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Current Research in Food Science 2023As a novel protein resource, the low digestibility of protein (SPP) limits its large-scale application. From the perspective of food processing methods, different...
As a novel protein resource, the low digestibility of protein (SPP) limits its large-scale application. From the perspective of food processing methods, different heating treatments were explored to improve the structure and digestibility of SPP. In this study, SPP was heated by water bath and microwave at the same heating rate and heating temperature. Microwave accelerated protein denaturation and structure unfolded as the heating intensity increases, causing more exposed hydrophobic residues and enhancing surface hydrophobicity. The data of free sulfhydryl group, particle size, and gel electrophoresis, showed that microwave treatment promoted the formation of protein aggregates. The structural changes can potentially improve the accessibility of digestive enzymes, promote the in vitro digestibility rate, and further accelerate the production of small molecular peptides and the release of free amino acids. This study provided an innovative approach to improve the digestibility and therefore the utilization efficiency of SPP.
PubMed: 37691697
DOI: 10.1016/j.crfs.2023.100581 -
The Journal of Physical Chemistry. B Feb 2022Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the...
Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.
Topics: Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Protein Denaturation; Protein Folding; Protein Unfolding; Proteins; Thermodynamics
PubMed: 35128921
DOI: 10.1021/acs.jpcb.1c10750 -
Scientific Reports Apr 2021The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering....
The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (T). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔT = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its T. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest T (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.
Topics: Amino Acid Sequence; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Engineering; Protein Stability; Protein Structure, Secondary; Proteins
PubMed: 33824364
DOI: 10.1038/s41598-021-86952-2 -
Biochemistry Jun 2023Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into...
Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood. We examined the folding landscape of monomeric caspases from two coral species over a broad pH range of 3-10.5. On an evolutionary timescale, the two coral caspases diverged from each other approximately 300 million years ago, and they diverged from human caspases about 600 million years ago. Our results indicate that both proteins have overall high stability, ∼15 kcal mol, near the physiological pH range (pH 6-8) and unfold via two partially folded intermediates, I and I, that are in equilibrium with the native and the unfolded state. Like the dimeric caspases, the monomeric coral caspases undergo a pH-dependent conformational change resulting from the titration of an evolutionarily conserved site. Data from molecular dynamics simulations paired with limited proteolysis and MALDI-TOF mass spectrometry show that the small subunit of the monomeric caspases is unstable and unfolds prior to the large subunit. Overall, the data suggest that all caspases share a conserved folding landscape, that a conserved allosteric site can be fine-tuned for species-specific regulation, and that the subfamily of stable dimers may have evolved to stabilize the small subunit.
Topics: Humans; Protein Folding; Caspases; Protein Denaturation
PubMed: 37337671
DOI: 10.1021/acs.biochem.3c00004 -
Frontiers in Immunology 2021Infection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during...
BACKGROUND
Infection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during infection. The direct involvement of N-protein in lung injury is poorly understood.
METHODS
Recombinant N-protein was pretreated with polymyxin B, a lipopolysaccharide (LPS)-neutralizing agent. C57BL/6, C3H/HeJ (resistant to LPS), and C3H/HeN (control for C3H/HeJ) mice were exposed to N-protein intratracheal administration to examine acute lung injury. , bone marrow-derived macrophages (BMDMs) were cultured with N-protein to study phosphorylation of nuclear factor kappa B (NF-ĸB) p65, macrophage polarization, and expression of proinflammatory cytokines.
RESULTS
N-protein produced acute lung injury in C57BL/6 mice, with elevated protein permeability, total cell count, neutrophil infiltration, and proinflammatory cytokines in the bronchioalveolar lavage. N-protein also induced lung injury in both C3H/HeJ and C3H/HeN mice, indicating that the effect could not be attributed to the LPS contamination. N-protein triggered phosphorylation of NF-ĸB p65 , which was abolished by both N-protein denaturation and treatment with an antibody for N-protein, demonstrating that the effect is N-protein specific. In addition, N-protein promoted M1 macrophage polarization and the expression of proinflammatory cytokines, which was also blocked by N-protein denaturation and antibody for N-protein. Furthermore, N-protein induced NF-ĸB p65 phosphorylation in the lung, while pyrrolidine dithiocarbamate, an NF-ĸB inhibitor, alleviated the effect of N-protein on acute lung injury.
CONCLUSIONS
SARS-CoV-2 N-protein itself is toxic and induces acute lung injury in mice. Both N-protein and NF-ĸB pathway may be therapeutic targets for treating multi-organ injuries in Coronavirus disease 2019 (COVID-19).
Topics: Acute Lung Injury; Animals; COVID-19; Coronavirus Nucleocapsid Proteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; NF-kappa B; Phosphoproteins; SARS-CoV-2
PubMed: 34950152
DOI: 10.3389/fimmu.2021.791753 -
Biomolecules Feb 2021The accumulation and propagation in the brain of misfolded proteins is a pathological hallmark shared by many neurodegenerative diseases such as Alzheimer's disease (Aβ... (Review)
Review
The accumulation and propagation in the brain of misfolded proteins is a pathological hallmark shared by many neurodegenerative diseases such as Alzheimer's disease (Aβ and tau), Parkinson's disease (α-synuclein), and prion disease (prion protein). Currently, there is no epidemiological evidence to suggest that neurodegenerative disorders are infectious, apart from prion diseases. However, there is an increasing body of evidence from experimental models to suggest that other pathogenic proteins such as Aβ and tau can propagate in vivo and in vitro in a prion-like mechanism, inducing the formation of misfolded protein aggregates such as amyloid plaques and neurofibrillary tangles. Such similarities have raised concerns that misfolded proteins, other than the prion protein, could potentially transmit from person-to-person as rare events after lengthy incubation periods. Such concerns have been heightened following a number of recent reports of the possible inadvertent transmission of Aβ pathology via medical and surgical procedures. This review will provide a historical perspective on the unique transmissible nature of prion diseases, examining their impact on public health and the ongoing concerns raised by this rare group of disorders. Additionally, this review will provide an insight into current evidence supporting the potential transmissibility of other pathogenic proteins associated with more common neurodegenerative disorders and the potential implications for public health.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Creutzfeldt-Jakob Syndrome; Humans; Mice; Neurodegenerative Diseases; Phenotype; Plaque, Amyloid; Prion Diseases; Prion Proteins; Prions; Protein Denaturation; Protein Folding; alpha-Synuclein; tau Proteins
PubMed: 33540845
DOI: 10.3390/biom11020207 -
International Journal of Molecular... Apr 2023Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a...
Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures ( 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from . IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNA during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.
Topics: Prokaryotic Initiation Factor-2; Cold Temperature; Protein Biosynthesis; Thermodynamics; Hot Temperature; Sodium Chloride; Sodium Chloride, Dietary; Protein Denaturation
PubMed: 37047761
DOI: 10.3390/ijms24076787 -
ACS Nano Jul 2022The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge...
The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge of single-cell proteomics, single-molecule technologies are being developed and improved. Most approaches, to date, rely on the analysis of polypeptides, resulting from digested proteins, either in solution or immobilized on a surface. Nanopore biosensing is an emerging single-molecule technique that circumvents surface immobilization and is optimally suited for the analysis of long biopolymers, as has already been shown for DNA sequencing. However, proteins, unlike DNA molecules, are not uniformly charged and harbor complex tertiary structures. Consequently, the ability of nanopores to analyze unfolded full-length proteins has remained elusive. Here, we evaluate the use of heat denaturation and the anionic surfactant sodium dodecyl sulfate (SDS) to facilitate electrokinetic nanopore sensing of unfolded proteins. Specifically, we characterize the voltage dependence translocation dynamics of a wide molecular weight range of proteins (from 14 to 130 kDa) through sub-5 nm solid-state nanopores, using a SDS concentration below the critical micelle concentration. Our results suggest that proteins' translocation dynamics are significantly slower than expected, presumably due to the smaller nanopore diameters used in our study and the role of the electroosmotic force opposing the translocation direction. This allows us to distinguish among the proteins of different molecular weights based on their dwell time and electrical charge deficit. Given the simplicity of the protein denaturation assay and circumvention of the tailor-made necessities for sensing protein of different folded sizes, shapes, and charges, this approach can facilitate the development of a whole proteome identification technique.
Topics: Nanopores; Proteome; DNA; Electroosmosis; Nanotechnology
PubMed: 35785960
DOI: 10.1021/acsnano.2c05391 -
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 -
Bioinformation 2022The most common intracanal medication is calcium hydroxide. Its efficacy can be affected by a number of factors, including pH, serum proteins, collagen, and dentin. It's...
The most common intracanal medication is calcium hydroxide. Its efficacy can be affected by a number of factors, including pH, serum proteins, collagen, and dentin. It's also ineffective against E. faecalis and fungus, lacks an anti-inflammatory component, and has mixed reviews when it comes to pain relief. Natural alternatives to synthetic intracanal medication are being researched at the moment. We evaluated the antioxidant and anti-inflammatory activity of green synthesized silver nanoparticle based intracanal medicaments. Silver nanoparticles integrated into calcium hydroxide and graphene oxide nanoparticles were the experimental groups and Calcium hydroxide served as the control. Antioxidant activity was determined using the DPPH and Nitric oxide assays, while anti-inflammatory activity was determined using the protein denaturation and Xanthine Oxidase Inhibition assays. Both experimental groups had higher antioxidant activity than the control group based on DPPH and Nitric oxide assays. Calcium hydroxide combined with silver nanoparticles demonstrated improved anti-inflammatory efficacy in a protein denaturation and Xanthine oxidase inhibition assay. Within the constraints of an in vitro study, it can be concluded that intracanal medicaments containing silver nanoparticles can be employed efficiently during root canal preparation. In comparison to standard calcium hydroxide-based intracanal medicaments, it has effective antioxidant and anti-inflammatory effects.
PubMed: 36945225
DOI: 10.6026/97320630018450