Effects of High-Intensity Ultrasound Pretreatment on Structure, Properties, and Enzymolysis of Walnut Protein Isolate.

The purpose of this paper was to investigate the effect of high-intensity ultrasonication (HIU) pretreatment before enzymolysis on structural conformations of walnut protein isolate (WPI) and antioxidant activity of its hydrolysates. Aqueous WPI suspensions were subjected to ultrasonic processing at different power levels (600-2000 W) and times (5-30 min), and then changes in the particle size, zeta (ζ) potential, and structure of WPI were investigated, and antioxidant activity of its hydrolysates was determined. The particle size of the particles of aqueous WPI suspensions was decreased after ultrasound, indicating that sonication destroyed protein aggregates. The ζ-potential values of a protein solution significantly changed after sonication, demonstrating that the original dense structure of the protein was destroyed. Fourier transform infrared spectroscopy indicated a change in the secondary structure of WPI after sonication, with a decrease in ß-turn and an increase in α-helix, ß-sheet, and random coil content. Two absorption peaks of WPI were generated, and the fluorescence emission intensity of the proteins decreased after ultrasonic treatment, indicating that the changes in protein tertiary structure occurred. Moreover, the degree of hydrolysis and the antioxidant activity of the WPI hydrolysates increased after sonication. These results suggest that HIU pretreatment is a potential tool for improving the functional properties of walnut proteins.

Denaturation of Lysozyme with Visible-light-responsive Photocatalysts of Ground Rhodium-doped and Ground Rhodium-antimony-co-doped Strontium Titanate.

Protein denaturants play an important role in medical and biological research, and development of new denaturants is widely explored to study aging and various diseases. In this research, we treated lysozyme, a model protein, with photocatalysts of ground Rh-doped SrTiO3 (g-STO:Rh) and ground Rh-Sb-co-doped SrTiO3 (g-STO:Rh/Sb) under visible light irradiation to explore the potential of those photocatalysts as denaturants. SDS-PAGE showed that photocatalysis with g-STO:Rh induced the fragmentation of lysozyme into unidentifiable decomposition products. BCA and Bradford protein assays indicated that the peptide bonds and basic, aromatic and N-terminal amino acid residues in lysozyme were denaturated by g-STO:Rh photocatalysis. The denaturation of those amino acids, as quantified by the decreased solubility of lysozyme, was estimated to be more severe by Bradford protein assay than by BCA protein assay. Circular dichroism (CD) spectra of lysozyme revealed that the secondary structure was denatured by g-STO:Rh photocatalysis, indicating that g-STO:Rh photocatalysis is especially effective against the amino acid residues that form the secondary structure via hydrogen bonds. Furthermore, the lytic activity of lysozyme was reduced by g-STO:Rh photocatalysis, owing to denaturation of the enzyme. The visible-light-responsive photocatalyst of g-STO:Rh/Sb accelerates the oxidation reaction and has stronger oxidizing power than g-STO:Rh. Lysozyme was denatured more quickly by g-STO:Rh/Sb photocatalysis than by g-STO:Rh according to analysis by SDS-PAGE, CD spectroscopy, BCA and Bradford protein assays, and lytic activity. These results suggest that higher photocatalytic activity induces more significant denaturation of lysozyme, implying that the main factor of photocatalytic denaturation of lysozyme is oxidation. It should be noted that, as far as we know, this is the first report for denaturation of protein using visible-light-responsive photocatalyst.

Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody.

Photostability testing of therapeutic proteins is a critical requirement in the development of biologics. Upon exposure to light, pharmaceutical proteins may undergo a change in structure, stability, and functional properties that could have a potential impact on safety and efficacy. In this work, we studied how exposure to light, according to ICH guidelines, leads to photo-oxidation of a therapeutic IgG1 mAb. We also tested the ability of five different excipients to prevent such oxidation. In samples that were exposed to light, we found that the CH2 domain was considerably destabilized but there were no major changes in the overall structure of the protein. Aggregation of the protein was observed because of light exposure. Mass spectrometry identified that light exposure oxidizes two key methionine residues in the Fc region of the protein. In terms of function, a loss in binding to the neonatal Fc receptor, decreased antibody-dependent cell-mediated cytotoxicity and cell proliferation activities of the protein were seen. Combined analysis of the photo-oxidation effects on the structure, stability, aggregation, and function of the mAb has identified the underlying unifying mechanism. Among the sugars and amino acids tested, methionine was the most effective in protecting mAb against photo-oxidation.

Microwave-radiation-induced molecular structural rearrangement of hen egg-white lysozyme.

We have investigated the nonthermal effect of 10 GHz/22 dBm microwave radiation on hen egg-white lysozyme (HEWL) over different irradiation times, ranging from 2 min to 1 h. To ensure a control over the radiation parameters, a pair of microwave rectangular waveguides is used to irradiate the samples. Optical spectroscopic measurements, which include UV-visible absorption spectroscopy, Raman spectroscopy, and far UV CD spectroscopy, reveal the exposure of the buried tryptophan (Trp) residues of the native molecule between 15 and 30 min of radiation. The higher duration of the perturbation leads to a compact structure of the protein and Trp residues are buried again. Interestingly, we do not find any change in the secondary structure of the protein even for 1 h duration of radiation. The relaxation dynamics of the irradiated molecules also has been discussed. We have shown that the molecules relax to their native configuration in 7-8 h after the radiation field is turned off. The structural rearrangement over the above timescale has further been probed by a model calculation, based on a modified Langevin equation. Our coarse-grained simulation approach utilizes the mean of atomic positions and net atomic charge of each amino acid of native HEWL to mimic the initial conformation of the molecule. The modified positions of the residues are then calculated for the given force fields. The simulation results reveal the nonmonotonous change in overall size of the molecule, as observed experimentally. The radiation parameters used in our experiments are very similar to those of some of the electronic devices we often come across. Thus, we believe that the results of our studies on a simple protein structure may help us in understanding the effect of radiation on complex biological systems as well.

Influence of Gamma Irradiation on Porcine Serum Albumin Structural Properties and Allergenicity.

Pork provides an ideal source of food energy; however, pork can elicit an allergic reaction, and porcine serum albumin (PSA) has been identified as a major allergen. This study examined the impact of gamma irradiation on the allergenicity and structural qualities of PSA; the PSA solution was gamma-irradiated at 1, 2, 4, 6, and 8 kGy. Allergenicity was investigated by immunoblotting and competitive indirect ELISA using serum from patients who are allergic to pork, and conformational changes in irradiated PSA were measured by circular dichroism, sulfhydryl group detection, and fluorescence emission spectra. The secondary and tertiary structures of gamma-irradiated PSA were altered, and the allergenicity of PSA was lowered by boosting the amount of irradiation. In addition, there is high correlation between depletion in the α-helix and immunoglobulin E-binding capability of PSA. The results show a new possibility in using gamma irradiation to reduce the allergenicity of pork products.

Protective effects of curcumin against gamma ray induced conformational change of human serum albumin.

This study explores the possibility for protection by curcumin during the molecular and structural changes of human serum albumin (HSA) exposed to gamma irradiation. We used a combination of spectroscopic methods to probe the conformational ensemble of the irradiated HSA and finally evaluated the extent of restoration by curcumin. SDS-PAGE study unfolded the formation of cross linked aggregates as a consequence of increasing exposure of gamma radiation. CD and FTIR spectroscopy indicated significant decrease in the alpha helix content of HSA from 57% to 15% with increasing radiation doses. Steady state and time resolved fluorescence studies complemented the spectroscopic measurements, when lifetime decay was significantly reduced from 6.35ns to 0.37ns. Hydrophobic study showed the effectiveness of curcumin for protection at low dose of gamma irradiated HSA samples. We integrated these observations to investigate protein aggregation under increasing gamma radiation and estimated the same in presence of curcumin. It was elucidated, that when HSA is irradiated at low dose of gamma radiation in presence of curcumin, it is capable of retaining the characteristic properties to a higher extent indicating stabilization of molecular structure of HSA by curcumin. A model for curcumin based protection has been proposed utilizing ThT assay.

The effect of microwave on the interaction of flavour compounds with G-actin from grass carp (Catenopharyngodon idella).

BACKGROUND: In order to investigate the influence of non-thermal effects of microwaves on the flavour of fish and meat products, the G-actin of grass carp in ice baths was exposed to different microwave powers (0, 100, 300 or 500 W); the surface hydrophobicity, sulfhydryl contents, secondary structures and adsorption capacity of G-actin to ketones were determined. RESULTS: As microwave power increased from 0 to 300 W, the surface hydrophobicity, total and reactive sulfhydryls increased; α-helix, ß-sheet and random coil fractions turned into ß-turn fractions. As microwave power increased from 300 to 500 W, however, hydrophobicity and sulfhydryl contents decreased; ß-turn and random coil fractions turned into α-helix and ß-sheet fractions. The tendencies of adsorbed capacity of ketones were similar to hydrophobicity and sulfhydryl contents. CONCLUSION: The increased adsorbing of ketones could be attributed to the unfolding of secondary structures by revealing new binding sites, including thiol groups and hydrophobic binding sites. The decreased binding capacity was related to the refolding and aggregation of protein. The results suggested that microwave powers had obvious effects on the flavour retention and proteins structures in muscle foods. © 2017 Society of Chemical Industry.

Changes in nail keratin observed by Raman spectroscopy after Nd:YAG laser treatment.

Lasers and photodynamic therapy have been considered a convergence treatment for onychomycosis, which is a fungal infection on the nail bed and nail plate. Laser therapies have shown satisfactory results without significant complications for onychomycosis; however, the mechanism of clearing remains unknown. In this work, we investigated changes in the chemical structure of nail keratin induced by Nd:YAG laser using Raman spectroscopy. Toe nails with onychomycosis were treated with 1064 nm Nd:YAG laser. After laser treatment, the disulfide band (490-590 cm-1 ) of nail keratin was rarely observed or was reduced in intensity. The amide I band (1500-1700 cm-1 ) also showed changes induced by the laser. The α-helical (1652 cm-1 ) structures dominated the ß-sheet (1673 cm-1 ) in nontreated nail, but the opposite phenomenon was observed after laser treatment.

Parallel ß-sheet vibration band increases with proteins dipole moment under exposure to 1765 MHz microwaves.

Effects of exposure of 4 h to mobile phones microwaves at 1765 MHz at a power density around 940 mW/m(2) on four typical proteins (hemoglobin in H2 O solution, and myoglobin, bovine serum albumin, and lysozyme in D2 O solution) were studied by means of Fourier Transform Infrared spectroscopy and Fourier self-deconvolution analysis. Increase in intensity of parallel ß-sheet component around 1635 cm(-1) was observed after exposure of hemoglobin, myoglobin, and bovine serum albumin, showing that a mechanism of unfolding occurred after exposure, whereas no appreciable change in the amide I region occurred after lysozyme exposure. In addition, a relationship between protein dipole moment and protein unfolding rate was demonstrated with a correlation coefficient r = 0.973 and 95% confidence interval.

Interactions of benzotriazole UV stabilizers with human serum albumin: Atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations.

Benzotriazole UV stabilizers (BZTs) belong to one prominent group of ultraviolet (UV) stabilizers and are widely used in various plastics materials. Their large production volumes, frequent detections in the environment and potential toxicities have raised increasing public concern. BZTs can be transported in vivo by transport proteins in plasma and the binding association to transport proteins may serve as a significant parameter to evaluate the bioaccumulative potential. We utilized a novel HSA biosensor, circular dichroism spectroscopy, fluorescence spectroscopy to detect the dynamic interactions of six BZTs (UV-326, UV-327, UV-328, UV-329, UV-P, and BZT) with human serum albumin (HSA), and characterized the corresponding structure-activity relationships (SAR) by molecular dynamics simulations. All test BZTs potently bind at Sudlow site I of HSA with a binding constant of 10(4) L/mol at 298 K. Minor changes in the moieties of BZTs affect their interactions with HSA and differently induce conformations of HSA. Their binding reduced electrochemical impedance spectra and α-helix content of HSA, caused slight red-shifted emission, and changed fluorescence lifetime components of HSA in a concentration-dependent mode. UV-327 and UV-329 form hydrogen bonds with HSA, while UV-329, UV-P and BZT bind HSA with more favorable electrostatic interactions. Our in vitro and in silico study offered a significant framework toward the understanding of risk assessment of BZTs and provides guide for future design of environmental benign BZTs-related materials.

Secondary Structure Alterations of Histones H2A and H2B in X-Irradiated Human Cancer Cells: Altered Histones Persist in Cells for at Least 24 Hours.

We measured and compared the circular dichroism (CD) spectra and secondary structures of histone proteins H2A, H2B and their variants extracted from X-irradiated and unirradiated human HeLa cells. Compared to unirradiated cells, a relative increase in α-helix structure and decrease in other secondary structures was observed in X-irradiated cells. These structural alterations persisted for at least 24 h, which is substantially longer than the 2 h generally known to be required for DNA double-strand break repair.

Effects of Very Low Dose Fast Neutrons on Cell Membrane And Secondary Protein Structure in Rat Erythrocytes.

The effects of ionizing radiation on biological cells have been reported in several literatures. Most of them were mainly concerned with doses greater than 0.01 Gy and were also concerned with gamma rays. On the other hand, the studies on very low dose fast neutrons (VLDFN) are rare. In this study, we have investigated the effects of VLDFN on cell membrane and protein secondary structure of rat erythrocytes. Twelve female Wistar rats were irradiated with neutrons of total dose 0.009 Gy (241Am-Be, 0.2 mGy/h) and twelve others were used as control. Blood samples were taken at the 0, 4th, 8th, and 12th days postirradiation. Fourier transform infrared (FTIR) spectra of rat erythrocytes were recorded. Second derivative and curve fitting were used to analysis FTIR spectra. Hierarchical cluster analysis (HCA) was used to classify group spectra. The second derivative and curve fitting of FTIR spectra revealed that the most significant alterations in the cell membrane and protein secondary structure upon neutron irradiation were detected after 4 days postirradiation. The increase in membrane polarity, phospholipids chain length, packing, and unsaturation were noticed from the corresponding measured FTIR area ratios. This may be due to the membrane lipid peroxidation. The observed band shift in the CH2 stretching bands toward the lower frequencies may be associated with the decrease in membrane fluidity. The curve fitting of the amide I revealed an increase in the percentage area of α-helix opposing a decrease in the ß-structure protein secondary structure, which may be attributed to protein denaturation. The results provide detailed insights into the VLDFN effects on erythrocytes. VLDFN can cause an oxidative stress to the irradiated erythrocytes, which appears clearly after 4 days postirradiation.

Identifying and quantifying radiation damage at the atomic level.

Radiation damage impedes macromolecular diffraction experiments. Alongside the well known effects of global radiation damage, site-specific radiation damage affects data quality and the veracity of biological conclusions on protein mechanism and function. Site-specific radiation damage follows a relatively predetermined pattern, in that different structural motifs are affected at different dose regimes: in metal-free proteins, disulfide bonds tend to break first followed by the decarboxylation of aspartic and glutamic acids. Even within these damage motifs the decay does not progress uniformly at equal rates. Within the same protein, radiation-induced electron density decay of a particular chemical group is faster than for the same group elsewhere in the protein: an effect known as preferential specific damage. Here, BDamage, a new atomic metric, is defined and validated to recognize protein regions susceptible to specific damage and to quantify the damage at these sites. By applying BDamage to a large set of known protein structures in a statistical survey, correlations between the rates of damage and various physicochemical parameters were identified. Results indicate that specific radiation damage is independent of secondary protein structure. Different disulfide bond groups (spiral, hook, and staple) show dissimilar radiation damage susceptibility. There is a consistent positive correlation between specific damage and solvent accessibility.

Bacteriorhodopsin: Would the real structural intermediates please stand up?

BACKGROUND: Bacteriorhodopsin (bR) is the simplest known light driven proton pump and has been heavily studied using structural methods: eighty four X-ray diffraction, six electron diffraction and three NMR structures of bR are deposited within the protein data bank. Twenty one X-ray structures report light induced structural changes and changes induced by mutation, changes in pH, thermal annealing or X-ray induced photo-reduction have also been examined. SCOPE OF REVIEW: We argue that light-induced structural changes that are replicated across several studies by independent research groups are those most likely to represent what is happening in reality. We present both internal distance matrix analyses that sort deposited bR structures into hierarchal trees, and difference Fourier analysis of deposited X-ray diffraction data. MAJOR CONCLUSIONS: An internal distance matrix analysis separates most wild-type bR structures according to their different crystal forms, indicating how the protein's structure is influenced by crystallization conditions. A similar analysis clusters eleven studies of illuminated bR crystals as one branch of a hierarchal tree with reproducible movements of the extracellular portion of helix C towards helix G, and of the cytoplasmic portion of helix F away from helices A, B and G. All crystallographic data deposited for illuminated crystals show negative difference density on a water molecule (Wat402) that forms H-bonds to the retinal Schiff Base and two aspartate residues (Asp85, Asp212) in the bR resting state. Other recurring difference density features indicated reproducible side-chain, backbone and water molecule displacements. X-ray induced radiation damage also disorders Wat402 but acts via cleaving the head-groups of Asp85 and Asp212. GENERAL SIGNIFICANCE: A remarkable level of agreement exists when deposited structures and crystallographic observations are viewed as a whole. From this agreement a unified picture of the structural mechanism of light-induced proton pumping by bR emerges. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation.

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the ß-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.

Using a non-invasive technique in nutrition: synchrotron radiation infrared microspectroscopy spectroscopic characterization of oil seeds treated with different processing conditions on molecular spectral factors influencing nutrient delivery.

Non-invasive techniques are a key to study nutrition and structure interaction. Fourier transform infrared microspectroscopy coupled with a synchrotron radiation source (SR-IMS) is a rapid, non-invasive, and non-destructive bioanalytical technique. To understand internal structure changes in relation to nutrient availability in oil seed processing is vital to find optimal processing conditions. The objective of this study was to use a synchrotron-based bioanalytical technique SR-IMS as a non-invasive and non-destructive tool to study the effects of heat-processing methods and oil seed canola type on modeled protein structure based on spectral data within intact tissue that were randomly selected and quantify the relationship between the modeled protein structure and protein nutrient supply to ruminants. The results showed that the moisture heat-related processing significantly changed (p<0.05) modeled protein structures compared to the raw canola (control) and those processing by dry heating. The moisture heating increased (p<0.05) spectral intensities of amide I, amide II, α-helices, and ß-sheets but decreased (p<0.05) the ratio of modeled α-helices to ß-sheet spectral intensity. There was no difference (p>0.05) in the protein spectral profile between the raw and dry-heated canola tissue and between yellow- and brown-type canola tissue. The results indicated that different heat processing methods have different impacts on the protein inherent structure. The protein intrinsic structure in canola seed tissue was more sensitive and more response to the moisture heating in comparison to the dry heating. These changes are expected to be related to the nutritive value. However, the current study is based on limited samples, and more large-scale studies are needed to confirm our findings.

Microwave irradiation induced changes in protein molecular structures of barley grains: relationship to changes in protein chemical profile, protein subfractions, and digestion in dairy cows.

The objectives of this study were to evaluate microwave irradiation (MIR) induced changes in crude protein (CP) subfraction profiles, ruminal CP degradation characteristics and intestinal digestibility of rumen undegraded protein (RUP), and protein molecular structures in barley (Hordeum vulgare) grains. Samples from hulled (n = 1) and hulless cultivars (n = 2) of barley, harvested from four replicate plots in two consecutive years, were evaluated. The samples were either kept as raw or irradiated in a microwave for 3 min (MIR3) or 5 min (MIR5). Compared to raw grains, MIR5 decreased the contents of rapidly degradable CP subfraction (from 45.22 to 6.36% CP) and the ruminal degradation rate (from 8.16 to 3.53%/h) of potentially degradable subfraction. As a consequence, the effective ruminal degradability of CP decreased (from 55.70 to 34.08% CP) and RUP supply (from 43.31 to 65.92% CP) to the postruminal tract increased. The MIR decreased the spectral intensities of amide 1, amide II, α-helix, and ß-sheet and increased their ratios. The changes in protein spectral intensities were strongly correlated with the changes in CP subfractions and digestive kinetics. These results show that MIR for a short period (5 min) with a lower energy input can improve the nutritive value and utilization of CP in barely grains.

Intramolecular interactions that induce helical rearrangement upon rhodopsin activation: light-induced structural changes in metarhodopsin IIa probed by cysteine S-H stretching vibrations.

Rhodopsin undergoes rearrangements of its transmembrane helices after photon absorption to transfer a light signal to the G-protein transducin. To investigate the mechanism by which rhodopsin adopts the transducin-activating conformation, the local environmental changes in the transmembrane region were probed using the cysteine S-H group, whose stretching frequency is well isolated from the other protein vibrational modes. The S-H stretching modes of cysteine residues introduced into Helix III, which contains several key residues for the helical movements, and of native cysteine residues were measured by Fourier transform infrared spectroscopy. This method was applied to metarhodopsin IIa, a precursor of the transducin-activating state in which the intramolecular interactions are likely to produce a state ready for helical movements. No environmental change was observed near the ionic lock between Arg-135 in Helix III and Glu-247 in Helix VI that maintains the inactive conformation. Rather, the cysteine residues that showed environmental changes were located around the chromophore, Ala-164, His-211, and Phe-261. These findings imply that the hydrogen bond between Helix III and Helix V involving Glu-122 and His-211 and the hydrophobic packing between Helix III and Helix VI involving Gly-121, Leu-125, Phe-261, and Trp-265 are altered before the helical rearrangement leading toward the active conformation.

Physicochemical properties of peanut protein isolate-glucomannan conjugates prepared by ultrasonic treatment.

Peanut protein isolate (PPI) was glycated with glucomannan through classical heating or ultrasound treatment in this work. The physicochemical properties of PPI-glucomannan conjugates prepared by ultrasound treatment were compared to those prepared by classical heating. Compared with classical heating, ultrasound treatment could accelerate the graft reaction between PPI and glucomannan and improve the concentration of available free amino groups of PPI. Solubility and emulsifying properties of the conjugates obtained by ultrasound treatment were both improved as compared to those obtained by classical heating and native PPI. Decreases of lysine and arginine contents during the graft reaction indicated that these two amino acid residues attended the covalent linkage between PPI and glucomannan. Structural feature analyses suggested that conjugates obtained by ultrasound treatment had less α-helix, more ß-structures and random coil, higher surface hydrophobicity and less compact tertiary structure as compared to those obtained by classical heating and native PPI.

Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin.

The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.