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Medecine Sciences : M/S Feb 2017Protein molecular aging corresponds to all modifications affecting proteins during their biological life, which lead to the alteration of their structural and functional... (Review)
Review
Protein molecular aging corresponds to all modifications affecting proteins during their biological life, which lead to the alteration of their structural and functional properties. This phenomenon participates in cell and tissue aging and is therefore involved in the aging of human organism. It is also amplified in various chronic diseases such as diabetes mellitus or chronic kidney disease, where it participates in the development of long-term complications. This review aims at describing the main reactions responsible for molecular aging, their impact on protein properties and the parameters which could influence this phenomenon. A general scheme explaining its role in physiopathology is also proposed.
Topics: Animals; Cellular Senescence; Disease; Glycosylation; Humans; Oxidation-Reduction; Protein Carbonylation; Protein Denaturation; Protein Processing, Post-Translational; Proteins
PubMed: 28240209
DOI: 10.1051/medsci/20173302013 -
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 -
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 -
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 -
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 -
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 -
Poultry Science Jul 2015The objective of this study was to determine the relationship between water-holding capacity (WHC) attributes and protein denaturation in broiler breast meat. Boneless...
The objective of this study was to determine the relationship between water-holding capacity (WHC) attributes and protein denaturation in broiler breast meat. Boneless skinless breast fillets (n = 72) were collected from a commercial processing plant at 2 h postmortem and segregated into low-WHC and high-WHC groups based on muscle pH and color (L*a*b*). At 6 and 24 h postmortem, brine uptake (%), cooking loss (%), and protein solubility (sarcoplasmic and myofibrillar) were measured and protein fractions were analyzed using SDS-PAGE. Drip loss accumulation (%) was measured after storage for 2 and 7 days postmortem. High-WHC fillets exhibited lower L*-lightness values and greater pH values at 2 and 24 h postmortem than low-WHC fillets. High-WHC fillets had greater brine uptake and less cooking loss at both 6 and 24 h postmortem compared to low-WHC fillets. Aging from 6 to 24 h postmortem increased brine uptake in high-WHC fillets, but did not affect cooking loss in either low-WHC or high-WHC fillets. Drip loss accumulation was greater in low-WHC fillets at both 2 and 7 days postmortem. Myofibrillar protein solubility decreased with postmortem time but was not different between low-WHC and high-WHC fillets. Sarcoplasmic protein solubility increased with postmortem time and was greater in high-WHC fillets. SDS-PAGE analysis indicated that low-WHC fillets exhibited more glycogen phosphorylase denaturation than high-WHC fillets as evidenced by a more extensive shift of the protein from the sarcoplasmic to the myofibrillar protein fraction. Correlation analysis revealed that overall protein solubility measurements were not related to WHC attributes but that the degree of glycogen phosphorylase denaturation was significantly correlated (|r| = 0.52 to 0.80) to measures of WHC. Data indicated that WHC differences in broiler breast fillets were not due to differences in myofibrillar protein denaturation and suggested that the denaturation of sarcoplasmic proteins onto myofibrils may influence WHC in breast meat.
Topics: Animals; Chickens; Cooking; Electrophoresis, Polyacrylamide Gel; Meat; Muscle Proteins; Myofibrils; Pectoralis Muscles; Protein Denaturation; Sarcoplasmic Reticulum; Solubility; Time Factors; Water
PubMed: 26009757
DOI: 10.3382/ps/pev120 -
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