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International Journal of Molecular... Jun 2021Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms...
Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180-250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.
Topics: Circular Dichroism; Free Radicals; Heating; Protein Denaturation; Proteins; Reactive Oxygen Species; Spectrum Analysis; Ultraviolet Rays; Water
PubMed: 34204483
DOI: 10.3390/ijms22126512 -
MAbs 2021Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of... (Comparative Study)
Comparative Study
Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of protein-based drugs can endanger patient safety due, for example, to increased immunogenicity. Aggregation can also inactivate protein drugs and prevent target engagement, and thus regulatory requirements are strict regarding drug stability monitoring during manufacturing and storage. Many of the current technologies for aggregation monitoring are time- and material-consuming and require specific instruments and expertise. These types of assays are not only expensive, but also unsuitable for larger sample panels. Here we report a label-free time-resolved luminescence-based method using an external Eu-conjugated probe for the simple and fast detection of protein stability and aggregation. We focused on monitoring the properties of IgG, which is a common format for biological drugs. The Protein-Probe assay enables IgG aggregation detection with a simple single-well mix-and-measure assay performed at room temperature. Further information can be obtained in a thermal ramping, where IgG thermal stability is monitored. We showed that with the Protein-Probe, trastuzumab aggregation was detected already after 18 hours of storage at 60°C, 4 to 8 days earlier compared to SYPRO Orange- and UV250-based assays, respectively. The ultra-high sensitivity of less than 0.1% IgG aggregates enables the Protein-Probe to reduce assay time and material consumption compared to existing techniques.
Topics: Antineoplastic Agents, Immunological; Drug Compounding; Europium; High-Throughput Screening Assays; Hot Temperature; Immunoglobulin G; Luminescent Agents; Luminescent Measurements; Organometallic Compounds; Protein Aggregates; Protein Binding; Protein Denaturation; Protein Stability; Time Factors; Trastuzumab
PubMed: 34455913
DOI: 10.1080/19420862.2021.1955810 -
Journal of Dairy Science Sep 2022Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We...
Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).
Topics: Animals; Gels; Hot Temperature; Hydrogen-Ion Concentration; Lactoglobulins; Milk Proteins; Osmolar Concentration; Protein Denaturation; Salts; Sodium Chloride; Temperature; Whey Proteins
PubMed: 35879172
DOI: 10.3168/jds.2021-21738 -
Role of freezing-induced myofibrillar protein denaturation in the generation of thaw loss: A review.Meat Science Aug 2022Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from... (Review)
Review
Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from ice crystallization and the freezing-induced denaturation of myofibrillar proteins, the latter of which has, however, not received much research focus. This review discusses the relationship between myofibrillar protein denaturation and water-holding capacity of meat in freezing-thawing with the aim to improve the understanding the relative importance of protein denaturation in the formation of thaw loss. The contribution of decreased pH and high ionic strength in the unfrozen water in freezing is emphasized and we hypothesize that these two factors are causing protein denaturation and conformational changes within muscle fibers, and consequently loss of water-holding capacity. Slow freezing produces more thaw loss than fast freezing, and this is discussed here in relation to the impacts on myofibrillar protein denaturation induced by the freezing rate.
Topics: Freezing; Meat; Protein Denaturation; Proteins; Water
PubMed: 35533633
DOI: 10.1016/j.meatsci.2022.108841 -
Molecules (Basel, Switzerland) Oct 2022The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be... (Review)
Review
The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.
Topics: Protein Denaturation; Protein Conformation; Deuterium Oxide; Aryldialkylphosphatase; Electrophoresis, Polyacrylamide Gel; Cholinesterases; Biological Products; Thermodynamics; Protein Folding
PubMed: 36296453
DOI: 10.3390/molecules27206861 -
Journal of Molecular Biology Oct 2023The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In...
The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In particular, understanding the thermodynamics and kinetics of the folding process is important for uncovering the mechanisms behind human disorders caused by protein misfolding. To address this issue, it is essential to collect and curate experimental kinetic and thermodynamic data on protein folding. K-Pro is a new database designed for collecting and storing experimental kinetic data on monomeric proteins, with a two-state folding mechanism. With 1,529 records from 62 proteins corresponding to 65 structures, K-Pro contains various kinetic parameters such as the logarithm of the folding and unfolding rates, Tanford's β and the ϕ values. When available, the database also includes thermodynamic parameters associated with the kinetic data. K-Pro features a user-friendly interface that allows browsing and downloading kinetic data of interest. The graphical interface provides a visual representation of the protein and mutants, and it is cross-linked to key databases such as PDB, UniProt, and PubMed. K-Pro is open and freely accessible through https://folding.biofold.org/k-pro and supports the latest versions of popular browsers.
Topics: Humans; Databases, Protein; Kinetics; Protein Denaturation; Protein Folding; Proteins; Thermodynamics; Mutant Proteins
PubMed: 37625584
DOI: 10.1016/j.jmb.2023.168245 -
Drug Discovery Today Feb 2016Biologics exist in equilibrium between native, partially denatured, and denatured conformational states. The population of any of these states is dictated by their Gibbs... (Review)
Review
Biologics exist in equilibrium between native, partially denatured, and denatured conformational states. The population of any of these states is dictated by their Gibbs energy and can be altered by changes in physical and solution conditions. Some conformations have a tendency to self-associate and aggregate, an undesirable phenomenon in protein therapeutics. Conformational equilibrium and self-association are linked thermodynamic functions. Given that any associative reaction is concentration dependent, conformational stability studies performed at different protein concentrations can provide early clues to future aggregation problems. This analysis can be applied to the selection of protein variants or the identification of better formulation solutions. In this review, we discuss three different aggregation situations and their manifestation in the observed conformational equilibrium of a protein.
Topics: Biological Products; Protein Conformation; Protein Denaturation; Proteins; Thermodynamics
PubMed: 26608889
DOI: 10.1016/j.drudis.2015.11.007 -
Biomolecules Feb 2020As a tribute to Professor Oleg B. Ptitsyn, we organized an interview with Professor Akiyoshi Wada held in Tokyo in the middle of September 2019. Both Professor A. Wada...
As a tribute to Professor Oleg B. Ptitsyn, we organized an interview with Professor Akiyoshi Wada held in Tokyo in the middle of September 2019. Both Professor A. Wada and the late Professor O. B. Ptitsyn greatly contributed to the field of protein biophysics, and they played leading roles in establishing the concept of the "Molten Globule state" 35-40 years ago. This editorial is intended to recount, as accurately as possible, some episodes during the early days of protein research that led to the discovery of this state, and how this concept was coined the "Molten Globule state" and came to be widely accepted by biophysicists, biochemists, and molecular biologists.
Topics: Amino Acid Sequence; Biophysical Phenomena; Circular Dichroism; History, 20th Century; Models, Molecular; Protein Biosynthesis; Protein Conformation; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 32050721
DOI: 10.3390/biom10020269 -
Chemical Reviews Jul 2016Water is an essential participant in the stability, structure, dynamics, and function of proteins and other biomolecules. Thermodynamically, changes in the aqueous... (Review)
Review
Water is an essential participant in the stability, structure, dynamics, and function of proteins and other biomolecules. Thermodynamically, changes in the aqueous environment affect the stability of biomolecules. Structurally, water participates chemically in the catalytic function of proteins and nucleic acids and physically in the collapse of the protein chain during folding through hydrophobic collapse and mediates binding through the hydrogen bond in complex formation. Water is a partner that slaves the dynamics of proteins, and water interaction with proteins affect their dynamics. Here we provide a review of the experimental and computational advances over the past decade in understanding the role of water in the dynamics, structure, and function of proteins. We focus on the combination of X-ray and neutron crystallography, NMR, terahertz spectroscopy, mass spectroscopy, thermodynamics, and computer simulations to reveal how water assist proteins in their function. The recent advances in computer simulations and the enhanced sensitivity of experimental tools promise major advances in the understanding of protein dynamics, and water surely will be a protagonist.
Topics: Hydrogen Bonding; Hydrostatic Pressure; Ion Channels; Molecular Structure; Muramidase; Phase Transition; Protein Denaturation; Proteins; Temperature; Terahertz Spectroscopy; Water
PubMed: 27186992
DOI: 10.1021/acs.chemrev.5b00664 -
Analytical Chemistry Mar 2020In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules,...
In modern biochemistry, protein stability and ligand interactions are of high interest. These properties are often studied with methods requiring labeled biomolecules, as the existing methods utilizing luminescent external probes suffer from low sensitivity. Currently available label-free technologies, e.g., thermal shift assays, circular dichroism, and differential scanning calorimetry, enable studies on protein unfolding and protein-ligand interactions (PLI). Unfortunately, the required micromolar protein concentration increases the costs and predisposes these methods for spontaneous protein aggregation. Here, we report a time-resolved luminescence method for protein unfolding and PLI detection with nanomolar sensitivity. The Protein-Probe method is based on highly luminescent europium chelate-conjugated probe, which is the key component in sensing the hydrophobic regions exposed to solution after protein unfolding. With the same Eu-probe, we also demonstrate ligand-interaction induced thermal stabilization with model proteins. The developed Protein-Probe method provides a sensitive approach overcoming the problems of the current label-free methodologies.
Topics: Ligands; Models, Molecular; Protein Binding; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Proteins; Temperature; Transition Temperature
PubMed: 32013400
DOI: 10.1021/acs.analchem.9b05712