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MethodsX Dec 2023The unwanted phenomenon of protein fibrillation is observed and during therapeutic protein development in the industry. Protein aggregation is associated with various...
The unwanted phenomenon of protein fibrillation is observed and during therapeutic protein development in the industry. Protein aggregation is associated with various degenerative disorders and might induce immune-related challenges post-administration of biopharmaceutics. A pipeline for early detection, identification, and removal of pre-formed fibrils is needed to improve the quality, efficacy, and effectiveness of the formulation. Protein fibril formation is accompanied by unfolding, secondary structural changes and the formation of larger aggregates. However, most detection processes come with extensive sample preparation steps and inefficient repeatability, incurring a financial burden on research. The current article summarizes and critically discusses six simple yet powerful methods to detect aggregation phenomena in the line of detecting fibrillar aggregates in heat-induced bovine serum albumin protein. Comparing the native and heat-induced protein samples would provide insights about aggregates. Easy, inexpensive and optimized protocols for detecting the fibrillation of proteins are explained. The procedures mentioned here detected the appearance of β-sheet-rich fibrils in the heat-induced protein sample. The aggregation is characterized by enhanced thioflavin-T fluorescence, alteration in the intrinsic fluorescence, decrease in helicity and subsequent increase in β-sheet and appearance of particles with larger hydrodynamic diameters. •This article summarizes various analytical techniques to easily characterize the fibrillation of proteins.•Various techniques to detect the formation of β-sheet rich structures, changes in the secondary structures and size of aggregates have been discussed.•The stated methodologies are validated on a model protein, albumin.
PubMed: 37928109
DOI: 10.1016/j.mex.2023.102445 -
The FEBS Journal Mar 2021Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss... (Review)
Review
Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss the inherent properties of multidomain proteins under a force vector from a structural and functional perspective. We then characterize three main systems where multidomain proteins could play major roles through mechanical unfolding: muscular contraction, cellular mechanotransduction, and bacterial adhesion. We analyze how key multidomain proteins for each system can produce a gain-of-function from the perspective of a fine-tuned quantized response, a molecular battery, delivery of mechanical work through refolding, elasticity tuning, protection and exposure of cryptic sites, and binding-induced mechanical changes. Understanding how mechanical unfolding and refolding affect function will have important implications in designing mechano-active drugs against conditions such as muscular dystrophy, cancer, or novel antibiotics.
Topics: Elasticity; Mechanotransduction, Cellular; Models, Molecular; Protein Binding; Protein Domains; Protein Folding; Protein Unfolding; Proteins; Stress, Mechanical; Thermodynamics
PubMed: 32761965
DOI: 10.1111/febs.15508 -
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 -
Toxins Aug 2022Staphylococcal food poisoning (SFP) is a common food-borne illness often associated with contamination during food handling. The genes for Staphylococcal enterotoxin...
Staphylococcal food poisoning (SFP) is a common food-borne illness often associated with contamination during food handling. The genes for Staphylococcal enterotoxin (SE) isoforms SEA and SEB are frequently detected in human nasal isolates and these toxins are commonly associated with SFP. Past studies described the resistance of preformed SE proteins to heat inactivation and their reactivation upon cooling in foods. Full thermodynamic analyses for these processes have not been reported, however. The thermal stabilities of SEA, SEB, and SEH and reversibility of unfolding in simple buffers were investigated at pH 4.5 and pH 6.8 using differential scanning calorimetry (DSC). SEA and SEB unfolding was irreversible at pH 6.8 and at least partially reversible at pH 4.5 while SEH unfolding was irreversible at pH 4.5 and reversible at pH 6.8. Additional studies showed maximum refolding for SEB at pH 3.5-4.0 and diminished refolding at pH 4.5 with increasing ionic strength. SE-stimulated secretion of interferon-gamma by human peripheral blood mononuclear cells was used to assess residual SE biological activity following heat treatments using conditions matching those used for DSC studies. The biological activities of SEB and SEH exhibited greater resistance to heat inactivation than that of SEA. The residual activities of heat-treated SEB and SEH were measurable but diminished further in the presence of reconstituted nonfat dry milk adjusted to pH 4.5 or pH 6.8. To different extents, the pH and ionic strengths typical for foods influenced the thermal stabilities of SEA, SEB, and SEH and their potentials to renature spontaneously after heat treatments.
Topics: Enterotoxins; Food Microbiology; Humans; Leukocytes, Mononuclear; Staphylococcal Food Poisoning; Staphylococcal Infections; Staphylococcus aureus
PubMed: 36006217
DOI: 10.3390/toxins14080554 -
Analytical Chemistry Nov 2022Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase () is an important method for the study of...
Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase () is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between and behavior, we use unbiased molecular dynamics (MD) to create models of unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded models for observed charge states of NTL9. The unfolding behavior replicates the behavior in-solution and is in line with the observations; however, the theoretical collision cross section (CCS) of the models was lower compared to that of the data, which may reflect reduced sampling.
Topics: Protons; Protein Unfolding; Molecular Dynamics Simulation; Proteins; Ions; Protein Conformation
PubMed: 36350278
DOI: 10.1021/acs.analchem.2c03352 -
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 -
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 -
SLAS Discovery : Advancing Life... Apr 2021Targeted protein degradation (TPD) is a recent strategy, utilizing the cell's proteostasis machinery to deplete specific proteins. This represents a paradigm shift in... (Review)
Review
Targeted protein degradation (TPD) is a recent strategy, utilizing the cell's proteostasis machinery to deplete specific proteins. This represents a paradigm shift in early drug discovery, away from occupancy-driven to event-driven mechanisms.Recent efforts have focused on the development of proteolysis-targeting chimeras (PROTACs). These heterobifunctional molecules combine a target-specific binding moiety linked to an E3 ligase ligand and trigger selective ubiquitination of the target protein, marking it for proteasomal degradation. While these molecules can be highly efficacious, they generally have unfavorable physicochemical properties due to their large size.In contrast, smaller molecules that induce degradation could represent an attractive, simple option to overcoming the limitations of both traditional modulators and PROTACs. These molecules may have a range of mechanisms: recruitment of an E3 ligase (molecular glues), introduction of hydrophobic areas, or inducing local unfolding, each of which triggers degradation.We recently completed a high-throughput screen of 111,000 compounds in a cellular HiBiT assay in an effort to identify such molecules. Preliminary analysis indicates that we have been able to identify alternative small-molecule degraders. We highlight methods for triage, characterization, selectivity, and mode of action. In summary, we believe that these types of small-molecule degraders, which may possibly have more acceptable physicochemical properties than the inherently larger heterobifunctional molecules, are an exciting approach for inducing TPD, and we illustrate that a general screening approach can be successful in identifying useful start points for developing such molecules.
Topics: Drug Discovery; Eukaryotic Cells; High-Throughput Screening Assays; Humans; Hydrophobic and Hydrophilic Interactions; Ligands; Molecular Targeted Therapy; Proteasome Endopeptidase Complex; Protein Binding; Protein Processing, Post-Translational; Protein Unfolding; Proteolysis; Proteomics; Proteostasis; Small Molecule Libraries; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 33632029
DOI: 10.1177/2472555221991104 -
Frontiers in Pharmacology 2019Neurodegenerative diseases share the fact that they derive from altered proteins that undergo an unfolding process followed by formation of β-structures and a... (Review)
Review
Neurodegenerative diseases share the fact that they derive from altered proteins that undergo an unfolding process followed by formation of β-structures and a pathological tendency to self-aggregate in neuronal cells. This is a characteristic of tau protein in Alzheimer's disease and several tauopathies associated with tau unfolding, α-synuclein in Parkinson's disease, and huntingtin in Huntington disease. Usually, the self-aggregation products are toxic to these cells, and toxicity spreads all over different brain areas. We have postulated that these protein unfolding events are the molecular alterations that trigger several neurodegenerative disorders. Most interestingly, these events occur as a result of neuroinflammatory cascades involving alterations in the cross-talks between glial cells and neurons as a consequence of the activation of microglia and astrocytes. The model we have hypothesized for Alzheimer's disease involves damage signals that promote glial activation, followed by nuclear factor NF-kβ activation, synthesis, and release of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and IL-12 that affect neuronal receptors with an overactivation of protein kinases. These patterns of pathological events can be applied to several neurodegenerative disorders. In this context, the involvement of innate immunity seems to be a major paradigm in the pathogenesis of these diseases. This is an important element for the search for potential therapeutic approaches for all these brain disorders.
PubMed: 31572186
DOI: 10.3389/fphar.2019.01008 -
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