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Cold Spring Harbor Perspectives in... Jan 2020The proteasome, the most complex protease known, degrades proteins that have been conjugated to ubiquitin. It faces the unique challenge of acting enzymatically on... (Review)
Review
The proteasome, the most complex protease known, degrades proteins that have been conjugated to ubiquitin. It faces the unique challenge of acting enzymatically on hundreds and perhaps thousands of structurally diverse substrates, mechanically unfolding them from their native state and translocating them vectorially from one specialized compartment of the enzyme to another. Moreover, substrates are modified by ubiquitin in myriad configurations of chains. The many unusual design features of the proteasome may have evolved in part to endow this enzyme with a robust ability to process substrates regardless of their identity. The proteasome plays a major role in preserving protein homeostasis in the cell, which requires adaptation to a wide variety of stress conditions. Modulation of proteasome function is achieved through a large network of proteins that interact with it dynamically, modify it enzymatically, or fine-tune its levels. The resulting adaptability of the proteasome, which is unique among proteases, enables cells to control the output of the ubiquitin-proteasome pathway on a global scale.
Topics: Adenosine Triphosphate; Animals; Caenorhabditis elegans; Cryoelectron Microscopy; Cytoplasm; DNA-Binding Proteins; Gene Expression Regulation; Homeostasis; Humans; Models, Molecular; Molecular Conformation; Nuclear Respiratory Factor 1; Proteasome Endopeptidase Complex; Protein Denaturation; Protein Engineering; Protein Folding; Protein Processing, Post-Translational; Protein Transport; Saccharomyces cerevisiae Proteins; Transcription Factors; Ubiquitin; Ubiquitin Thiolesterase
PubMed: 30833452
DOI: 10.1101/cshperspect.a033985 -
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 -
Semi-industrial production of a minimally processed infant formula powder using membrane filtration.Journal of Dairy Science May 2021Infant formula (IF) is submitted to several heat treatments during production, which can lead to denaturation or aggregation of proteins and promote Maillard reaction....
Infant formula (IF) is submitted to several heat treatments during production, which can lead to denaturation or aggregation of proteins and promote Maillard reaction. The objective of this study was to investigate innovative minimal processing routes for the production of first-age IF powder, thus ensuring microbial safety with minimal level of protein denaturation. Three nutritionally complete IF powders were produced at a semi-industrial scale based on ingredients obtained by fresh bovine milk microfiltration (0.8 and 0.1-µm pore size membranes). Low-temperature vacuum evaporation (50°C) and spray-drying (inlet and outlet temperatures of 160 and 70°C, respectively) were conducted to produce the T- formula with no additional heat treatment. The T+ formula was produced with a moderate heat treatment (75°C for 2 min) applied before spray-drying, whereas the T+++ formula received successive heat treatments (72°C for 30 s on the milk; 90°C for 2-3 s before evaporation; 85°C for 2 min before spray-drying), thus mimicking commercial powdered IF. Protein denaturation and Maillard reaction products were followed throughout the production steps and the physicochemical properties of the powders were characterized. The 3 IF powders presented satisfactory physical properties in terms of a, free fat content, glass transition temperature, and solubility index, as well as satisfactory bacteriological quality with a total flora <10 cfu/g and an absence of pathogens when a high level of bacteriological quality of the ingredients was ensured. Protein denaturation occurred mostly during the heat treatments of T+ and T+++ and was limited during the spray-drying process. The IF powder produced without heat treatment (T-) presented a protein denaturation extent (6 ± 4%) significantly lower than that in T+++ (58 ± 0%), but not significantly different from that in T+ (10 ± 4%). Although T- tended to contain less Maillard reaction products than T+ and T+++, the Maillard reaction products did not significantly discriminate the infant formulas in the frame of this work. The present study demonstrated the feasibility of producing at a semi-industrial scale an infant formula being bacteriologically safe and containing a high content of native proteins. Application of a moderate heat treatment before spray-drying could further guarantee the microbiological quality of the IF powders while maintaining a low protein denaturation extent. This study opens up new avenues for the production of minimally processed IF powders.
Topics: Animals; Cattle; Desiccation; Infant Formula; Powders; Solubility; Temperature
PubMed: 33685709
DOI: 10.3168/jds.2020-19529 -
Polymers Nov 2021Polyacrylamide gel electrophoresis is widely used for studying proteins and protein-containing objects. However, it is employed most frequently as a qualitative method...
Polyacrylamide gel electrophoresis is widely used for studying proteins and protein-containing objects. However, it is employed most frequently as a qualitative method rather than a quantitative one. This paper shows the feasibility of routine digital image acquisition and mathematical processing of electropherograms for protein quantification when using vertical gel electrophoresis and Chrom & Spec software. Both the well-studied model protein molecules (bovine serum albumin) and more complex real-world protein-based products (casein-containing isolate for sports nutrition), which were subjected to mechanical activation in a planetary ball mill to obtain samples characterized by different protein denaturation degrees, were used as study objects. Protein quantification in the mechanically activated samples was carried out. The degree of destruction of individual protein was shown to be higher compared to that of the protein-containing mixture after mechanical treatment for an identical amount of time. The methodological approach used in this study can serve as guidance for other researchers who would like to use electrophoresis for protein quantification both in individual form and in protein mixtures. The findings prove that photographic imaging of gels followed by mathematical data processing can be applied for analyzing the electrophoretic data as an affordable, convenient and quick tool.
PubMed: 34833270
DOI: 10.3390/polym13223971 -
Proceedings of the National Academy of... Aug 2021The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift...
The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τ The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Models, Molecular; Protein Denaturation; Protein Folding; Protein Unfolding; Solvents; Thermodynamics; src Homology Domains
PubMed: 34404723
DOI: 10.1073/pnas.2112021118 -
The Journal of Biological Chemistry Mar 2024The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when... (Review)
Review
The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.
Topics: Bacterial Proteins; Copper; Homeostasis; Oxidation-Reduction; Oxidoreductases; Salmonella; Sulfhydryl Compounds; Carrier Proteins
PubMed: 38309504
DOI: 10.1016/j.jbc.2024.105710 -
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 -
Current Research in Food Science 2023Food applications involving plant proteins require modification of their functionality to mimic the unique properties of animal proteins. Enzymatic hydrolysis is...
Food applications involving plant proteins require modification of their functionality to mimic the unique properties of animal proteins. Enzymatic hydrolysis is commonly used to alter the functionality of plant proteins, particularly to improve their solubility near the isoelectric point. Current methodological approaches mostly indicate improved solubility upon hydrolysis. However, published methods include the removal of insoluble material before analysis, and calculations are based on only the solubilized material as a percentage of the filtered protein. This approach artificially increases solubility estimation and gives an incorrect assessment of the efficacy of hydrolysis. By using the total amount of protein, this study aims to determine the effect of two microbial proteases, Flavourzyme and Alcalase, on the solubility and structural and thermal properties of soy and chickpea proteins. Protein isolates were first extracted from soy and chickpea flour and hydrolyzed from 0 to 3 h. Then, their degree of hydrolysis and solubility at a range of pHs were determined using the o-phthaldialdehyde (OPA) and Lowry methods, respectively. Proteins' electrophoretic mobility, protein-protein interactions, thermal properties, and protein secondary structures were also determined. Solubility decreased over time though the solubility of the hydrolysate improved near the isoelectric point. Soy Flavourzyme hydrolysates remained the most soluble and chickpea Flavourzyme hydrolysates showed the least solubility. Thermal data suggested that Alcalase reduced the protein denaturation temperature, leading to a loss of solubility upon thermal enzyme inactivation. The loss of solubility of hydrolysates was strongly associated with hydrogen bonding, which may result from the formation of polar peptide termini. These results challenge commonly accepted beliefs that hydrolysis inevitably improves solubility of plant proteins. Instead, it is shown that hydrolysis causes structural changes that result in aggregation, thus potentially limiting the application of enzymatic hydrolysis without the addition of further processing methods.
PubMed: 37065430
DOI: 10.1016/j.crfs.2023.100487 -
Molecules (Basel, Switzerland) Apr 2022In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and...
In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and integrity influence drug efficacy, and ultimately patient safety. Existing analytical methodologies solely rely on relative changes in optical properties such as fluorescence or scattering upon thermal or chemical perturbation. Here, we present an absolute analytical method for assessing protein stability, structure, and unfolding utilizing Taylor dispersion analysis (TDA) and LED-UV fluorescence detection. The developed TDA method measures the change in size (hydrodynamic radius) and intrinsic fluorescence of a protein during in-line denaturation with guanidinium hydrochloride (GuHCl). The conformational stability of the therapeutic antibody adalimumab and human serum albumin were characterized as a function of pH. The simple workflow and low sample consumption (40 ng protein per data point) of the methodology make it ideal for assessing protein characteristics related to stability in early drug development or when having a scarce amount of sample available.
Topics: Guanidine; Humans; Hydrodynamics; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Serum Albumin, Human
PubMed: 35458703
DOI: 10.3390/molecules27082506 -
Foods (Basel, Switzerland) Apr 2023Plants have been recognized as renewable and sustainable sources of proteins. However, plant protein extraction is challenged by the plant's recalcitrant cell wall. The...
Plants have been recognized as renewable and sustainable sources of proteins. However, plant protein extraction is challenged by the plant's recalcitrant cell wall. The conventional extraction methods make use of non-reusable strong alkali chemicals in protein-denaturing extraction conditions. In this study, soy protein was extracted using NHOH, a weak, recoverable, and reusable alkali. The extraction conditions were optimized using response surface methodology (RSM). A central composite design (CCD) with four independent variables: temperature (25, 40, 55, 70, and 85 °C); NHOH concentration (0.5, 1, and 1.5%); extraction time (6, 12, 18, and 24 h) and solvent ratio (1:5, 1:10, 1:15 and 1:20 /) were used to study the response variables (protein yield and amine concentration). Amine concentration indicates the extent of protein hydrolysis. The RSM model equation for the independent and response variables was computed and used to create the contour plots. A predicted yield of 64.89% protein and 0.19 mM amine revealed a multiple R-squared value of 0.83 and 0.78, respectively. The optimum conditions to obtain the maximum protein yield (65.66%) with the least amine concentration (0.14 Mm) were obtained with 0.5% NHOH concentration, 12 h extraction time, and a 1:10 (/) solvent ratio at 52.5 °C. The findings suggest that NHOH is suitable to extract soybean protein with little or no impact on protein denaturation.
PubMed: 37048336
DOI: 10.3390/foods12071515