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International Journal of Biological... May 2024In this paper, effects of preheating-induced denaturation of proteins and oleosomes on protein structure and soymilk quality were studied. The protein in soybeans baked...
In this paper, effects of preheating-induced denaturation of proteins and oleosomes on protein structure and soymilk quality were studied. The protein in soybeans baked at 55 °C (B-55) and 85 °C (B-85) showed an increase of β-sheet content by 3.2 % and a decrease of α-helix content by 3.3 %, indicating that proteins were gradually unfolded while oleosomes remained intact. The protein resisted thermal denaturation during secondary heating, and soymilks were stable as reflected by a small d (0.4 μm). However, raw soymilk from soybeans baked at 115 °C (B-115), steamed for 1 min (ST-1) and 5 min (ST-5) presented oleosomes destruction and lipids aggregates. The proteins were coated around the oil aggregates. The β-turn content from soybeans steamed for 10 min (ST-10) increased by 9.5 %, with a dense network where the OBs were tightly wrapped, indicating the serious protein denaturation. As a result, the soymilks B-115 or steamed ones were unstable as evidenced by the serious protein aggregation and larger d (5.65-12.48 μm). Furthermore, the soymilks were graininess and the protein digestion was delayed due to the formation of insoluble protein aggregates. The flavor and early-stage lipid digestion of soymilk from steamed soybeans was improved owing to lipid release.
Topics: Protein Denaturation; Soy Milk; Soybean Proteins; Hot Temperature; Lipid Droplets; Cooking
PubMed: 38697416
DOI: 10.1016/j.ijbiomac.2024.131999 -
Comprehensive Reviews in Food Science... Mar 2024Whey protein denaturation and aggregation have long been areas of research interest to the dairy industry, having significant implications for process performance and... (Review)
Review
Whey protein denaturation and aggregation have long been areas of research interest to the dairy industry, having significant implications for process performance and final product functionality and quality. As such, a significant number of analytical techniques have been developed or adapted to assess and characterize levels of whey protein denaturation and aggregation, to either maximize processing efficiency or create products with enhanced functionality (both technological and biological). This review aims to collate and critique these approaches based on their analytical principles and outline their application for the assessment of denaturation and aggregation. This review also provides insights into recent developments in process analytical technologies relating to whey protein denaturation and aggregation, whereby some of the analytical methods have been adapted to enable measurements in-line. Developments in this area will enable more live, in-process data to be generated, which will subsequently allow more adaptive processing, enabling improved product quality and processing efficiency. Along with the applicability of these techniques for the assessment of whey protein denaturation and aggregation, limitations are also presented to help assess the suitability of each analytical technique for specific areas of interest.
Topics: Whey Proteins; Whey; Protein Denaturation; Hydrogen-Ion Concentration
PubMed: 38343297
DOI: 10.1111/1541-4337.13289 -
Current Molecular Pharmacology 2024Ischemia-Reperfusion Injury (IRI) is a paradoxical phenomenon where removing the source of injury can cause additional damage. Ischemia reduces ATP production and... (Review)
Review
Ischemia-Reperfusion Injury (IRI) is a paradoxical phenomenon where removing the source of injury can cause additional damage. Ischemia reduces ATP production and intracellular pH, reducing oxidative reactions, increasing lactic acid release, and activating anaerobic metabolism. Reperfusion restores aerobic respiration and increases ROS production, leading to malfunction of transmembrane transport, activation of proteases, DNA dissolution, and protein denaturation, leading to apoptotic cell death. Nrf2 is a transcription factor that regulates cellular inflammation and oxidative responses. It is activated by oxidants and electrophiles and enhances detoxifying enzyme expression, maintaining redox homeostasis. It also activates ARE, which activates several ARE-regulated genes that favor cell survival by exhibiting resistance to oxidants and electrophiles. Nrf2 regulates the antioxidant defense system by producing phase II and antioxidant defense enzymes, including HO-1, NQO-1, gglutamylcysteine synthetase, and rate-limiting enzymes for glutathione synthesis. Nrf2 protects mitochondria from damage and supports mitochondrial function in stress conditions. Resveratrol is a stilbene-based compound with a wide variety of health benefits for humans, including antioxidant, anticarcinogenic, antitumor, and estrogenic/antiestrogenic. Resveratrol protects against IRI through several signaling pathways, including the Nrf2/ARE pathway. Here, we review the studies that investigated the mechanisms of resveratrol protection against IRI through modulation of the Nrf2 signaling pathway.
Topics: Humans; Resveratrol; Antioxidants; NF-E2-Related Factor 2; Reperfusion Injury; Oxidants
PubMed: 38389416
DOI: 10.2174/0118761429246578231130064830 -
AMB Express Jul 2023Natural products, such as enzymatic hydrolysates and bioactive peptides from dietary sources, are safe alternatives to synthetic compounds linked to various deleterious...
Natural products, such as enzymatic hydrolysates and bioactive peptides from dietary sources, are safe alternatives to synthetic compounds linked to various deleterious effects. The purpose of this study is to determine the in vitro bioactivities (antioxidant and anti-inflammatory activities) of Garcinia kola seeds enzymatic hydrolysates (GKPHs) at different enzyme (pepsin)-substrate ratios. G. kola protein, isolated by alkaline solubilization and acid precipitation, was hydrolyzed with pepsin at varying enzyme-substrate (E:S) ratios. The antioxidant parameters investigated include 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging, hydrogen peroxide scavenging and ferrous ion (Fe) chelating activities. For anti-inflammatory properties, membrane stabilization and protein denaturation activities tests were used. GKPH produced at 1:32 had the highest degree of hydrolysis (66.27 ± 4.21%). All GKPHs had excellent in vitro anti-inflammatory properties. However, only enzymatic hydrolysates produced at 1:16 (E:S) ratio chelated iron (II) and as well had the highest percentage hemolysis inhibition of 84.45 ± 0.007%, percentage protein denaturation inhibition of 53.36 ± 0.01% at maximum concentration and exhibited highest DPPH scavenging activity (87.24 ± 0.10%). The enzymatic hydrolysates had excellent solubility, emulsifying and foaming properties. It could be deduced from this study that pepsin at a ratio of 1:16 of G. kola protein produced the most effective enzymatic hydrolysates in terms of their antioxidant and anti-inflammatory activities. G. kola pepsin enzymatic hydrolysates, thus, have potential in development as functional foods and as therapeutics pharmaceutical industries in the management of diseases associated with oxidative stress and inflammation owing to their excellent functional, antioxidant and anti-inflammatory properties.
PubMed: 37495834
DOI: 10.1186/s13568-023-01583-2 -
Pharmaceutics Nov 2023In recent years, biosynthesized zinc oxide nanoparticles (ZnONPs) have gained tremendous attention because of their safe and non-toxic nature and distinctive biomedical... (Review)
Review
In recent years, biosynthesized zinc oxide nanoparticles (ZnONPs) have gained tremendous attention because of their safe and non-toxic nature and distinctive biomedical applications. A diverse range of microbes (bacteria, fungi and yeast) and various parts (leaf, root, fruit, flower, peel, stem, etc.) of plants have been exploited for the facile, rapid, cost-effective and non-toxic synthesis of ZnONPs. Plant extracts, microbial biomass or culture supernatant contain various biomolecules including enzymes, amino acids, proteins, vitamins, alkaloids, flavonoids, etc., which serve as reducing, capping and stabilizing agents during the biosynthesis of ZnONPs. The biosynthesized ZnONPs are generally characterized using UV-VIS spectroscopy, TEM, SEM, EDX, XRD, FTIR, etc. Antibiotic resistance is a serious problem for global public health. Due to mutation, shifting environmental circumstances and excessive drug use, the number of multidrug-resistant pathogenic microbes is continuously rising. To solve this issue, novel, safe and effective antimicrobial agents are needed urgently. Biosynthesized ZnONPs could be novel and effective antimicrobial agents because of their safe and non-toxic nature and powerful antimicrobial characteristics. It is proven that biosynthesized ZnONPs have strong antimicrobial activity against various pathogenic microorganisms including multidrug-resistant bacteria. The possible antimicrobial mechanisms of ZnONPs are the generation of reactive oxygen species, physical interactions, disruption of the cell walls and cell membranes, damage to DNA, enzyme inactivation, protein denaturation, ribosomal destabilization and mitochondrial dysfunction. In this review, the biosynthesis of ZnONPs using microbes and plants and their characterization have been reviewed comprehensively. Also, the antimicrobial applications and mechanisms of biosynthesized ZnONPs against various pathogenic microorganisms have been highlighted.
PubMed: 38004613
DOI: 10.3390/pharmaceutics15112634 -
Biophysical Journal Jul 2023The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric...
The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric and filamentous actin. Recent work on the protein actophorin from the amoeba Acanthamoeba castellani identified a series of site-directed mutations that increase the thermal stability of the protein by 22°C. Here, we expand this observation by showing that the mutant protein is also significantly stable to both equilibrium and kinetic chemical denaturation, and employ computer simulations to account for the increase in thermal or chemical stability through an accounting of atomic-level interactions. Specifically, the potential of mean force (PMF) can be obtained from steered molecular dynamics (SMD) simulations in which a protein is unfolded. However, SMD can be inefficient for large proteins as they require large solvent boxes, and computationally expensive as they require increasingly many SMD trajectories to converge the PMF. Adaptive steered molecular dynamics (ASMD) overcomes the second of these limitations by steering the particle in stages, which allows for convergence of the PMF using fewer trajectories compared with SMD. Use of the telescoping water scheme within ASMD partially overcomes the first of these limitations by reducing the number of waters at each stage to only those needed to solvate the structure within a given stage. In the PMFs obtained from ASMD, the work of unfolding Acto-2 was found to be higher than the Acto-WT by approximately 120 kCal/mol and reflects the increased stability seen in the chemical denaturation experiments. The evolution of the average number of hydrogen bonds and number of salt bridges during the pulling process provides a mechanistic view of the structural changes of the actophorin protein as it is unfolded, and how it is affected by the mutation in concert with the energetics reported through the PMF.
Topics: Acanthamoeba; Actins; Amoeba; Molecular Dynamics Simulation; Solvents; Protein Denaturation
PubMed: 36461639
DOI: 10.1016/j.bpj.2022.11.2941 -
RSC Advances May 2024An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of...
An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of 5-amino-7-(substituted)--(4-sulfamoylphenyl)-4,7-dihydro-[1,2,4]-triazolo[1,5-][1,3,5]triazine-2-carboxamide derivatives generated the reaction of 2-hydrazinyl--(4-sulfamoylphenyl)-2-thioxoacetamide, cyanoguanidine and a variety of aldehydes was achieved under green conditions. This experiment was conducted to evaluate the anti-inflammatory effect of the newly synthesized compounds using indomethacin as a reference medication; all compounds were tested for anti-inflammatory activity using the inhibition of albumin denaturation, RBC hemolysis technique and COX inhibition assay. The results showed that all evaluated compounds exhibited significant anti-inflammatory efficacy leading to excellently effective RBC membrane stabilization, inhibition of protein denaturation, and inhibition of COX enzymes when compared to those of indomethacin. At concentrations of 50, 100, 200, and 300 μg ml, these compounds decreased COX-1 and COX-2 activities more than indomethacin and have IC values in the range of 40.04-87.29 μg ml for COX-1 and 27.76-42.3 μg ml for COX-2 while indomethacin showed IC = 91.57 for COX-1 and 42.66 μg ml for COX-2. The anti-inflammatory findings show the need for more investigation to define the properties underlying the evaluated compounds' anti-inflammatory abilities. The enzyme cyclooxygenase-2 (COX 2) (PDB ID: 5IKT) was docked with ten synthetic substances. With docking scores () of -8.82, -7.82, and -7.76 kcal mol, 7-furan triazolo-triazine (4), 7-(2-hydroxy phenyl) triazolo-triazine (11), and 7-(4-dimethylamino phenyl) triazolo-triazine (12) had the greatest binding affinities, respectively. Therefore, these substances have COX-2 (PDB ID: 5IKT) inhibitory capabilities and hence may be investigated for COX 2 targeting development. Furthermore, both the top-ranked compounds (4 and 11) and the standard indomethacin were subjected to DFT analysis. The HOMO - LUMO energy difference (Δ) of the mentioned compounds was found to be less than that of indomethacin.
PubMed: 38832248
DOI: 10.1039/d4ra02970d -
Analytical Chemistry Oct 2023LC-MS based peptide mapping, i.e., proteolytic digestion followed by LC-MS/MS analysis, is the method of choice for protein primary structural characterization. Manual...
LC-MS based peptide mapping, i.e., proteolytic digestion followed by LC-MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC-MS peptide mapping. The method generates LC-MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC-MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC-MS analysis.
Topics: Chromatography, Liquid; Peptide Mapping; Proteolysis; Tandem Mass Spectrometry; Chromatography, High Pressure Liquid; Peptide Hydrolases
PubMed: 37816151
DOI: 10.1021/acs.analchem.3c01554 -
Molecules (Basel, Switzerland) Nov 2023MDM2 is an E3 ubiquitin ligase which is crucial for the degradation and inhibition of the key tumor-suppressor protein p53. In this work, we explored the stability and...
MDM2 is an E3 ubiquitin ligase which is crucial for the degradation and inhibition of the key tumor-suppressor protein p53. In this work, we explored the stability and the conformational features of the N-terminal region of MDM2 (N-MDM2), through which it binds to the p53 protein as well as other protein partners. The isolated domain possessed a native-like conformational stability in a narrow pH range (7.0 to 10.0), as shown by intrinsic and 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, far-UV circular dichroism (CD), and size exclusion chromatography (SEC). Guanidinium chloride (GdmCl) denaturation followed by intrinsic and ANS fluorescence, far-UV CD and SEC at physiological pH, and differential scanning calorimetry (DSC) and thermo-fluorescence experiments showed that (i) the conformational stability of isolated N-MDM2 was very low; and (ii) unfolding occurred through the presence of several intermediates. The presence of a hierarchy in the unfolding intermediates was also evidenced through DSC and by simulating the unfolding process with the help of computational techniques based on constraint network analysis (CNA). We propose that the low stability of this protein is related to its inherent flexibility and its ability to interact with several molecular partners through different routes.
Topics: Protein Folding; Tumor Suppressor Protein p53; Protein Denaturation; Protein Conformation; Circular Dichroism; Hydrogen-Ion Concentration; Spectrometry, Fluorescence; Calorimetry, Differential Scanning
PubMed: 38005300
DOI: 10.3390/molecules28227578 -
National Science Review Nov 2023A single-domain protein catenane refers to two mechanically interlocked polypeptide rings that fold synergistically into a compact and integrated structure, which is...
A single-domain protein catenane refers to two mechanically interlocked polypeptide rings that fold synergistically into a compact and integrated structure, which is extremely rare in nature. Here, we report a single-domain protein catenane of dihydrofolate reductase (-DHFR). This design was achieved by rewiring the connectivity between secondary motifs to introduce artificial entanglement and synthesis was readily accomplished through a series of programmed and streamlined post-translational processing events in cells without any additional reactions. The target molecule contained few exogenous motifs and was thoroughly characterized using a combination of ultra-performance liquid chromatography-mass spectrometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protease cleavage experiments and ion mobility spectrometry-mass spectrometry. Compared with the linear control, -DHFR retained its catalytic capability and exhibited enhanced stability against thermal or chemical denaturation due to conformational restriction. These results suggest that linear proteins may be converted into their concatenated single-domain counterparts with almost identical chemical compositions, well-preserved functions and elevated stabilities, representing an entirely new horizon in protein science.
PubMed: 38188024
DOI: 10.1093/nsr/nwad304