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Nature Methods Jul 2019One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of... (Review)
Review
One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.
Topics: Benchmarking; Mass Spectrometry; Protein Denaturation; Protein Processing, Post-Translational; Proteins; Proteomics
PubMed: 31249407
DOI: 10.1038/s41592-019-0457-0 -
Poultry Science May 2017Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical...
Precipitation and ultimate pH effect on chemical and gelation properties of protein prepared by isoelectric solubilization/precipitation process from pale, soft, exudative (PSE)-like chicken breast meat1.
Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical and gelation properties of PSE-like chicken breast meat paste were studied. These pastes were prepared by the pH adjustment method and protein isolation using the isoelectric solubilization/precipitation (ISP) process from PSE-like chicken meat. The ISP-isolated samples were solubilized at pH 11.0 and recovered at pH 5.5 and 6.2. The ultimate pH of the ISP-isolated protein and meat paste was adjusted to 6.2 and 7.0. The ultimate pH in this article referred to the final pH of the extracted protein and meat paste. Higher reactive sulfhydryl content and surface hydrophobicity were found in the precipitation at pH 6.2 than at pH 5.5. However, various ultimate pH values showed no significant influence on the surface hydrophobicity. The hardness of gel, as measured by textural profile analysis, was improved using 6.2 as the precipitation pH compared with pH 5.5. The viscoelastic modulus (G΄) of gel pastes prior to the thermal gelation was higher with ISP treatment. However, lower G΄ was seen after thermal gelation compared with the control. Dynamic rheological measurement demonstrated a different gel-forming mechanism for protein precipitated at pH values of 5.5 and 6.2 compared with the meat paste. The cooking loss showed that the recovered protein failed to form a gel with good water-retention capacity unless the ultimate pH was adjusted to 7.0. Gels made from protein extracted by the ISP method had higher yellowness and lower redness values, probably due to protein denaturation. Precipitation at pH 6.2 formed a harder gel with lower water-retention ability than that at pH 5.5, and this result was possibly due to higher surface hydrophobicity and S-S bridge formation. Overall, network characteristics of ISP-treated protein gels were strongly dependent on precipitation pH and ultimate pH.
Topics: Animals; Avian Proteins; Chemical Precipitation; Chickens; Cooking; Food Handling; Gels; Hydrogen-Ion Concentration; Pectoralis Muscles; Poultry Products; Protein Denaturation
PubMed: 27837115
DOI: 10.3382/ps/pew412 -
F1000Research 2019Human aldose reductase (hAR) is the first and rate-limiting enzyme of the polyol pathway. For the development of secondary complications of diabetes in chronic...
Human aldose reductase (hAR) is the first and rate-limiting enzyme of the polyol pathway. For the development of secondary complications of diabetes in chronic hyperglycemic conditions, one of the critical factors is the increased flux of glucose through the polyol pathway. Due to this clinical implication, hAR attracted considerable attention from the drug discovery perspective. In spite of extensive characterization in the context of biochemical and structural aspects, we know very little about the unfolding behavior of hAR. This study reports equilibrium unfolding studies of hAR. We carried out thermal denaturation and chemical-induced equilibrium unfolding studies of hAR monitored by circular dichroism and fluorescence spectroscopy. Thermal denaturation studies presented a classical picture of two-state unfolding from native to the denatured state. The data was used to derive thermodynamic parameters and study the thermostability of hAR. Chemical induced equilibrium unfolding studies led us to discover an intermediate state, which gets populated at 3.5-4.0 M and 0.7-2.0 M of urea and GuHCl, respectively. Thermodynamic parameters derived from chemical-induced unfolding are in agreement with those obtained from thermal denaturation of hAR. This study revealed that aldose reductase unfolds from native to the unfolded state via an intermediate. Assessment of the thermodynamic stability of native, intermediate, and unfolded states shows that significant energy barriers separate these states, which ensures the cooperativity of unfolding. As hAR functions in cells that are under osmotic and oxidative stress, these findings may have implications for its native conformation under the physiological state.
Topics: Aldehyde Reductase; Guanidine; Humans; Hydrogen-Ion Concentration; Protein Denaturation; Protein Folding
PubMed: 31723418
DOI: 10.12688/f1000research.18963.2 -
Nature Communications Apr 2022Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses...
Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings.
Topics: Animals; Censuses; Circular Dichroism; Cysteine; Kinetics; Ligands; Mammals; Protein Conformation; Protein Denaturation; Protein Folding; Proteome; Thermodynamics; Urea
PubMed: 35422070
DOI: 10.1038/s41467-022-29661-2 -
Molecules (Basel, Switzerland) Jan 2021The α and polyproline II (PPII) basins are the two most populated regions of the Ramachandran map when constructed from the protein coil library, a widely used... (Review)
Review
The α and polyproline II (PPII) basins are the two most populated regions of the Ramachandran map when constructed from the protein coil library, a widely used denatured state model built from the segments of irregular structure found in the Protein Data Bank. This indicates the α and PPII conformations are dominant components of the ensembles of denatured structures that exist in solution for biological proteins, an observation supported in part by structural studies of short, and thus unfolded, peptides. Although intrinsic conformational propensities have been determined experimentally for the common amino acids in short peptides, and estimated from surveys of the protein coil library, the ability of these intrinsic conformational propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs), an increasingly important class of proteins in cell function, has thus far proven elusive to establish. Recently, we demonstrated that the sequence dependence of the mean hydrodynamic size of IDPs in water and the impact of heat on the coil dimensions, provide access to both the sequence dependence and thermodynamic energies that are associated with biases for the α and PPII backbone conformations. Here, we compare results from peptide-based studies of intrinsic conformational propensities and surveys of the protein coil library to those of the sequence-based analysis of heat effects on IDP hydrodynamic size, showing that a common structural and thermodynamic description of the protein denatured state is obtained.
Topics: Amino Acid Sequence; Intrinsically Disordered Proteins; Models, Molecular; Peptides; Protein Conformation; Protein Denaturation; Thermodynamics; Water
PubMed: 33530506
DOI: 10.3390/molecules26030634 -
European Journal of Pharmaceutics and... Nov 2023Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates...
Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage. In this work, we measured the aggregation rates of two antibodies in a broad temperature range (from 60 °C to -25 °C), using an isochoric cooling method to prevent freezing of the formulations below 0 °C. Both antibodies evidenced increasing aggregation rates when approaching extreme temperatures, because of hot and cold denaturation. This behavior was modelled using Arrhenius and Gibbs-Helmholtz equations, which enabled to deconvolute the contribution of unfolding from the protein association kinetics. This approach made possible to model the aggregation rates at refrigeration temperature (5 °C) in a relatively short timeframe (1-2 weeks) and using standard characterization techniques (SEC-HPLC and DLS).
Topics: Protein Stability; Cold Temperature; Temperature; Freezing; Antibodies; Protein Denaturation
PubMed: 37832611
DOI: 10.1016/j.ejpb.2023.10.009 -
Protein Science : a Publication of the... Jun 2024Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning...
Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high-throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate-scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per-plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single-chain variable fragment of nivolumab, harvesting the melting temperature (T) ranging from -9.3 up to +10.8°C compared to the wild-type sequence. Our findings will allow for data-driven stabilization in protein design and streamlining the rational approaches.
Topics: Protein Stability; Thermodynamics; Protein Denaturation; High-Throughput Screening Assays; Brevibacillus
PubMed: 38801228
DOI: 10.1002/pro.5029 -
FEBS Open Bio Aug 2019Fibroblast growth factors (FGFs) regulate embryonic development and homeostasis, including tissue and organ repair and specific aspects of metabolism. The basic FGF and...
Fibroblast growth factors (FGFs) regulate embryonic development and homeostasis, including tissue and organ repair and specific aspects of metabolism. The basic FGF and acidic FGF, now known as FGF2 and FGF1, are widely used protein drugs for tissue repair. However, they are susceptible to denaturation at ambient temperatures and during long-time storage, which will reduce their biological activity. The interaction of FGFs with the sulfated domains of heparan sulfate and heparin is essential for their cellular signaling and stability. Therefore, we analyzed the interactions of FGF1 and FGF2 with four sulfated polysaccharides: heparin, dextran sulfate (DXS), λ-carrageenan, and chondroitin sulfate. The results of thermal stability and cell proliferation assays demonstrate that heparin, DXS, and λ-carrageenan bound to both FGFs and protected them from denaturation. Our results suggest heparin, DXS, and λ-carrageenan are potential formulation materials that bind and stabilize FGFs, and which may also potentiate their activity and control their delivery.
Topics: Carrageenan; Chondroitin Sulfates; Dextran Sulfate; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Glycosaminoglycans; HEK293 Cells; Heparin; Humans; Protein Denaturation; Sulfates
PubMed: 31271519
DOI: 10.1002/2211-5463.12696 -
Proteins Nov 2017The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic...
The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (∼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.
Topics: Animals; Calorimetry; Chickens; Muramidase; Protein Aggregates; Protein Denaturation; Protein Stability; Thermodynamics
PubMed: 28722205
DOI: 10.1002/prot.25354 -
Molecules (Basel, Switzerland) Jun 2022Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced...
Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qβ host factor (Hfq) from mesophilic (). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower.
Topics: Circular Dichroism; Mutation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Pseudomonas aeruginosa; Thermodynamics; Tryptophan; Urea
PubMed: 35744948
DOI: 10.3390/molecules27123821