-
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 -
The Journal of Physical Chemistry. B Mar 2021One-third of the reported cases of light chain amyloidosis are related to the germ line λ6 family; remarkably, healthy individuals express this type of protein in just...
One-third of the reported cases of light chain amyloidosis are related to the germ line λ6 family; remarkably, healthy individuals express this type of protein in just 2% of the peripheral blood and bone marrow B-cells. The appearance of the disease has been related to the inherent properties of this protein family. A recombinant representative model for λ6 proteins called 6aJL2 containing the amino acid sequence encoded by the 6a and JL2 germ line genes was previously designed and synthesized to study the properties of this family. Previous work on 6aJL2 suggested a simple two-state folding model at 25 °C; no intermediate could be identified either by kinetics or by fluorescence and circular dichroism equilibrium studies, although the presence of an intermediate that is populated at ∼2.4 M urea was suggested by size exclusion chromatography. In this study we employed classic equilibrium and kinetic experiments and analysis to elucidate the detailed folding mechanism of this protein. We identify species that are kinetically accessible and/or are populated at equilibrium. We describe the presence of intermediate and native-like species and propose a five-species folding mechanism at 25 °C at short incubation times, similar to and consistent with those observed in other proteins of this fold. The formation of intermediates in the mechanism of 6aJL2 is faster than that proposed for a light chain, which could be an important distinction in the amyloidogenic potential of both germ lines.
Topics: Amino Acid Sequence; Amyloidosis; Circular Dichroism; Humans; Kinetics; Protein Denaturation; Protein Folding
PubMed: 33620231
DOI: 10.1021/acs.jpcb.0c08534 -
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 -
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 -
Journal of Agricultural and Food... Aug 2022Thermal treatment applied during the cooking of pulses leads to denaturation and even aggregation of the proteins, which may impact protein digestibility. Thermal...
Thermal treatment applied during the cooking of pulses leads to denaturation and even aggregation of the proteins, which may impact protein digestibility. Thermal transitions of lentil, chickpea, and bean proteins were studied using differential scanning calorimetry (DSC). Protein-enriched samples were obtained by dry air classification of dehulled seeds and were heated to 160 °C, with water contents ranging from 0.2 to 4 kg/kg on a dry basis. The DSC peaks of the resulting endotherms were successfully modeled as overlapping Gaussian functions. The denaturation temperatures were modeled as a function of the temperature according to the Flory-Huggins theory. The modeling allows for the calculation of the degree of protein transition for any temperature and moisture condition. The denaturation diagrams reflect the different protein compositions of lentil, chickpea, and bean (particularly the 11S/7S globulin ratio). Chickpea proteins were more thermally stable than those from lentil and bean. Proteins underwent an irreversible transition, suggesting that unfolding and aggregation were coupled.
Topics: Calorimetry, Differential Scanning; Cicer; Fabaceae; Lens Plant; Protein Denaturation; Water
PubMed: 35921686
DOI: 10.1021/acs.jafc.2c03553 -
Biomacromolecules Oct 2022Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely...
Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.
Topics: Alginates; Phosphoglycerate Kinase; Polymers; Protein Denaturation; Protein Folding; Protein Stability
PubMed: 36054903
DOI: 10.1021/acs.biomac.2c00297 -
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 -
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 -
Journal of the American Chemical Society Apr 2022Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the...
Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the freezing point of water. Understanding the mechanisms that determine cold denaturation and the rules that permit its observation is an important challenge. A way to approach them is to be able to induce cold denaturation in an otherwise stable protein by means of mutations. Here, we studied CyaY, a relatively stable bacterial protein with no detectable cold denaturation and a high melting temperature of 54 °C. We have characterized for years the yeast orthologue of CyaY, Yfh1, a protein that undergoes cold and heat denaturation at 5 and 35 °C, respectively. We demonstrate that, by transferring to CyaY the lessons learnt from Yfh1, we can induce cold denaturation by introducing a restricted number of carefully designed mutations aimed at destabilizing the overall fold and inducing electrostatic frustration. We used molecular dynamics simulations to rationalize our findings and demonstrate the individual effects observed experimentally with the various mutants. Our results constitute the first example of rationally designed cold denaturation and demonstrate the importance of electrostatic frustration on the mechanism of cold denaturation.
Topics: Cold Temperature; Hot Temperature; Molecular Dynamics Simulation; Protein Denaturation; Proteins; Thermodynamics
PubMed: 35427450
DOI: 10.1021/jacs.1c13355