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Plant Molecular Biology Jun 2024The red and far-red light photoreceptor phytochrome B (phyB) transmits light signals following cytosol-to-nuclear translocation to regulate transcriptional networks...
The red and far-red light photoreceptor phytochrome B (phyB) transmits light signals following cytosol-to-nuclear translocation to regulate transcriptional networks therein. This necessitates changes in protein-protein interactions of phyB in the cytosol, about which little is presently known. Via introduction of a nucleus-excluding G767R mutation into the dominant, constitutively active phyB (YHB) allele, we explore the functional consequences of expressing a cytosol-localized YHB variant in transgenic Arabidopsis seedlings. We show that YHB elicits selective constitutive photomorphogenic phenotypes in dark-grown phyABCDE null mutants, wild type and other phy-deficient genotypes. These responses include light-independent apical hook opening, cotyledon unfolding, seed germination and agravitropic hypocotyl growth with minimal suppression of hypocotyl elongation. Such phenotypes correlate with reduced PIF3 levels, which implicates cytosolic targeting of PIF3 turnover or PIF3 translational inhibition by YHB. However, as expected for a cytoplasm-tethered phyB, YHB elicits reduced light-mediated signaling activity compared with similarly expressed wild-type phyB in phyABCDE mutant backgrounds. YHB also interferes with wild-type phyB light signaling, presumably by formation of cytosol-retained and/or otherwise inactivated heterodimers. Our results suggest that cytosolic interactions with PIFs play an important role in phyB signaling even under physiological conditions.
Topics: Phytochrome B; Arabidopsis; Cytosol; Arabidopsis Proteins; Signal Transduction; Basic Helix-Loop-Helix Transcription Factors; Hypocotyl; Plants, Genetically Modified; Light; Mutation; Gene Expression Regulation, Plant; Seedlings; Phenotype
PubMed: 38874897
DOI: 10.1007/s11103-024-01469-2 -
Journal of the Science of Food and... Jun 2024The effect of oleogels prepared with peanut oil and different concentrations of γ-oryzanol and β-sitosterol mixture (γ/β; 20, 40, 60, 80 and 100 g kg) on the...
BACKGROUND
The effect of oleogels prepared with peanut oil and different concentrations of γ-oryzanol and β-sitosterol mixture (γ/β; 20, 40, 60, 80 and 100 g kg) on the physicochemical and gel properties of myofibrillar protein (MP) was investigated.
RESULTS
The solubility and average particle size of MP first decreased and then increased with increasing γ/β concentration. Peanut oil or oleogels could induce the exposure of hydrophobic amino acids and the unfolding of MP, thus significantly increasing the surface hydrophobicity, sulfhydryl content and absolute value of zeta potential, which reached maximum values when the γ/β concentration was 60 g kg (P < 0.05). The addition of peanut oil decreased the gel strength and water holding capacity of MP gel. However, oleogels prepared with 60 g kg γ/β could significantly increase the hydrophobic interactions and disulfide bond content of MP gel (P < 0.05), which promoted the crosslinking and aggregation of MP, enhancing the gel properties. Peanut oil had no significant influence on the secondary structure of MP, while oleogels promoted the transition of MP conformation from α-helix to β-sheet structure. The results of light microscopy and confocal laser scanning microscopy indicated that oleogels prepared with 60 g kg γ/β filled in the pores of MP gel network to form denser and more uniform structure.
CONCLUSION
Oleogels prepared with 60 g kg γ/β could effectively improve the quality of MP gel and have promising application prospects in surimi products. © 2024 Society of Chemical Industry.
PubMed: 38872574
DOI: 10.1002/jsfa.13642 -
Food Chemistry Jun 2024This study aimed to explore how pulsed electric field (PEF) treatment affects the structural, physicochemical, and emulsification properties of porcine-derived...
Mechanisms underlying the changes in the structural, physicochemical, and emulsification properties of porcine myofibrillar proteins induced by prolonged pulsed electric field treatment.
This study aimed to explore how pulsed electric field (PEF) treatment affects the structural, physicochemical, and emulsification properties of porcine-derived myofibrillar proteins (MPs). Increasing PEF treatment induced partial polarization and protein unfolding, resulting in notable denaturation that affected both the secondary and tertiary structures. PEF treatment also improved the solubility and emulsification ability of MPs by reducing their pH and surface hydrophobicity. Confocal laser scanning microscopy confirmed the effective adsorption of MPs and PEF-treated MPs at the oil/water interface, resulting in well-fabricated Pickering emulsions. A weak particle network increased the apparent viscosity in short-term PEF-treated Pickering emulsions. Conversely, in emulsions with long-term PEF-treated MP, rheological variables decreased, and dispersion stability increased. These results endorse the potential application of PEF-treated porcine-derived MPs as efficient Pickering stabilizers, offering valuable insights into the creative use of PEF for enhancing high-quality meat products, meeting the increasing demand for clean-label choices.
PubMed: 38870818
DOI: 10.1016/j.foodchem.2024.140024 -
The Journal of Physical Chemistry. B Jun 2024Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated -type ferric heme were...
Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated -type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast -1 cytochrome to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins.
Topics: Molecular Dynamics Simulation; Lysine; Cytochromes c; Heme; Density Functional Theory; Saccharomyces cerevisiae; Ligands; Saccharomyces cerevisiae Proteins
PubMed: 38864552
DOI: 10.1021/acs.jpcb.4c00625 -
Analytical Chemistry Jun 2024Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment...
Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.
Topics: Immunoglobulin Fc Fragments; Recombinant Fusion Proteins; Mass Spectrometry; Protein Unfolding; Interleukin-10; Ion Mobility Spectrometry; Protein Stability; Humans; Immunoglobulin G
PubMed: 38853531
DOI: 10.1021/acs.analchem.4c01408 -
Biochimica Et Biophysica Acta. Proteins... Jun 2024The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we...
The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant Plumbago zeylanica, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant K ∼ 10 M)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of β-sheet (∼18%) and disordered (∼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native β-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.
PubMed: 38849109
DOI: 10.1016/j.bbapap.2024.141028 -
Advanced Healthcare Materials Jun 2024The endoplasmic reticulum (ER) plays an important role in protein synthesis and its disruption can cause protein unfolding and misfolding. Accumulation of such proteins...
The endoplasmic reticulum (ER) plays an important role in protein synthesis and its disruption can cause protein unfolding and misfolding. Accumulation of such proteins leads to ER stress, which ultimately promotes many diseases. Routine screening of ER activity in immune cells can flag serious conditions at early stages, but the current clinically used bio-probes have limitations. Herein, an ER-specific fluorophore based on a biocompatible benzothiadiazole-imine cage (BTD-cage) with excellent photophysical properties is developed. The cage outperforms commercially available ER stains in long-term live cell imaging with no fading or photobleaching over time. The cage is responsive to different levels of ER stress where its fluorescence increases accordingly. Incorporating the bio-probe into an immune disorder model, a 6-, 21-, and 48-fold increase in intensity is shown in THP-1, Raw 246.7, and Jurkat cells, respectively (within 15 min). These results strongly support that this system can be used for rapid visual and selective detection of ER stress. It is envisaged that tailoring molecular interactions and molecular recognition using supramolecular improved fluorophores can expand the library of biological probes for enhanced selectivity and targetability toward cellular organelles.
PubMed: 38848965
DOI: 10.1002/adhm.202401117 -
Chemical Science Jun 2024Protein aggregation is a key process in the development of many neurodegenerative disorders, including dementias such as Alzheimer's disease. Significant progress has...
Protein aggregation is a key process in the development of many neurodegenerative disorders, including dementias such as Alzheimer's disease. Significant progress has been made in understanding the molecular mechanisms of aggregate formation in pure buffer systems, much of which was enabled by the development of integrated rate laws that allowed for mechanistic analysis of aggregation kinetics. However, in order to translate these findings into disease-relevant conclusions and to make predictions about the effect of potential alterations to the aggregation reactions by the addition of putative inhibitors, the current models need to be extended to account for the altered situation encountered in living systems. In particular, , the total protein concentrations typically do not remain constant and aggregation-prone monomers are constantly being produced but also degraded by cells. Here, we build a theoretical model that explicitly takes into account monomer production, derive integrated rate laws and discuss the resulting scaling laws and limiting behaviours. We demonstrate that our models are suited for the aggregation-prone Huntington's disease-associated peptide HttQ45 utilizing a system for continuous monomer production and the aggregation of the tumour suppressor protein P53. The aggregation-prone HttQ45 monomer was produced through enzymatic cleavage of a larger construct in which a fused protein domain served as an internal inhibitor. For P53, only the unfolded monomers form aggregates, making the unfolding a rate-limiting step which constitutes a source of aggregation-prone monomers. The new model opens up possibilities for a quantitative description of aggregation in living systems, allowing for example the modelling of inhibitors of aggregation in a dynamic environment of continuous protein synthesis.
PubMed: 38846392
DOI: 10.1039/d4sc00088a -
International Journal of Biological... Jun 2024Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of...
Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of allosteric and template-mediated bridging mechanisms have been proposed to explain the differences in rate acceleration and specificity. The difficulty in working with heterogeneous heparin species has rendered a crystallographic interpretation of the differences in antithrombin activation between mimetics and natural heparin inaccessible. In this study, we examine the allosteric changes in antithrombin caused by binding fondaparinux, enoxaparin and depolymerized natural heparins using millisecond hydrogen deuterium exchange mass spectrometry (TRESI-HDX MS) and relate these conformational changes to complex stability in the gas phase using collision induced unfolding (CIU). This exploration reveals that in addition to the dynamic changes caused by fondaparinux, long chain heparins reduce structural flexibility proximal to Arg393, the cleavable residue in the reactive centre loop of the protein. These local changes in protein dynamics are associated with an increase in overall complex stability that increases with heparin chain length. Ultimately, these results shed light on the molecular mechanisms underlying differences in activity and specificity between heparin mimetics and natural heparins.
PubMed: 38838881
DOI: 10.1016/j.ijbiomac.2024.132868 -
Journal of Colloid and Interface Science May 2024Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded...
HYPOTHESIS
Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded state. However, further studies are required to refine our understanding of the underlying mechanism of the refolding process. While interactions between nonionic surfactants and tightly folded proteins are not anticipated, we hypothesized that intermediate stages of surfactant-induced unfolding could define new interaction mechanisms by which nonionic surfactants can further alter protein conformation.
EXPERIMENTS
In this work, the behavior of three model proteins (human growth hormone, bovine serum albumin, and β-lactoglobulin) was investigated in the presence of the anionic surfactant sodium dodecylsulfate, the nonionic surfactant β-dodecylmaltoside, and mixtures of both surfactants. The transitions occurring to the proteins were determined using intrinsic fluorescence spectroscopy and far-UV circular dichroism. Based on these results, we developed a detailed interaction model for human growth hormone. Using nuclear magnetic resonance and contrast-variation small-angle neutron scattering, we studied the amino acid environment and the conformational state of the protein.
FINDINGS
The results demonstrate the key role of surfactant cooperation in defining the conformational state of the proteins, which can shift away or toward the folded state depending on the nonionic-to-ionic surfactant ratio. Dodecylmaltoside, initially a non-interacting surfactant, can unexpectedly associate with sodium dodecylsulfate-unfolded proteins to further impact their conformation at low nonionic-to-ionic surfactant ratio. When this ratio increases, the protein begins to retrieve the folded state. However, the native conformation cannot be fully recovered due to remnant surfactant molecules still adsorbed to the protein. This study demonstrates that the conformational landscape of the protein depends on a delicate interplay between the surfactants, ultimately controlled by the ratio between them, resulting in unpredictable changes in the protein conformation.
PubMed: 38838632
DOI: 10.1016/j.jcis.2024.05.157