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Angewandte Chemie (International Ed. in... Aug 2017A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force,...
A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week-long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.
Topics: Antioxidants; Mechanical Phenomena; Protein Denaturation; Protein Folding; Proteins
PubMed: 28470663
DOI: 10.1002/anie.201703630 -
International Journal of Molecular... Jun 2021Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and...
Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.
Topics: Enzyme Assays; Peptide Hydrolases; Protein Denaturation; Proteins; Substrate Specificity; Temperature
PubMed: 34198602
DOI: 10.3390/ijms22126362 -
Biophysical Journal Nov 2018A growing number of proteins have been identified as knotted in their native structures, with such entangled topological features being expected to play stabilizing...
A growing number of proteins have been identified as knotted in their native structures, with such entangled topological features being expected to play stabilizing roles maintaining both the global fold and the nature of proteins. However, the molecular mechanism underlying the stabilizing effect is ambiguous. Here, we combine unbiased and mechanical atomistic molecular dynamics simulations to investigate how a protein is stabilized by an inherent knot by directly comparing chemical, thermal, and mechanical denaturing properties of two proteins having the same sequence and secondary structures but differing in the presence or absence of an inherent knot. One protein is YbeA from Escherichia coli, containing a deep trefoil knot within the sequence, and the other is the modified protein with the knot of YbeA being removed. Under certain chemical denaturing conditions, the unknotted protein fully unfolds whereas the knotted protein does not, suggesting a higher intrinsic stability for the protein having a knot. Both proteins unfold under enhanced thermal fluctuations but at different rates and with distinct pathways. Opening the hydrophobic core via separation between two α-helices is identified as a crucial step initiating the protein unfolding, which, however, is restrained for the knotted protein by topological and geometrical frustrations. Energy barriers for denaturing the protein are reduced by removing the knot, as evidenced by mechanical unfolding simulations. Finally, yet importantly, no obvious change in size or location of the knot was observed during denaturing processes, indicating that YbeA may remain knotted for a relatively long time during and after denaturation.
Topics: Biomechanical Phenomena; Escherichia coli Proteins; Methyltransferases; Molecular Dynamics Simulation; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Temperature
PubMed: 30314655
DOI: 10.1016/j.bpj.2018.09.015 -
International Journal of Molecular... Mar 2021The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon...
The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.
Topics: Algorithms; Bacillus subtilis; Enzyme Stability; Histidine Kinase; Hydrogen-Ion Concentration; Models, Chemical; Models, Molecular; Phosphorylation; Protein Conformation; Protein Denaturation; Protein Multimerization; Spectrum Analysis; Structure-Activity Relationship; Temperature
PubMed: 33810099
DOI: 10.3390/ijms22063231 -
Molecules (Basel, Switzerland) May 2023In this article, we present the first detailed analysis of the hydro-distilled essential oil (HDEO) of the inflorescence heads of Boiss. (Asteraceae) from the flora of...
In this article, we present the first detailed analysis of the hydro-distilled essential oil (HDEO) of the inflorescence heads of Boiss. (Asteraceae) from the flora of Jordan, offering observations at different growth (pre-flowering, full-flowering and post-flowering) stages. Additionally, we investigated the methanolic extract obtained from the aerial parts of the plant material at the full flowering stage in order to determine its inhibitory activity in terms of COX and protein denaturation and evaluate its antimicrobial effects against (Gram-positive) and (Gram-negative) bacteria. Performing GC/MS analysis of HDEO, obtained from the fresh inflorescence heads at the different growth stages, resulted in the identification of 192 constituents. The main class of compounds detected in these three stages comprised aliphatic hydrocarbons and their derivatives, which amounted to 50.04% (pre-flower), 40.28% (full-flower) and 41.34% (post-flower) of the total composition. The oils also contained appreciable amounts of oxygenated terpenoids, primarily sesquiterpenoids and diterpenoids. The pre-flowering stage was dominated by (2E)-hexenal (8.03%) in addition to the oxygenated diterpene (6,10)-pseudo phytol (7.54%). The full-flowering stage primarily contained (6,10)-pseudo phytol (7.84%), β-bisabolene (7.53%, SH) and the diterpene hydrocarbon dolabradiene (5.50%). The major constituents detected in the HDEO obtained at the post-flowering stage included the oxygenated sesquiterpenoid intermedeol (5.53%), the sesquiterpene hydrocarbon ()-caryophyllene (5.01%) and (6,10)-pseudo phytol (4.47%). The methanolic extract obtained from air-dried aerial parts of displayed more COX-2 inhibition than COX-1 inhibition at a concentration level of 200 µg/mL. The extract exhibited a capacity to inhibit protein denaturation that was comparable with respect to the activity of diclofenac sodium and displayed moderate levels of antimicrobial activity against both bacterial species. The current results demonstrate the need to perform further detailed phytochemical investigations to isolate and characterize active constituents.
Topics: Oils, Volatile; Tenrecidae; Jordan; Protein Denaturation; Escherichia coli; Staphylococcus aureus; Diterpenes; Sesquiterpenes; Gram-Negative Bacteria; Phytol; Anti-Infective Agents; Plant Extracts; Microbial Sensitivity Tests
PubMed: 37241978
DOI: 10.3390/molecules28104238 -
Chembiochem : a European Journal of... Mar 2019In all intracellular processes, protein structure and dynamics are subject to the influence of macromolecular crowding (MC). Here, the impact of MC agents of different...
In all intracellular processes, protein structure and dynamics are subject to the influence of macromolecular crowding (MC). Here, the impact of MC agents of different types and sizes on the model protein Bacillus subtilis Cold shock protein B (BsCspB) during both thermal and chemical denaturation have been comprehensively investigated. We consistently reveal a distinct stabilization of BsCspB in a manner dependent on the MC concentration but not on viscosity, polarity, or size of the MC agent used. This general stabilization has been decoded by use of NMR spectroscopy, through monitoring of chemical shift (CS) perturbations and the intramolecular hydrogen-bonding networks, as well as local protection of amide protons against exchange with solvent protons. Whereas CSs and hydrogen-bonding networks are not systematically affected in the presence of MC, we detected a pronounced reduction in exchange in loop regions of BsCspB. We conclude that this reduced accessibility of solvent protons is a key parameter for the increases in protein stability seen under MC.
Topics: Bacillus subtilis; Bacterial Proteins; Nuclear Magnetic Resonance, Biomolecular; Protein Denaturation; Protein Stability; Protons; Temperature; Thermodynamics; Water
PubMed: 30508270
DOI: 10.1002/cbic.201800679 -
Journal of Dairy Science Jun 2021Milk protein concentrates (MPC) are typically dried high-protein powders with functional and nutritional properties that can be tailored through modification of...
Milk protein concentrates (MPC) are typically dried high-protein powders with functional and nutritional properties that can be tailored through modification of processing conditions, including temperature, pH, filtration, and drying. However, the effects of processing conditions on the structure-function properties of liquid MPC (fluid ultrafiltered milk), specifically, are understudied. In this report, the pH of liquid MPC [13% protein (70% protein DM basis), pH 6.7] was adjusted to 6.5 or 6.9, and samples at pH 6.5, 6.7, and 6.9 were subjected to heat treatment at either 85°C for 5 min or 125°C for 15 s. Sodium dodecyl sulfate PAGE was used to determine the distribution of caseins and denatured whey proteins in the soluble and micellar phases, and HPLC was used to quantify native whey proteins as a measure of denaturation, based on the processing conditions. Both heat treatments resulted in substantial whey protein denaturation at each pH, with β-lactoglobulin denatured more extensively than α-lactalbumin. Changes in liquid MPC physicochemical properties were monitored at d 1, 5, and 8 during storage at 4°C. Viscosity increased after heat treatment and also over time, regardless of pH and heating conditions, suggesting the role of whey protein denaturation and aggregation, and their interactions with casein micelles. The MPC samples processed at pH 6.9 had a significantly higher viscosity than those heated at pH 6.5 or 6.7, for both temperature and time conditions; and samples processed at 85°C for 5 min had higher viscosity than those heated at 125°C for 15 s. Particle size analysis indicated the presence of larger particles after 5 and 8 d of MPC storage after heating at pH 6.9. Acid-induced gelation of the liquid MPC led to significantly higher gel firmness after processing at 85°C for 5 min, compared with 125°C for 15 s. Also, gels made from MPC adjusted to pH 6.5 had higher storage moduli, with both time and temperature combinations, demonstrating the role of pH-dependent association of denatured whey proteins with casein micelles in gel network formation. These findings enable a better understanding of the processing factors contributing to structural and functional properties of liquid MPC and can be helpful in tailoring milk protein ingredient functionality for a variety of food products.
Topics: Animals; Caseins; Hot Temperature; Hydrogen-Ion Concentration; Micelles; Milk Proteins; Protein Denaturation; Whey Proteins
PubMed: 33773779
DOI: 10.3168/jds.2020-19355 -
Journal of Pharmaceutical Sciences Dec 2021The purpose of this investigation was to highlight the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic...
The purpose of this investigation was to highlight the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic antibodies. In this case study, we compared results from isothermal chemical denaturation (ICD) and NMR with standard methods to relate conformational states of a model monoclonal antibody (mAb1) with protein-protein interactions (PPI) that lead to self - association in concentrated solutions. The increase in aggregation rate and relative viscosity for mAb1 was found to be both concentration and pH dependent. The free energy of unfolding (∆G⁰) from ICD and thermal analysis in dilute solutions indicated that although the native state predominated between pH 4 - pH 7, it was disrupted at the CH and unfolded noncooperatively under acidic conditions. One-dimensional (1D) H NMR and two-dimensional (2D) C-H NMR performed, in concentrated solutions, confirmed that PPI between pH 4-7 occurred while mAb1 was in the native state. NMR corroborated that mAb1 maintained a dominant native state at formulation-relevant conditions at the tested pH range, had increased global molecular tumbling dynamics at lower pH and confirmed increased PPI at higher pH conditions. This report aligns and compares typical characterization of an IgG1 with assessment of structure by NMR and provided a more precise assessment and deeper insight into the conformation of an IgG1 in concentrated solutions.
Topics: Antibodies, Monoclonal; Hydrogen-Ion Concentration; Immunoglobulin G; Magnetic Resonance Spectroscopy; Protein Conformation; Protein Denaturation; Viscosity
PubMed: 34506864
DOI: 10.1016/j.xphs.2021.09.005 -
Proceedings of the National Academy of... Mar 2022SignificanceUnderstanding the molecular forces driving the unfolded polypeptide chain to self-assemble into a functional native structure remains an open question....
SignificanceUnderstanding the molecular forces driving the unfolded polypeptide chain to self-assemble into a functional native structure remains an open question. However, identifying the states visited during protein folding (e.g., the transition state between the unfolded and native states) is tricky due to their transient nature. Here, we introduce calorimetric force spectroscopy in a temperature jump optical trap to determine the enthalpy, entropy, and heat capacity of the transition state of protein barnase. We find that the transition state has the properties of a dry molten globule, that is, high free energy and low configurational entropy, being structurally similar to the native state. This experimental single-molecule study characterizes the thermodynamic properties of the transition state in funneled energy landscapes.
Topics: Bacterial Proteins; Calorimetry; Optical Tweezers; Protein Conformation; Protein Denaturation; Protein Folding; Ribonucleases; Single Molecule Imaging; Thermodynamics
PubMed: 35271392
DOI: 10.1073/pnas.2112382119 -
Molecules (Basel, Switzerland) Jul 2022Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of... (Review)
Review
Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn's hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.
Topics: Circular Dichroism; Protein Conformation; Protein Denaturation; Protein Folding; Proteins
PubMed: 35889244
DOI: 10.3390/molecules27144361