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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 -
Marine Drugs Sep 2022This study aimed to compare the anti-inflammatory effects of fucoidans from brown seaweeds ( (SJ), (FV), (FD), (FS), and (AN)), and determine the relationship...
This study aimed to compare the anti-inflammatory effects of fucoidans from brown seaweeds ( (SJ), (FV), (FD), (FS), and (AN)), and determine the relationship between composition and biological activity. The anti-inflammatory activity was tested in vitro. It is believed that inflammation could be triggered by free radicals. Fucoidans from (FV1 and FV3) showed the strongest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with an IC = 0.05 mg/mL. In the total antioxidant capacity (TAC) test, the activity was concentration-dependent. Notable, the TAC of fucoidans except samples of FV2 and SJ (which have a lower phenolic content) was higher than that of phloroglucinol. The TAC of fucoidans strongly and positively correlated with polyphenol content. A weak correlation was associated with xylose content. The synergistic effect for fucoidans was calculated for the first time using carbohydrates and polyphenols as model mixtures. The synergy in the DPPH test was found only for FV1 and FV3 (mixture effect ME = 2.68 and 2.04, respectively). The ME strongly positively correlated with polyphenols. The relationship of ME with fucose content was positive but moderate. It was first established that the anti-inflammatory effects of fucoidan could be mediated via the inhibition of protein denaturation. The inhibition was concentration-dependent and strongly correlated with the fucose content and moderate with sulfate content. The purified fucoidan FV2 showed the most promising activity (IC 0.20 mg/mL vs. IC 0.37 mg/mL for diclofenac sodium). Similar relations were also observed in the membrane protection model. Fucoidans were able to stabilize the cell membrane integrity of human red blood corpuscles (HRBC). The results of our study support the rationality of fucoidan use as a promising agent for the treatment of inflammatory-related diseases via mechanisms of radical scavenging, antioxidant activity, inhibition of protein denaturation, and HRBC membrane stabilization.
Topics: Humans; Phaeophyceae; Antioxidants; Polyphenols; Fucose; Diclofenac; Xylose; Polysaccharides; Seaweed; Fucus; Anti-Inflammatory Agents; Sulfates; Phloroglucinol
PubMed: 36286430
DOI: 10.3390/md20100606 -
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 -
Biomimetics (Basel, Switzerland) Jul 2023The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model...
The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group's reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific β-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal β-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.
PubMed: 37504169
DOI: 10.3390/biomimetics8030281 -
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 -
Current Research in Food Science 2022The development of novel and sustainable food products, such as cheese- and meat analogues, requires a better understanding of the use of less refined ingredients. We...
The development of novel and sustainable food products, such as cheese- and meat analogues, requires a better understanding of the use of less refined ingredients. We investigated the distribution of water between the protein and starch phase of doughs and heat-induced gels made from air-classified faba bean fractions by developing a method suited for investigation of such multi-component ingredients. The moisture contents of the protein and starch phases in the dough were determined using a method based on partial sorption isotherms of mixed doughs of protein- and starch-rich fractions at high water activity. Water content of the protein phase is higher than that of the starch phase in dough, showing that protein takes up more water than starch at room temperature. Also, the moisture content of the protein phase in the gels was calculated using a model based on the denaturation temperature of legumin. From the experiments and the modelling, it became evident that the moisture content of the protein phase in the gel is lower than the moisture content of the protein phase in the dough, showing the importance of considering moisture migration from the protein to the starch during heating.
PubMed: 35497777
DOI: 10.1016/j.crfs.2022.03.013 -
Frontiers in Microbiology 2022Trimethylamine N-oxide (TMAO), an important intestinal flora-derived metabolite, plays a role in the development of cardiovascular disease and tumor immunity. Here, we...
Trimethylamine N-oxide (TMAO), an important intestinal flora-derived metabolite, plays a role in the development of cardiovascular disease and tumor immunity. Here, we determined the minimum inhibitory concentration (MIC) of antibiotics against under gradient concentrations of TMAO and performed a bacterial killing analysis. Overall, TMAO (in the range of 10 ~ 100 mM) increased the MIC of quinolones, aminoglycosides, and β-lactams in a concentration-dependent manner, and increased the lethal dose of antibiotics against . It implies that TMAO is a potential risk for failure of anti-infective therapy, and presents a case for the relationship between intestinal flora-derived metabolites and antibiotic resistance. Further data demonstrated that the inhibition of antibiotic efficacy by TMAO is independent of the downstream metabolic processes of TMAO and the typical bacterial resistance mechanisms ( motif and efflux pump). Interestingly, TMAO protects from high-protein denaturant (urea) stress and improves the viability of bacteria following treatment with two disinfectants (ethanol and hydrogen peroxide) that mediate protein denaturation by chemical action or oxidation. Since antibiotics can induce protein inactivation directly or indirectly, our work suggests that disruption of protein homeostasis may be a common pathway for different stress-mediated bacterial growth inhibition/cell death. In addition, we further discuss this possibility, which provides a different perspective to address the global public health problem of antibiotic resistance.
PubMed: 35875516
DOI: 10.3389/fmicb.2022.956673 -
International Journal of Inflammation 2021Bergapten, a furocoumarin found in many medicinal plants, is used for the management of various conditions. The present study evaluated the ability of bergapten to...
Bergapten, a furocoumarin found in many medicinal plants, is used for the management of various conditions. The present study evaluated the ability of bergapten to prevent human erythrocyte hemolysis and protein denaturation. Bergapten administered at 10, 30, and 100 g/ml exhibited a significant concentration-dependent protection on the erythrocyte membrane exposed to hypotonicity and heat-induced hemolysis. The concentration at which bergapten inhibited 50% of the cells from hemolysis (IC) was determined on a dose-response curve, plotted as logarithmic (concentration) against percentage inhibition, keeping the hemolysis produced within the control group at 100%. Bergapten treatment produced an IC value of 7.71 ± 0.27 g/ml and 4.23 ± 0.42 g/ml for hypotonicity and heat-induced hemolysis, respectively. Diclofenac sodium at similar concentrations produced an IC value of 12.22 ± 0.30 g/ml and 9.44 ± 0.23 g/ml in the hypotonicity and heat-induced hemolysis, respectively. The ability of bergapten to inhibit protein denaturation was studied as part of an investigation on its mechanism of action. The results showed a significant concentration-dependent reduction in protein denaturation. When administered at 10, 30, and 100 g/ml, bergapten produced a concentration-dependent reduction in albumin denaturation. Bergapten inhibited protein denaturation with IC values of 5.34 ± 0.30 g/ml and 12.18 ± 0.20 g/ml in the heat-treated egg albumin and bovine serum albumin denaturation experiments, respectively. Diclofenac sodium (10, 30, and 100 g/ml) exhibited a similar protection against heat-treated egg albumin and bovine serum albumin denaturation experiments with IC values of 8.93 ± 0.17 g/ml and 12.72 ± 0.11 g/ml, respectively. Taken together, data from this study show that the pharmacological properties of bergapten may in part be related to its membrane-stabilizing and antidenaturation properties.
PubMed: 34970434
DOI: 10.1155/2021/1279359