-
Cold Spring Harbor Protocols Feb 2020The only way to solubilize many antigens for immunoprecipitation is by denaturation. This cell lysis protocol is ideally suited for this purpose to release proteins from...
The only way to solubilize many antigens for immunoprecipitation is by denaturation. This cell lysis protocol is ideally suited for this purpose to release proteins from complex structures or reveal antibody epitopes hidden within native proteins. Short linear epitopes may not be accessible to antibodies within the native tertiary and quaternary protein structures, but they become exposed upon the unraveling of proteins, exposing their secondary structure. Antibodies otherwise not suitable for the immunoprecipitation of proteins prepared under nondenaturing conditions are now able to bind these antigens of interest in cell lysates prepared under denaturing conditions. These antibodies may also work well for immunoblotting purposes when the protein target is completely denatured. Harvested cells in this protocol are washed in tris-buffered saline (TBS) before lysis in 2% sodium dodecyl sulfate (SDS)-containing Lysis buffer for 10 min at 100°C. The resulting sample is diluted 20-fold in TBS to reduce the SDS concentration to ≤0.1% before the addition of an antibody for immunoprecipitation. Addition of 2% bovine serum albumin (BSA) or 0.1% Nonidet P-40 to the TBS before an immunoprecipitation, respectively, ensures either removal of SDS from the target protein or retaining denatured proteins in solution.
Topics: Animals; Detergents; Hot Temperature; Humans; Immunoblotting; Immunoprecipitation; Protein Denaturation; Proteins; Sodium Dodecyl Sulfate; Tromethamine
PubMed: 32015005
DOI: 10.1101/pdb.prot098616 -
International Journal of Biological... May 2024In this paper, effects of preheating-induced denaturation of proteins and oleosomes on protein structure and soymilk quality were studied. The protein in soybeans baked...
In this paper, effects of preheating-induced denaturation of proteins and oleosomes on protein structure and soymilk quality were studied. The protein in soybeans baked at 55 °C (B-55) and 85 °C (B-85) showed an increase of β-sheet content by 3.2 % and a decrease of α-helix content by 3.3 %, indicating that proteins were gradually unfolded while oleosomes remained intact. The protein resisted thermal denaturation during secondary heating, and soymilks were stable as reflected by a small d (0.4 μm). However, raw soymilk from soybeans baked at 115 °C (B-115), steamed for 1 min (ST-1) and 5 min (ST-5) presented oleosomes destruction and lipids aggregates. The proteins were coated around the oil aggregates. The β-turn content from soybeans steamed for 10 min (ST-10) increased by 9.5 %, with a dense network where the OBs were tightly wrapped, indicating the serious protein denaturation. As a result, the soymilks B-115 or steamed ones were unstable as evidenced by the serious protein aggregation and larger d (5.65-12.48 μm). Furthermore, the soymilks were graininess and the protein digestion was delayed due to the formation of insoluble protein aggregates. The flavor and early-stage lipid digestion of soymilk from steamed soybeans was improved owing to lipid release.
Topics: Protein Denaturation; Soy Milk; Soybean Proteins; Hot Temperature; Lipid Droplets; Cooking
PubMed: 38697416
DOI: 10.1016/j.ijbiomac.2024.131999 -
Analytical Biochemistry Aug 2021There have been numerous studies of the temperature denaturation of monoclonal antibodies (mAbs) using differential scanning calorimetry (DSC). In general, mAbs are...
There have been numerous studies of the temperature denaturation of monoclonal antibodies (mAbs) using differential scanning calorimetry (DSC). In general, mAbs are characterized by complex temperature denaturation transitions in which the various domains (CH2, CH3, Fab) give rise to different peaks in the heat capacity function. The complexity and overall irreversibility of the temperature denaturation transition is well known and has limited the number of publications with an in-depth analysis of the data. Here we report that the temperature denaturation of the CH2 domain is reversible and only becomes irreversible after denaturation of the Fab domain, which is intrinsically irreversible. For these studies we have used the HIV neutralizing monoclonal antibody 17b. To account for the experimental heat capacity function, a mixed denaturation model that combines multiple reversible and irreversible transitions has been developed. This model accounts well for the DSC data and for the pH dependence of the heat capacity function of 17b and other monoclonal antibodies for which data is available in the literature. It is expected that a more detailed analysis of the stability of monoclonal antibodies will contribute to the development of better approaches to understand and optimize the structural viability of these therapeutic macromolecules.
Topics: Antibodies, Monoclonal; Calorimetry, Differential Scanning; Hydrogen-Ion Concentration; Protein Denaturation; Temperature; Thermodynamics
PubMed: 33964250
DOI: 10.1016/j.ab.2021.114240 -
Comprehensive Reviews in Food Science... Mar 2024Whey protein denaturation and aggregation have long been areas of research interest to the dairy industry, having significant implications for process performance and... (Review)
Review
Whey protein denaturation and aggregation have long been areas of research interest to the dairy industry, having significant implications for process performance and final product functionality and quality. As such, a significant number of analytical techniques have been developed or adapted to assess and characterize levels of whey protein denaturation and aggregation, to either maximize processing efficiency or create products with enhanced functionality (both technological and biological). This review aims to collate and critique these approaches based on their analytical principles and outline their application for the assessment of denaturation and aggregation. This review also provides insights into recent developments in process analytical technologies relating to whey protein denaturation and aggregation, whereby some of the analytical methods have been adapted to enable measurements in-line. Developments in this area will enable more live, in-process data to be generated, which will subsequently allow more adaptive processing, enabling improved product quality and processing efficiency. Along with the applicability of these techniques for the assessment of whey protein denaturation and aggregation, limitations are also presented to help assess the suitability of each analytical technique for specific areas of interest.
Topics: Whey Proteins; Whey; Protein Denaturation; Hydrogen-Ion Concentration
PubMed: 38343297
DOI: 10.1111/1541-4337.13289 -
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 -
Proceedings of the National Academy of... Oct 2019Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously...
Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å.
Topics: Bacteriophage T4; Muramidase; Nuclear Magnetic Resonance, Biomolecular; Pressure; Protein Conformation; Protein Denaturation; Protein Folding; Proteins
PubMed: 31570587
DOI: 10.1073/pnas.1911181116 -
Methods in Molecular Biology (Clifton,... 2023Understanding how point mutations affect the performance of protein stability has been the focus of several studies all over the years. Intrinsic fluorescence is...
Understanding how point mutations affect the performance of protein stability has been the focus of several studies all over the years. Intrinsic fluorescence is commonly used to follow protein unfolding since during denaturation, progressive redshifts on tryptophan fluorescence emission are observed. Since the unfolding process (achieved by chemical or physical denaturants) can be considered as two-state N➔D, it is possible to utilize the midpoint unfolding curves (fU = 50%) as a parameter to evaluate if the mutation destabilizes wild-type protein. The idea is to determine the [D] or T values from both wild type and mutant and calculate the difference between them. Positive values indicate the mutant is less stable than wild type.
Topics: Protein Denaturation; Circular Dichroism; Protein Stability; Protein Unfolding; Tryptophan
PubMed: 36413321
DOI: 10.1007/978-1-0716-2784-6_16 -
Methods in Molecular Biology (Clifton,... 2020CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding...
CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein-ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC.This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand.The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules to measure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein-ligand interactions and protein photostability.
Topics: Circular Dichroism; Ligands; Protein Binding; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Proteins; Radiation; Synchrotrons
PubMed: 31773649
DOI: 10.1007/978-1-0716-0163-1_6 -
Scientific Reports Apr 2021The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering....
The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (T). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔT = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its T. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest T (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.
Topics: Amino Acid Sequence; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Engineering; Protein Stability; Protein Structure, Secondary; Proteins
PubMed: 33824364
DOI: 10.1038/s41598-021-86952-2 -
Food Chemistry Mar 2021Quinoa protein possesses great amino acid profiles and can be a potential food ingredient with broad applications. The objective of this study was to investigate the...
Quinoa protein possesses great amino acid profiles and can be a potential food ingredient with broad applications. The objective of this study was to investigate the effect of different drying methods, namely freeze drying, spray drying, and vacuum drying on the functional and physicochemical properties of quinoa protein isolate, e.g., morphology, amino acid composition, SDS-PAGE profile, sulfhydryl/disulfide content, secondary structure, surface hydrophobicity, and thermal stability. The freeze-dried protein exhibited the highest emulsification capacity and stability and oil binding capacity, which was contributed to its higher surface hydrophobicity, while the spray-dried sample had the highest solubility and water absorption capacity at pH 7. Gels (8%) prepared with the freeze-dried protein had higher elastic and viscous modulus than that from others. The freeze-dried protein had the highest maximal denaturation temperature but lowest enthalpy, which may be attributed to its higher amount of random coil but lower percent of regular α-helix and β-sheet structures. Overall, quinoa protein isolate from different processing methods demonstrated distinct functional properties. This information will be useful to optimize quinoa protein production and benefit its applications.
Topics: Amino Acids; Chenopodium quinoa; Desiccation; Electrophoresis, Polyacrylamide Gel; Emulsions; Freeze Drying; Gels; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Plant Proteins; Plant Proteins, Dietary; Protein Denaturation; Protein Structure, Secondary; Solubility; Temperature; Vacuum; Viscosity
PubMed: 32829242
DOI: 10.1016/j.foodchem.2020.127823