groups delivers proteins with multiple sites for monitoring by F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa peptidyl-prolyl isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2,4)-5-fluoroleucine, (2,4)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The F chemical shifts correlate with the three-bond H-F couplings (), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the F chemical shifts of CHF groups. Individual CHF groups can be confined to single rotameric states by the protein environment, but most CHF groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CHF rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the F chemical shift to the rotameric state of the CHF groups potentially renders them particularly sensitive for detecting allosteric effects.
Topics: Peptidylprolyl Isomerase; Escherichia coli; Escherichia coli Proteins; Ligands; Nuclear Magnetic Resonance, Biomolecular; Leucine; Fluorine
PubMed: 38753308
DOI: 10.1021/acs.biochem.4c00080 -
Frontiers in Molecular Biosciences 2024The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the...
The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon it is capable of functioning over a wide range of temperatures. Existing X-ray structures were determined at temperatures where the protein is least active. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2°C-65°C) and metal ion binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbations with and without magnesium and manganese. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature. Hydrogen exchange protection factors and temperature coefficients reveal that both the thumb and finger domains are very dynamic relative to the palm and little-finger (LF) domains. These trends remain true at high temperature with dynamics increasing as temperatures increase from 35°C to 50°C. Notably, NMR spectra show that the Dbh tertiary structure cold denatures beginning at 25°C and increases in denaturation as the temperature is lowered to 5°C with little change observed by CD. Above 35°C, chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, without DNA bound. In contrast, these bound metals are not apparent in any Dbh crystal structures of the protein without DNA. Two ion binding sites are confirmed to be near the active site, as reported in other Y-family polymerases, and we report a novel ion binding site in the LF domain. Thus, the solution-state structure of the Dbh polymerase is distinct from that of the solid-state structures and shows an unusually high cold denaturation temperature.
PubMed: 38745908
DOI: 10.3389/fmolb.2024.1364068 -
Journal of Biomedical Research May 2024The tumor suppressor p53 is a transcription factor with a powerful antitumor activity that is controlled by its negative regulator murine double minute 2 (MDM2, also...
The tumor suppressor p53 is a transcription factor with a powerful antitumor activity that is controlled by its negative regulator murine double minute 2 (MDM2, also termed HDM2 in humans) through a feedback mechanism. At the same time, is the most frequently mutated gene in human cancers. Mutant p53 proteins lose wild-type p53 tumor suppression functions but acquire new oncogenic properties, among which are deregulating cell proliferation, increasing chemoresistance, disrupting tissue architecture, and promoting migration, invasion and metastasis as well as several other pro-oncogenic activities. The oncogenic p53 mutation Y220C creates an extended surface crevice in the DNA-binding domain destabilizing p53 and causing its denaturation and aggregation. This cavity accommodates stabilizing small molecules that have therapeutic values. The development of suitable small-molecule stabilizers is one of the therapeutic strategies for reactivating the Y220C mutant protein. In this review, we summarize approaches that target p53-Y220C, including reactivating this mutation with small molecules that bind Y220C to the hydrophobic pocket and developing immunotherapies as the goal for the near future, which target tumor cells that express the p53-Y220C neoantigen.
PubMed: 38738269
DOI: 10.7555/JBR.37.20230093 -
Bio-protocol May 2024Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the...
Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].
PubMed: 38737506
DOI: 10.21769/BioProtoc.4981 -
The Journal of Physical Chemistry. B May 2024The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to... (Comparative Study)
Comparative Study
The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to explain this, the stabilization mechanism is still far from being fully understood. This study compares the stabilizing properties of trehalose with those of the structurally similar disaccharide sucrose. The stability has been evaluated for the two proteins, lysozyme and myoglobin, at both low and high temperatures by determining the glass transition temperature, , and the denaturation temperature, . The results show that the sucrose-containing samples exhibit higher than the corresponding trehalose-containing samples, particularly at low water contents. The better stabilizing effect of sucrose at high temperatures may be explained by the fact that sucrose, to a greater extent, binds directly to the protein surface compared to trehalose. Both sugars show elevation with an increasing sugar-to-protein ratio, which allows for a more complete sugar shell around the protein molecules. Finally, no synergistic effects were found by combining trehalose and sucrose. Conclusively, the exact mechanism of protein stabilization may vary with the temperature, as influenced by temperature-dependent interactions between the protein, sugar, and water. This variability can make trehalose to a superior stabilizer under some conditions and sucrose under others.
Topics: Trehalose; Sucrose; Muramidase; Calorimetry, Differential Scanning; Myoglobin; Protein Stability; Animals; Temperature
PubMed: 38733344
DOI: 10.1021/acs.jpcb.4c00022 -
Polymers Apr 2024Meat quality and shelf life are important parameters affecting consumer perception and safety. Several factors contribute to the deterioration and spoilage of meat... (Review)
Review
Meat quality and shelf life are important parameters affecting consumer perception and safety. Several factors contribute to the deterioration and spoilage of meat products, including microbial growth, chemical reactions in the food's constituents, protein denaturation, lipid oxidation, and discoloration. This study reviewed the development of functional packaging biomaterials that interact with food and the environment to improve food's sensory properties and consumer safety. Bioactive packaging incorporates additive compounds such as essential oils, natural extracts, and chemical substances to produce composite polymers and polymer blends. The findings showed that the incorporation of additive compounds enhanced the packaging's functionality and improved the compatibility of the polymer-polymer matrices and that between the polymers and active compounds. Food preservatives are alternative substances for food packaging that prevent food spoilage and preserve quality. The safety of food contact materials, especially the flavor/odor contamination from the packaging to the food and the mass transfer from the food to the packaging, was also assessed. Flavor is a key factor in consumer purchasing decisions and also determines the quality and safety of meat products. Novel functional packaging can be used to preserve the quality and safety of packaged meat products.
PubMed: 38732702
DOI: 10.3390/polym16091232 -
International Journal of Molecular... Apr 2024Yamogenin is a steroidal saponin occurring in plant species such as , , , and sp. In this study, we evaluated in vitro cytotoxic, antioxidant, and antimicrobial...
An In Vitro Study on the Cytotoxic, Antioxidant, and Antimicrobial Properties of Yamogenin-A Plant Steroidal Saponin and Evaluation of Its Mechanism of Action in Gastric Cancer Cells.
Yamogenin is a steroidal saponin occurring in plant species such as , , , and sp. In this study, we evaluated in vitro cytotoxic, antioxidant, and antimicrobial properties of yamogenin. The cytotoxic activity was estimated on human colon cancer HCT116, gastric cancer AGS, squamous carcinoma UM-SCC-6 cells, and human normal fibroblasts with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The amount of apoptotic and dead AGS cells after treatment with yamogenin was estimated with flow cytometry. Also, in yamogenin-treated AGS cells we investigated the reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activity level of caspase-8 and -9, and gene expression at mRNA level with flow cytometry, luminometry, and RT-PCR, respectively. The antioxidant properties of yamogenin were assessed with DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays. The antimicrobial potential of the compound was estimated on , , , , , , , , , , and bacteria strains. Yamogenin showed the strongest cytotoxic effect on AGS cells (IC 18.50 ± 1.24 µg/mL) among the tested cell lines. This effect was significantly stronger in combinations of yamogenin with oxaliplatin or capecitabine than for the single compounds. Furthermore, yamogenin induced ROS production, depolarized mitochondrial membrane, and increased the activity level of caspase-8 and -9 in AGS cells. RT-PCR analysis revealed that this sapogenin strongly up-regulated expression at the mRNA level. These results indicate that yamogenin induced cell death via the extrinsic and intrinsic way of apoptosis. Antioxidant study showed that yamogenin had moderate in vitro potential (IC 704.7 ± 5.9 µg/mL in DPPH and 631.09 ± 3.51 µg/mL in ABTS assay) as well as the inhibition of protein denaturation properties (with IC 1421.92 ± 6.06 µg/mL). Antimicrobial test revealed a weak effect of yamogenin on bacteria strains, the strongest one being against (with MIC value of 350 µg/mL). In conclusion, yamogenin may be a potential candidate for the treatment and prevention of gastric cancers.
Topics: Humans; Antioxidants; Saponins; Stomach Neoplasms; Cell Line, Tumor; Apoptosis; Reactive Oxygen Species; Anti-Infective Agents; Membrane Potential, Mitochondrial; Plant Extracts; Antineoplastic Agents, Phytogenic
PubMed: 38731847
DOI: 10.3390/ijms25094627 -
Foods (Basel, Switzerland) Apr 2024Antifreeze peptides have become effective antifreeze agents for frozen products, but their low quantity of active ingredients and high cost limit large-scale...
Antifreeze peptides have become effective antifreeze agents for frozen products, but their low quantity of active ingredients and high cost limit large-scale application. This study used the glycosylation of fish collagen peptides with glucosamine hydrochloride catalyzed by transglutaminase to obtain a transglutaminase-catalyzed glycosylation product (TGP) and investigate its antifreeze effect on tilapia. Compared with the blank group, the freshness (pH value of 6.31, TVB-N value of 21.7 mg/100 g, whiteness of 46.28), textural properties (especially hardness and elasticity), and rheological properties of the TGP groups were significantly improved. In addition, the protein structures of the samples were investigated using UV absorption and fluorescence spectroscopy. The results showed that the tertiary structure of the TGP groups changed to form a dense polymer. Therefore, this approach can reduce the denaturation and decomposition of muscle fibers and proteins in fish meat more effectively and has a better protective effect on muscle structure and protein aggregation, improving the stability of fish meat. This study reveals an innovative method for generating antifreeze peptides by enzymatic glycosylation, and glycosylated fish collagen peptide products can be used as new and effective green antifreeze agents in frozen foods.
PubMed: 38731690
DOI: 10.3390/foods13091319