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Biochemistry May 2019Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the...
Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the degree of structure and the compactness of denatured states populated in these experiments are still open questions. Here, building on previous works, we show that thermally and chemically denatured protein states are distinct from the viewpoint of far-ultraviolet circular dichroism experiments that report on the local conformational status of peptide bonds. The differences identified are independent of protein length, structural class, or experimental conditions, highlighting the presence of two sub-ensembles within the denatured states. The sub-ensembles, U and U for thermally induced and denaturant-induced unfolded states, respectively, can exclusively exchange populations as a function of temperature at high chemical denaturant concentrations. Empirical analysis suggests that chemically denatured states are ∼50% more expanded than the thermally denatured chains of the same protein. Our observations hint that the strength of protein backbone-backbone interactions and identity versus backbone-solvent/co-solvent interactions determine the conformational distributions. We discuss the implications for protein folding mechanisms, the heterogeneity in relaxation rates, and folding diffusion coefficients.
Topics: Circular Dichroism; DNA-Binding Proteins; Escherichia coli Proteins; Hot Temperature; Kinetics; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Folding; Repressor Proteins; Urea
PubMed: 31083972
DOI: 10.1021/acs.biochem.9b00089 -
The Journal of Physical Chemistry. B Jun 2020Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we...
Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the protein main chain. Because chemical shifts (CS's) measured by NMR spectroscopy are extremely sensitive to the local atomic environment, CS has served as a local probe of thermal unfolding of proteins by varying the position of the atomic isotope along the amino acid sequence. The variation of the CS of each C atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We demonstrate that these local heat-induced denaturation curves mirror the local protein nativeness defined by the free energy landscape of the local curvature and torsion of the protein main chain described by the CC virtual bonds. Comparison between molecular dynamics simulations and CS data of the gpW protein demonstrates that some local native states defined by the local curvature and torsion of the main chain, mainly located in secondary structures, are coupled to each other whereas others, mainly located in flexible protein segments, are not. Consequently, CS's of some residues are faithful reporters of global protein unfolding, with heat-induced denaturation curves similar to the average global one, whereas other residues remain silent about the protein unfolded state. For the latter, the local deformation of the protein main chain, characterized by its local curvature and torsion, is not cooperatively coupled to global unfolding.
Topics: Amino Acid Sequence; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Unfolding; Thermodynamics
PubMed: 32392067
DOI: 10.1021/acs.jpcb.0c01230 -
ELife Apr 2019Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in...
Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.
Topics: Adsorption; Air; Cryoelectron Microscopy; Electron Microscope Tomography; Fatty Acid Synthases; Protein Denaturation; Saccharomyces cerevisiae Proteins; Water
PubMed: 30932812
DOI: 10.7554/eLife.42747 -
Biophysical Chemistry Jan 2016Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common... (Review)
Review
Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results. It would always be preferable to measure the whole stability curve. The calculation of this curve is greatly facilitated whenever it is possible to observe cold denaturation. Using Yfh1, one of the few proteins whose cold denaturation occurs at neutral pH and low ionic strength, we could measure the variation of its full stability curve under several environmental conditions. Here we show the advantages of gauging stability as a function of external variables using stability curves.
Topics: Cold Temperature; Hydrogen-Ion Concentration; Iron-Binding Proteins; Models, Molecular; Osmolar Concentration; Protein Denaturation; Protein Stability; Temperature; Frataxin
PubMed: 26026885
DOI: 10.1016/j.bpc.2015.05.007 -
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 -
Scientific Reports Mar 2023In order to reduce infection risk of novel coronavirus (SARS-CoV-2), we developed nano-photocatalysts with nanoscale rutile TiO (4-8 nm) and CuO (1-2 nm or less)....
In order to reduce infection risk of novel coronavirus (SARS-CoV-2), we developed nano-photocatalysts with nanoscale rutile TiO (4-8 nm) and CuO (1-2 nm or less). Their extraordinarily small size leads to high dispersity and good optical transparency, besides large active surface area. Those photocatalysts can be applied to white and translucent latex paints. Although CuO clusters involved in the paint coating undergo gradual aerobic oxidation in the dark, the oxidized clusters are re-reduced under > 380 nm light. The paint coating inactivated the original and alpha variant of novel coronavirus under irradiation with fluorescent light for 3 h. The photocatalysts greatly suppressed binding ability of the receptor binding domain (RBD) of coronavirus (the original, alpha and delta variants) spike protein to the receptor of human cells. The coating also exhibited antivirus effects on influenza A virus, feline calicivirus, bacteriophage Qβ and bacteriophage M13. The photocatalysts would be applied to practical coatings and lower the risk of coronavirus infection via solid surfaces.
Topics: Humans; SARS-CoV-2; COVID-19; Protein Denaturation; Spike Glycoprotein, Coronavirus
PubMed: 36899059
DOI: 10.1038/s41598-023-30690-0 -
Analytical Chemistry Feb 2023The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for...
The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.
Topics: Humans; Protein Denaturation; Serum Albumin, Bovine; Hot Temperature; Fluorescence Polarization
PubMed: 36696963
DOI: 10.1021/acs.analchem.2c03912 -
Analytical Chemistry Mar 2020Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a...
Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.
Topics: Animals; Chromatography, Gel; Heart; Horses; Hydrogen-Ion Concentration; Myoglobin; Protein Denaturation; Spectrometry, Mass, Electrospray Ionization
PubMed: 32107919
DOI: 10.1021/acs.analchem.9b04961 -
International Journal of Molecular... Oct 2021A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism...
A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.
Topics: Biological Assay; Buffers; Circular Dichroism; Free Radicals; Ligands; Protein Denaturation; Protein Stability; Proteins; Reactive Oxygen Species; Synchrotrons; Ultraviolet Rays
PubMed: 34768758
DOI: 10.3390/ijms222111325 -
Protein Science : a Publication of the... Dec 2015We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard...
We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.
Topics: Circular Dichroism; Computational Biology; Models, Molecular; Protein Denaturation; Protein Stability; Proteins; Software; Spectrometry, Fluorescence; Temperature
PubMed: 26402034
DOI: 10.1002/pro.2809