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The effects of salt–enzyme–alkali progressive tenderization treatments on porcine cortical conformation and collagen properties were investigated, and their effectiveness and mechanisms were analyzed. The tenderization treatment comprised three treatment stages: CaCl2 (25 °C/0–30 min), papain (35 °C/30–78 min), and Na2CO3 (25 °C/78–120 min). The textural, microscopic, and collagenous properties (content, solubility, and structure) of pork skin were determined at the 0th, 30th, 60th, 90th, and 120th min of the treatment process. The results showed that the shear force, hardness, and chewability of the skin decreased significantly (p < 0.05), and the elasticity exhibited a gradual increase with the progression of tenderization. The content and solubility of collagen showed no significant change at the CaCl2 treatment stage. However, the soluble collagen content increased, the insoluble collagen content decreased, and the collagen solubility increased by 18.04% during the subsequent treatment with papain and Na2CO3. Meanwhile, the scanning electron microscopy results revealed that the regular, wavy structure of the pig skin collagen fibers gradually disappeared during the CaCl2 treatment stage, the overall structure revealed expansion, and the surface microscopic pores gradually increased during the papain and Na2CO3 treatment stages. The findings of the Fourier transform infrared spectroscopy analysis indicated that the hydrogen bonding interactions between the collagen molecules and the C=O, N-H and C-N bonds in the subunit structure of collagen were substantially altered during treatment and that the breakage of amino acid chains and reduction in structural ordering became more pronounced with prolonged treatment. In the tertiary structure, the maximum emission wavelength was blue-shifted and then red-shifted, and the fluorescence intensity was gradually weakened. The surface hydrophobicity was slowly increased. The salt–enzyme–alkali tenderization treatment considerably improved the physical properties and texture of edible pork skins by dissolving collagen fibers and destroying the structure of collagen and its interaction force. [ABSTRACT FROM AUTHOR]

Protein-protein and protein-water hydrogen bonding interactions play essential roles in the way a protein passes through the transition state during folding or unfolding, but the large number of these interactions in molecular dynamics (MD) simulations makes them difficult to analyze. Here, we introduce a state space representation and associated "rarity" measure to identify and quantify transition state passage (transit) events. Applying this representation to a long MD simulation trajectory that captured multiple folding and unfolding events of the GTT WW domain, a small protein often used as a model for the folding process, we identified three transition categories: Highway (faster), Meander (slower), and Ambiguous (intermediate). We developed data sonification and visualization tools to analyze hydrogen bond dynamics before, during, and after these transition events. By means of these tools, we were able to identify characteristic hydrogen bonding patterns associated with "Highway" versus "Meander" versus "Ambiguous" transitions and to design algorithms that can identify these same folding pathways and critical protein-water interactions directly from the data. Highly cooperative hydrogen bonding can either slow down or speed up transit. Furthermore, an analysis of protein-water hydrogen bond dynamics at the surface of WW domain shows an increase in hydrogen bond lifetime from folded to unfolded conformations with Ambiguous transitions as an outlier. In summary, hydrogen bond dynamics provide a direct window into the heterogeneity of transits, which can vary widely in duration (by a factor of 10) due to a complex energy landscape. [ABSTRACT FROM AUTHOR]

Predicting protein function is crucial for understanding biological life processes, preventing diseases and developing new drug targets. In recent years, methods based on sequence, structure and biological networks for protein function annotation have been extensively researched. Although obtaining a protein in three-dimensional structure through experimental or computational methods enhances the accuracy of function prediction, the sheer volume of proteins sequenced by high-throughput technologies presents a significant challenge. To address this issue, we introduce a deep neural network model DeepSS2GO (Secondary Structure to Gene Ontology). It is a predictor incorporating secondary structure features along with primary sequence and homology information. The algorithm expertly combines the speed of sequence-based information with the accuracy of structure-based features while streamlining the redundant data in primary sequences and bypassing the time-consuming challenges of tertiary structure analysis. The results show that the prediction performance surpasses state-of-the-art algorithms. It has the ability to predict key functions by effectively utilizing secondary structure information, rather than broadly predicting general Gene Ontology terms. Additionally, DeepSS2GO predicts five times faster than advanced algorithms, making it highly applicable to massive sequencing data. The source code and trained models are available at https://github.com/orca233/DeepSS2GO. [ABSTRACT FROM AUTHOR]

The limited solubility and stability of pea proteins hinder their utilization in liquid formulations. In this study, protein glutaminase (PG) was employed to modify pea protein isolates (PPIs) and obtain deamidated PPI with varying degrees of deamidation (DD, 10–25%). The solubility and thermal stability of these deamidated PPI samples were assessed, and a comprehensive analysis, including SDS-PAGE, zeta potential, FTIR, surface hydrophobicity, and intrinsic fluorescence, was conducted to elucidate the mechanism behind the improvement in their functional properties. The results reveal that PG modification greatly enhances the solubility and heat stability of PPI, with the most notable improvements observed at higher DD (>20%). PG modification increases the net charge of PPI, leading to the unfolding and extension of the protein structures, thus exposing more hydrophobic groups. These structural changes are particularly pronounced when DD exceeds 20%. This increased electrostatic repulsion between carboxyl groups would promote protein unfolding, enhancing interactions with water and hindering the aggregation of unfolded protein in the presence of salts at elevated temperatures (supported by high-performance size exclusion chromatography and transmission electron microscopy). Accordingly, PG-mediated deamidation shows promise in enhancing the functional properties of PPI. [ABSTRACT FROM AUTHOR]

This study mainly evaluated the effect of different energies of pulsed light (PL) treatment (100, 200, 300, 400, and 500 J/pulse) on myofibrillar protein (MP) of large yellow croaker during refrigerated storage. The results showed that PL treatment would cause a certain degree of oxidation to the MP of large yellow croaker at the initial stage, which showed that the total sulfhydryl content of the protein decreased, the carbonyl content and the average particle size increased, and the β‐sheet to β‐turn transformation, the tertiary structure of the protein unfolds, and the hydrophobic groups were exposed, causing the reduction of intrinsic fluorescence intensity. However, subsequent storage studies found that PL treatment could slow down the oxidation rate of MP. The decrease rate of total sulfhydryl content and the increase rate of carbonyl content in the 300 J/pulse group were both reduced by about 1.7 times compared with the control group. At the same time, the PL treatment with this intensity could also better protect the secondary structure, tertiary structure, and microstructure of MP. This study provided theoretical basis and reference for analyzing the quality change rule and mechanism of large yellow croaker during refrigerated storage after PL treatment. Studies have shown that PL treatment can reduce the adverse changes of MP in large yellow croaker during cold storage. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of RNA molecules plays a critical role in a wide range of cellular processes encompassing functions from riboswitches to epigenetic regulation. These RNA structures are incredibly dynamic and can indeed be described aptly as an ensemble of structures that shifts in distribution depending on different cellular conditions. Thus, the computational prediction of RNA structure poses a unique challenge, even as computational protein folding has seen great advances. In this review, we focus on a variety of machine learning-based methods that have been developed to predict RNA molecules' secondary structure, as well as more complex tertiary structures. We survey commonly used modeling strategies, and how many are inspired by or incorporate thermodynamic principles. We discuss the shortcomings that various design decisions entail and propose future directions that could build off these methods to yield more robust, accurate RNA structure predictions. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this work we use Raman spectroscopy for protein characterization in the frozen state. We investigate the behavior of frozen therapeutic monoclonal antibody IgG1 formulation upon thawing by Raman spectroscopy. Secondary and tertiary structure of the protein in three different mab formulations in the frozen state are followed through observation of marker bands for α-helix, β-sheet and random coil. We identify the tyrosine intensity ratio I 856 /I 830 as a marker for mab aggregation. Upon fast cooling (40 °C/min) to –80 °C we observe a significant increase of random coil and α –helical structures, while this is not the case for slower cooling (20 °C/min) to –80 °C. Most changes in the protein's secondary structure are observed in the course of thawing in the range up to -20 °C, when passing through the glass transitions and cold-crystallization of the two types of freeze-concentrated solutions formed through macro- and microcryoconcentration. An increase of protein concentration and the addition of mannitol suppress secondary structural changes but do no impact on aggregation. [ABSTRACT FROM AUTHOR]

The limited solubility and stability of pea proteins hinder their utilization in liquid formulations. In this study, protein glutaminase (PG) was employed to modify pea protein isolates (PPIs) and obtain deamidated PPI with varying degrees of deamidation (DD, 10–25%). The solubility and thermal stability of these deamidated PPI samples were assessed, and a comprehensive analysis, including SDS-PAGE, zeta potential, FTIR, surface hydrophobicity, and intrinsic fluorescence, was conducted to elucidate the mechanism behind the improvement in their functional properties. The results reveal that PG modification greatly enhances the solubility and heat stability of PPI, with the most notable improvements observed at higher DD (>20%). PG modification increases the net charge of PPI, leading to the unfolding and extension of the protein structures, thus exposing more hydrophobic groups. These structural changes are particularly pronounced when DD exceeds 20%. This increased electrostatic repulsion between carboxyl groups would promote protein unfolding, enhancing interactions with water and hindering the aggregation of unfolded protein in the presence of salts at elevated temperatures (supported by high-performance size exclusion chromatography and transmission electron microscopy). Accordingly, PG-mediated deamidation shows promise in enhancing the functional properties of PPI.

Emerging nonthermal processing techniques are gaining much attention due to their numerous advantages over traditional methods. Thus, in this study, high-voltage cold atmospheric plasma (HV-CAP) was employed separately and synergetically with ultrasound on lysozyme to investigate its activity and structure. The HV-CAP was operated at 120, 140, and 160 kV for 3 min at a frequency of 120 Hz. A significant difference was observed between the untreated and both treatments in lysozyme activity, with a more pronounced result in the ultrasound-assisted HV-CAP treatment. Both treatments resulted in the modification of the tertiary structure of the lysozyme as lipid oxidation, carbonyl content, UV spectra, intrinsic fluorescence, hydrophobicity, and sulphhydryl groups increased with increased voltage. Particle size distribution of lysozyme shows that noncovalent interactions affected the tertiary structure. The effects of both treatments on the secondary structure of lysozyme were demonstrated by CD and relative molar ellipticity. The results indicated that plasma-generated reactive species and ultrasound-induced cavitation influenced the unfolding of tertiary and secondary structures of the lysozyme. The CD spectra postulated that lysozyme inactivation was closely linked to the α-helix and β-sheet components of the secondary structure. This study presents an original novel knowledge on the modification of activity and structure of lysozyme subjected to ultrasound-assisted HV-CAP treatment that has been missing from the literature. [ABSTRACT FROM AUTHOR]

The effect of the chemical structure of selected phenolic acids on the molecular organization of gliadins was investigated with the application of Fourier Transform Infrared (FTIR) technique, steady-state, and time-resolved fluorescence spectroscopy. Hydroxybenzoic (4-hydroxybenzoic, protocatechuic, vanillic, and syringic) and hydroxycinnamic (coumaric, caffeic, ferulic, sinapic) acids have been used as gliadins modifiers. The results indicated that hydroxybenzoic acids due to their smaller size incorporate into spaces between two polypeptide chains and form a hydrogen bond with them leading to aggregation. Additionally, syringic acids could incorporate into hydrophobic pockets of protein. Whereas hydroxycinnamic acids, due to their higher stiffness and larger size, separated polypeptide chains leading to gliadin disaggregation. These acids did not incorporate into hydrophobic pockets. [ABSTRACT FROM AUTHOR]

In this paper, effects of pectin varying in overall charge (OCH) and local charge density (LCD) on the structure and thermal stability of Lysozyme (lys) are studied. The secondary and local tertiary structures of lys undergo at pH 5.1 a two-step change upon binding with increasing amounts of pectin, in which the peak of fluorescence first increases and shows a blue shift and the amount of helix conformation drops at a pectin/lys mass ratio (q) below or equal to that corresponding to maximal complexation qmax. With increasing amount of pectin, the native like state is recovered at q > qmax. The decrease in helix content depends mainly on the OCH of pectin. Analysis of the thermodenaturation behavior of lys reveals that affinity of pectin with denatured lys is higher than for the native state of lys. At the same OCH of pectin, unfolding of lys is more significant in case of a high LCD (blockwise distribution of charges) of the former. At q = qmax (first stage of binding) the key factors stabilizing lys within its complexes with pectin against thermo-aggregation are a high LCH of pectin and, to a lesser degree, a low OCH. At q > > qmax (second stage of binding), a high LCH of pectin is a key factor inhibiting thermo-aggregation of lys. [ABSTRACT FROM AUTHOR]

Protamine is a promising natural preservative protein, and the extraction process can affect the physicochemical properties and function of the protein. In this study, ultrasound-assisted (UA) acid extraction was used to extract Coregonus peled protamine (CPP). Changes in the extraction, physicochemical and functional properties of CPP with ultrasound time (10, 20, 30, 40 and 50 min) were investigated. The Peleg model was used to describe the extraction kinetics. The results showed that 30 min UA treatment resulted in the highest protein yield (6.81%) (P ≤ 0.05). In addition, the secondary and tertiary structures of CPP were modified, the content of α-helix and β-sheet increased, the content of β-turn decreased, and the fluorescence intensity, solubility and surface morphology were altered. In addition, the degree of oxidation, bacteriostatic and biological activities of CPP were also affected. In conclusion, the study suggests that acidic UA extraction for a moderate time could potentially increase the protein yield of CPP and improve its functional properties. • Ultrasound-assisted acid extraction was effective in extracting Coregonus peled protamine (CPP). • Peleg's model was a suitable model to represent the extraction kinetics of CPP. • UA acid extraction altered the secondary structure, tertiary structure and surface morphology of CPP. • UA extraction caused a certain degree of oxidation during CPP extraction. • Functionalities of CPP were enhanced by ultrasound-assisted (UA) extraction. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This study aimed to determine the transcription profile of NK-lysin gene in native chickens. Moreover, it was targeted toward determining the primary, three-dimensional, and molecular dynamic structures of NK-lysin and granulysin peptides to understand their mode of action and intracellular transduction pathways using in silico analysis. The results revealed that NK-lysin gene in native chickens and Gallus gallus were closely related to those of other avian species. However, there was a low sequence homology when aligned with the mammalian peptides. The coding region of NK-lysin peptide in native chickens encoded 140 amino acids as found in G. gallus with a homology of 98% that declined to 20%, particularly in mammalian species. The results revealed that the NK-lysin in native chickens was closely related to that in avian species at a range of 71–76%. However, it was different from that of other mammalians in terms of nucleotide and amino acid identities. The mRNA transcripts of NK-lysin had high and moderate expression levels in the testis and pancreas, respectively. Nonetheless, the small intestine, kidney, spleen, and liver had a low expression level. The NK-lysin peptides contained more than 50% of the total AA with a nonpolar feature, whereas polar AA constituted up to 30% of AA. The results also indicated that the hydrophilic regions and positively charged amino acids were predominant on the surface of the investigated peptides. The NK-lysin was folded in 4–5 helical units and 3–4 loop structures in their saposin domain. The third helical peptide was long in both avian and bovine species (104–123 residues). However, the fourth helical peptide was short in humans, pigs, and chimpanzees (101–123, 104–123, and 102–124 residues, respectively), with the helical unit residues of 95–97, 96–99, and 96–98, respectively. The obtained results can be helpful in developing novel approaches that could be used as alternatives or adjuncts to the existing means of control.

The aim of this paper was to study the effect of infrared radiation (IR) on the activity and conformation of polyphenol oxidase (PPO) in Acetes chinensis. In this paper, the specific activity of PPO was increased from 21.2 to 643.4 U/mg by a four‐step purification. The results showed that IR treatment had greater effect on the enzyme activity and conformation of PPO than hot air (HA) treatment. After IR treatment at 70°C, the relative enzyme activity of PPO was 9.28%, the surface hydrophobicity index increased by 80.42%, and the content of sulfhydryl group decreased to 96.99% of the control group. The results of circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) showed that the α‐helix of PPO treated by IR decreased and the random coil increased. The intrinsic fluorescence intensity of PPO decreased after IR treatment, indicating that the tertiary structure of PPO was destroyed. Scanning electron microscopy (SEM) results showed that the surface microstructure of PPO after IR treatment became clear and compact. In conclusion, IR treatment can completely destroy the secondary structure and tertiary structure of PPO and cause enzyme inactivation. This study provides a treatment for reducing the activity of PPO from A. chinensis during the production and processing. Practical Application: This study shows that IR treatment has a better inhibitory effect on the activity of PPO than HA treatment. It provides a better treatment method for inactivating the activity of PPO from Acetes chinensis during the production and processing. [ABSTRACT FROM AUTHOR]

This study aimed to determine the transcription profile of NK-lysin gene in native chickens. Moreover, it was targeted toward determining the primary, three-dimensional, and molecular dynamic structures of NK-lysin and granulysin peptides to understand their mode of action and intracellular transduction pathways using in silico analysis. The results revealed that NK-lysin gene in native chickens and Gallus gallus were closely related to those of other avian species. However, there was a low sequence homology when aligned with the mammalian peptides. The coding region of NK-lysin peptide in native chickens encoded 140 amino acids as found in G. gallus with a homology of 98% that declined to 20%, particularly in mammalian species. The results revealed that the NK-lysin in native chickens was closely related to that in avian species at a range of 71–76%. However, it was different from that of other mammalians in terms of nucleotide and amino acid identities. The mRNA transcripts of NK-lysin had high and moderate expression levels in the testis and pancreas, respectively. Nonetheless, the small intestine, kidney, spleen, and liver had a low expression level. The NK-lysin peptides contained more than 50% of the total AA with a nonpolar feature, whereas polar AA constituted up to 30% of AA. The results also indicated that the hydrophilic regions and positively charged amino acids were predominant on the surface of the investigated peptides. The NK-lysin was folded in 4–5 helical units and 3–4 loop structures in their saposin domain. The third helical peptide was long in both avian and bovine species (104–123 residues). However, the fourth helical peptide was short in humans, pigs, and chimpanzees (101–123, 104–123, and 102–124 residues, respectively), with the helical unit residues of 95–97, 96–99, and 96–98, respectively. The obtained results can be helpful in developing novel approaches that could be used as alternatives or adjuncts to the existing means of control. [ABSTRACT FROM AUTHOR]

Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide. [ABSTRACT FROM AUTHOR]

The application of electrospray to treat enzyme solution can produce electrically charged micro- or nanoparticles. Peroxidase is usually regarded as a typical enzyme indicating quality changes. In this study, we evaluated the effect of voltage on peroxidase inactivation under electrospray, and the structural changes caused by increasing voltages were analyzed. The results showed that peroxidase activity decreased with the increasing applied voltages. The relative residual activity of peroxidase reduced to 7.7% by electrospray applied with the voltage of 25 kV. The alteration of particle size and zeta potential indicated that the applied voltage caused aggregation or dissociation of peroxidase, affecting the activity of the peroxidase. From the analysis of spectra for electrospray treatment and control (no voltage), the former had a significant effect (p < 0.05) on the damage of secondary structure (α-helix) stability and has changed the spatial position of tertiary structure. This study provides a sound basis for the application of electrospray in enzyme inactivation and advanced food processing. [ABSTRACT FROM AUTHOR]

The higher-order structure (HOS) of protein therapeutics is a critical quality attribute directly related to their function. Traditionally, the HOS of protein therapeutics has been characterized by methods with low to medium structural resolution such as Fourier-transform infrared (FTIR), circular dichroism (CD), and intrinsic fluorescence spectroscopy, and differential scanning calorimetry (DSC). Recently, high-resolution nuclear magnetic resonance (NMR) methods have emerged as powerful tools for HOS characterization. NMR is a multi-attribute method with unique capabilities to provide information about all the structural levels of proteins in solution. We have in this study compared 1 D 1H Profile NMR with the established biophysical methods for HOS assessments using a set of blended samples of the monoclonal antibodies belonging to the subclasses IgG1 and IgG2. The study shows that Profile NMR can distinguish between most sample combinations (93%), DSC can differentiate 61% of the sample combinations, and near-ultraviolet CD spectroscopy can differentiate 52% of the sample combinations, whereas no significant distinction could be made between any samples using FTIR or intrinsic fluorescence. Our data therefore show that NMR has superior ability to address differences in HOS, a feature that could be directly applicable in comparability and similarity assessments. [ABSTRACT FROM AUTHOR]

• Different freezing treatment temperatures (−5, −20, −40 and −80 ℃) lead to changes in the secondary and tertiary structures as well as the micromorphology of SPI proteins. • Emulsification properties of soy protein isolate by freeze treatment was investigated. • Decrease in soy protein isolate emulsification properties was explained by the protein structure change. • The freezing treatment at −80 ℃ can induce aggregation of soy protein isolate. Different freezing temperatures (−5, −20, −40 and −80 ℃) could change soy protein isolate (SPI) structure and emulsion properties. After freezing at −5 ℃ and −20 ℃, the structure of the SPI loosened, the fluorescence intensity was red shifted, and the proportion of Phe, Tyr and Trp exposed increased. With decreasing temperature, the surface hydrophobicity (H 0 × 100), the number of sulfhydryl groups and the number of disulfide bonds all rose, then fell (−40 ℃), and rose again (−80 ℃). The β-sheet content in the protein secondary structure increased from 32.71% (control) to 50.66% (−40 ℃) and then decreased to 37.05% (−80 ℃), while the β-turn and random coil contents showed the opposite pattern, which also confirmed aggregation. The emulsification performance of SPI after freezing treatment was decreased. The results of this study provide theoretical support for future production of frozen foods with added SPI. [ABSTRACT FROM AUTHOR]

Water soluble chlorophyll-binding proteins (WSCPs) of higher plants differ from most proteins containing chlorophyll or bacteriochlorophyll in that they are soluble in watr and are neither embedded in the lipid membrane nor directly involved in the process of photosynthesis. Chlorophyll molecules in WSCPs ensembles are packed in dimers within the hydrophobic zone of the protein matrix, similar to the structure of a chlorophyll "special pair" in the reaction centers of phototrophs. This fact together with the detected photosensitizing activity of WSCPs makes it possible to consider these proteins as a promising object for modelling the evolutionary prototypes of the photosynthetic apparatus, as well as for developing the artificial solar energy converters. There are two classes of proteins in the WSCP family, class I and class II the representatives of these classes have a weak degree of homology in the primary structure, but a high degree of similarity in the tertiary and quaternary structure. One of the features of class I WSCPs is photoconversion, that is, to change the structure and spectral properties of the chromophore under the action of light. The functions of WSCPs in the plant are thought to be associated with stress protection. [ABSTRACT FROM AUTHOR]

Presenilins (PS1 and PS2) exhibit similar γ-secretase-dependent and −independent functions with subtle variations. In this study, we established a cost-effective process to overexpress and purify full-length human PS2 in sufficient quantities and quality for structural studies. Upon optimization, milligram quantities of homogeneous trimeric hisPS2 were purified, which enabled the preliminary characterization of human hisPS2 zymogen. Far-UV and near-UV CD as well as fluorescence spectroscopy revealed that purified hisPS2 contained the expected secondary structure and was folded into a defined tertiary structure. Thermal stability analysis revealed a T m value of ∼55 °C for secondary structure while cholesterol significantly increased the stability. The low melting temperature of ∼34 °C for the tertiary structure was able to explain the purity and aggregation problems observed during purification. Additionally, the occurrence of calcium ions induced structural changes to different extents for PS2WT and PS2-D263A/D366A was observed, which is consistent with previous studies. [ABSTRACT FROM AUTHOR]

Putative elements of secondary, tertiary and quaternary structure were examined for polylactic acid chains, attempting a parallel with secondary structure elements known from protein biology and also attempting an estimate, based on accurate atomic-level calculations, of interaction energies between polylactic acid chains. Spectroscopic parameters were predicted for all types of structure examined, in an attempt to aid our on-going efforts in synthesis and characterization of polylactic acid variants. [ABSTRACT FROM AUTHOR]

All-atom explicit solvent molecular dynamics was used to study the process of unfolding of hen egg white lysozyme in water and mixtures of water with dimethyl sulfoxide at different compositions. We have determined the kinetic parameters of unfolding at a constant temperature 450 K. For each run, the time of disruption of the tertiary structure of lysozyme t u was defined as the moment when a certain structural criterion computed from the trajectory reaches its critical value. A good agreement is observed between the results obtained using several different criteria. The secondary structure according to DSSP calculations is found to be partially unfolded to the moment of disruption of tertiary structure, but some of its elements keep for a long time after that. The values of t u averaged over ten 30 ns-long trajectories for each solvent composition are shown to decrease very rapidly with addition of dimethyl sulfoxide, and rather small amounts of dimethyl sulfoxide are found to change the pathway of unfolding. In pure water, despite the loss of tertiary contacts and disruption of secondary structure elements, the protein preserves its compact globular state at least over 130 ns of simulation, while even at 5 mol percents of dimethyl sulfoxide it loses its compactness within 30 ns. The proposed methodology is a generally applicable tool to quantify the rate of protein unfolding in simulation studies. [ABSTRACT FROM AUTHOR]

Pre-mRNA molecules can form a variety of structures, and both secondary and tertiary structures have important effects on processing, function and stability of these molecules. The prediction of RNA secondary structure is a challenging problem and various algorithms that use minimum free energy, maximum expected accuracy and comparative evolutionary based methods have been developed to predict secondary structures. However, these tools are not perfect, and this remains an active area of research. The secondary structure of pre-mRNA molecules can have an enhancing or inhibitory effect on pre-mRNA splicing. An example of enhancing structure can be found in a novel class of introns in zebrafish. About 10% of zebrafish genes contain a structured intron that forms a bridging hairpin that enforces correct splice site pairing. Negative examples of splicing include local structures around splice sites that decrease splicing efficiency and potentially cause mis-splicing leading to disease. Splicing mutations are a frequent cause of hereditary disease. The transcripts of disease genes are significantly more structured around the splice sites, and point mutations that increase the local structure often cause splicing disruptions. Post-splicing, RNA secondary structure can also affect the stability of the spliced intron and regulatory RNA interference pathway intermediates, such as pre-microRNAs. Additionally, RNA secondary structure has important roles in the innate immune defense against viruses. Finally, tertiary structure can also play a large role in pre-mRNA splicing. One example is the G-quadruplex structure, which, similar to secondary structure, can either enhance or inhibit splicing through mechanisms such as creating or obscuring RNA binding protein sites. [ABSTRACT FROM AUTHOR]

To determine whether non-thermal effect exist during the process of enzyme inactivation by microwave radiation, the conformation and activity of wheat germ lipase (WGL) treated by microwaves and conventional heating were investigated, respectively. The results indicated that, when the WGL was maintained at 20 °C, its Tyr, Trp residues, the secondary and tertiary structures did not changed, even though the microwave was sustained for 1,800 s with power of higher than 80 W. While, when the temperatures were up to 45 and 60 °C, the activity of WGL decreased 60% and 100%, respectively, and the tertiary structure of WGL was totally changed by microwave at 60 °C. Moreover, the inactivation efficient of microwave radiation was about 10% more than that of conventional heating based on the thermal effects. In conclusion, microwave inactive WGL based on thermal effects rather than non-thermal effect, and was more effective than conventional heating. Practical Applications Microwave has been widely used in food thawing, drying, sterilization, enzyme inactivation and et al. In cereal industry, microwave is usually used to inactivate wheat germ lipase (WGL), which plays an important role in the storage of wheat products. However, because the effects of microwave was strongly interferes by the possible non-thermal effect and cannot be separated easily, the potential inactivation mechanism of microwave on enzyme was not clear enough to guide the inactivation process of lipase by microwave. In this study, the effects of microwave and conventional heating on the stability of WGL were investigated to detect the possibility of non-thermal effects during the inactivation process of lipase by microwave. The results make sure that, the microwave inactivated WGL based on thermal effect, rather than non-thermal effect. Thus, we can focus on the factors that influence the heating efficiency to improve the effect of inactivation of WGL or other lipase by microwave. [ABSTRACT FROM AUTHOR]

The evaluation of the structural stability and bioactivity of monoclonal antibodies (mAb) is a crucial step in the development of mAb therapeutic based products, since immunogenicity needs to be avoided. In the present work, a study was carried out to understand the changes on the structure and bioactivity of mAbs induced by different pH and temperature values. Structural changes of bevacizumab were monitored using fluorescence spectroscopy, circular dichroism (CD) and Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The secondary and tertiary structural content was monitored at six different pH values and at room temperature, upon heating up to 85 °C and upon cooling down to 20 °C. Furthermore, the temperature induced conformational changes were continuously monitored from 20 °C to 85 °C using fluorescence spectroscopy and circular dichroism, allowing to monitor the melting temperature of the protein at different pH values. The results showed that the thermal denaturation of bevacizumab was irreversible at all pH value. The conformational changes induced by pH were higher at extreme pH values (5, 9 and 10) than neutral pH. Thermal stability studies showed that pH 6 was the pH that confer bevacizumab the highest structural stability. These studies were confirmed by in vitro studies, where bevacizumab's bioactivity was measured by cell viability/proliferation at all pH values at room temperature, and it was found a higher bioactivity for pH 6. Biophysical and biological studies were correlated in order to understand the importance of the modifications in bevacizumab structural content on its bioactivity. However, a decrease in bevacizumab's bioactivity was observed for pH 8, 9 and 10. Overall, this work demonstrated the usefulness of the spectroscopy techniques for estimating the stability of therapeutic mAb during formulation development. [ABSTRACT FROM AUTHOR]

In the gas phase, protein ions can adopt a broad range of structures, which have been investigated extensively in the past using ion mobility-mass spectrometry (IM-MS)-based methods. Compact ions with low number of charges undergo a Coulomb-driven transition to partially folded species when the charge increases, and finally form extended structures with presumably little or no defined structure when the charge state is high. However, with respect to the secondary structure, IM-MS methods are essentially blind. Infrared (IR) spectroscopy, on the other hand, is sensitive to such structural details and there is increasing evidence that helices as well as β-sheet-like structures can exist in the gas phase, especially for ions in low charge states. Very recently, we showed that also the fully extended form of highly charged protein ions can adopt a distinct type of secondary structure that features a characteristic C-type hydrogen bond pattern. Here we use a combination of IM-MS and IR spectroscopy to further investigate the influence of the initial, native conformation on the formation of these structures. Our results indicate that when intramolecular Coulomb-repulsion is large enough to overcome the stabilization energies of the genuine secondary structure, all proteins, regardless of their sequence or native conformation, form C-type hydrogen bond structures. Furthermore, our results suggest that in highly charged proteins the positioning of charges along the sequence is only marginally influenced by the basicity of individual residues. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance spectroscopy is a powerful technique that helps to determine the structures of biomolecules. Since the 1980s when nuclear magnetic resonance began to be applied in the determination of nucleic acid structures, it has been used to study aptamer structures and aptamer-target interactions. Nuclear magnetic resonance spectroscopy has revealed that aptamers adopt characteristic conformations and bind specifically to their targets. It is not easy to determine the structure of aptamers by nuclear magnetic resonance, especially in the case of aptamer-large target molecule complexes. However, nuclear magnetic resonance provides useful information about aptamers, even when their structure cannot be determined. This review includes the studies in which nuclear magnetic resonance spectroscopy was employed recently to analyse aptamers in several ways in addition to analysing them for structure determination. [ABSTRACT FROM AUTHOR]

• Ovotransferrin was phosphorylated by sodium tripolyphosphate under wet-heating. • The interaction site between ovotransferrin and STPP was studied by LC-MS/MS. • The thermal tolerance of ovotransferrin against pasteurization depended on the phosphorylation-induced conformational. • Phosphorylation improved thermal tolerance of ovotransferrin against pasteurization. • Phosphorylation at pH 8 is a practical method to improve thermostability. Ovotransferrin is the most heat-sensitive protein in egg white which alters the processing suitability. To improve thermostability, phosphorylation modification was performed under different pH values (5.0, 6.0, 7.0, 8.0, and 9.0), and the reasons for which were explored via physicochemical changes in this study. SEM and particle size results showed that phosphorylation diminished the average particle diameter and the number of attached particles. CD results revealed that the α-helix content of phosphorylated ovotransferrin increased and the random coil decreased significantly (P < 0.05). Tertiary structure, surface hydrophobicity and ζ-potential analyses showed that phosphorylation made more hydrophobic amino acids buried inside ovotransferrin. These changes might be because most of the phosphorylation occurred in α-helix and β- sheet to promote the thermal tolerance of phosphorylated ovotransferrin by improving its structure orderliness and decreasing surface hydrophobicity. The results indicated that phosphorylation was a practical method to enhance the thermal stability of heat-sensitive ovotransferrin. [ABSTRACT FROM AUTHOR]

Characterization of the higher order structure (HOS) of protein-based biopharmaceutical products is an important aspect of their development. Opinions vary about how best to apply biophysical methods, in which contexts to use these methods, and how to use the resulting data to make technical decisions as drug candidates are commercialized [Gabrielson JP, Weiss WF IV. J Pharm Sci . 2015;104(4):1240-1245]. The aim of this commentary is to provide guidance for the development and implementation of a robust and comprehensive HOS characterization strategy. We first consider important concepts involved in developing a strategy that is appropriately suited to a particular biologic, and then discuss ways industry can partner with academia, technology companies, government laboratories, and regulatory agencies to improve the consistency with which HOS characterization is applied across the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

The free energy of an RNA fold is a combination of favorable base pairing and stacking interactions competing with entropic costs of forming loops. Here we show how loop entropy, surprisingly, can promote tertiary order. A general formula for the free energy of forming multibranch and other RNA loops is derived with a polymer-physics based theory. We also derive a formula for the free energy of coaxial stacking in the context of a loop. Simulations support the analytic formulas. The effects of stacking of unpaired bases are also studied with simulations.

[Display omitted] • Ultrasound shortened detoxification time by 75 % & 33.3 % compared to thermal, soaking + thermal treatment. • Higher weight and protein loss was seen in soaking + thermal treatment. • Detoxification results in fading of the band ranging from 45 to 55 kDa. • Detoxified Meghalayan cherry kernel protein isolates were thermally stable. Valorizing food wastes and by-products can improve economic and environmental sustainability of the food production chain. In this regard, Meghalayan cherry kernels are a good source of proteins, but the presence of toxic compounds like amygdalin, makes them underutilized. Therefore, the present study was focused on detoxifying Meghalayan cherry kernel using thermal, soaking and ultrasound treatments and studying their impact on the structural and thermal characteristics of protein isolate. The results showed that all three treatments significantly reduced amygdalin content, with complete detoxification achieved after 30 and 60 min at 70 °C and 60 °C, respectively, in ultrasound, and after 90 and 120 min at 70 °C and 60 °C, respectively, in soaking + thermal treatment. The detoxification treatments significantly affected the protein content and weight-loss of Meghalayan cherry kernel. Fluorescence spectroscopy and FTIR showed alterations in Meghalayan cherry kernel protein isolate (MCKPI) secondary and tertiary structure. The fluorescence intensity was observed at 340 nm for native and detoxified protein isolate, and the lowest peak for MCKPI-US depicts conformational changes. The fading of bands in SDS-PAGE confirms structural changes due to thermal and sonication effects. SEM images demonstrated that more cracks and porous structures were seen in treated MCKPI than native MCKPI. Detoxification treatment increased thermal stability, resulting in lesser weight loss and higher denaturation temperature than native MCKPI. In this study, ultrasound treatment demonstrated the most pronounced effects on the detoxification of MCKPI and its thermal and structural properties, suggesting that Meghalayan cherry kernel is one of the most promising substrates for zero-waste bioprocess development. [ABSTRACT FROM AUTHOR]

• RF heating inactivates HRP at 70 °C and 90 °C. • RF heating changes the secondary and tertiary structures of HRP. • Conformational deterioration is responsible for the enzyme inactivation. • Conformational changes of enzyme vary with temperature and electrode gap. The effects of radio frequency (RF) heating on horseradish peroxidase (HRP) activity and its structure were investigated in this paper. The HRP was heated to 50 °C, 70 °C and 90 °C at different electrode gaps (100, 110 and 120 mm). The relative enzyme activity was 105.33 %–113.73 % at 50 °C, 91.11 %–93.05 % at 70 °C and 47.05 %–68.17 % at 90 °C. Ultraviolet–visible, circular dichroism and fluorescence spectra were used to monitor the variation in secondary and tertiary structure. The results showed that RF heating at the electrode gaps of 120 mm contributed to more severe enzyme inactivation and conformational destruction, which can be explained by the changes in Soret band, secondary structure content and tryptophan fluorescence intensity. This study revealed that enzyme inactivation by RF heating was associated with loss of helical structure, unfolding of enzyme protein and ejection of heme group. [ABSTRACT FROM AUTHOR]

mRNA is a nexus entity between DNA and translating ribosomes. Recent developments in deep sequencing technologies coupled with structural probing have revealed new insights beyond the classic role of mRNA and place it more centrally as a direct effector of a variety of processes, including translation, cellular localization, and mRNA degradation. Here, we highlight emerging approaches to probe mRNA secondary structure on a global transcriptome-wide level and compare their potential and resolution. Combined approaches deliver a richer and more complex picture. While our understanding on the effect of secondary structure for various cellular processes is quite advanced, the next challenge is to unravel more complex mRNA architectures and tertiary interactions. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs), which form a diverse class of RNAs, remain the least understood type of noncoding RNAs in terms of their nature and identification. Emerging evidence has revealed that a small number of newly discovered lncRNAs perform important and complex biological functions such as dosage compensation, chromatin regulation, genomic imprinting, and nuclear organization. However, understanding the wide range of functions of lncRNAs related to various processes of cellular networks remains a great experimental challenge. Structural versatility is critical for RNAs to perform various functions and provides new insights into probing the functions of lncRNAs. In recent years, the computational method of RNA structure prediction has been developed to analyze the structure of lncRNAs. This novel methodology has provided basic but indispensable information for the rapid, large-scale and in-depth research of lncRNAs. This review focuses on mainstream RNA structure prediction methods at the secondary and tertiary levels to offer an additional approach to investigating the functions of lncRNAs. [ABSTRACT FROM AUTHOR]

Mechanisms of folding of soluble proteins. The function of a protein is carried out by its three-dimensional structure. The rules for translation of the information encoded by DNAinto an amino acid sequence are well known. On the other hand, the rules governing the folding of an amino acid sequence into an univocal three-dimensional structure are still unknown. The speed at which this phenomenon takes place makes the experimental investigations difficult using biophysical methods. Various theoretical approaches have been set up with more or less success. None, nowadays, describes in a right way, the mechanisms involved. We present here the various hypotheses found in the literature about the early steps of the folding of soluble proteins.

Characterization of the higher order structure ( HOS) of biological products has been growing in importance in recent years. Scientists in the biopharmaceutical industry, academic researchers, and regulators are all increasingly aware of the critical role that HOS plays in maintaining the stability and intended biological function of biopharmaceutical products. We organized a consortium of scientists and researchers from industry and academic institutions to address how HOS data can be used most effectively to drive decisions during product development. In this commentary, we introduce the purpose, objectives, and scope of the consortium and then provide some brief points to consider in the context of characterizing HOS of biopharmaceutical products. Scientific advances in HOS analysis, as well as continued dialogue among academia, industry, and regulatory agencies will ensure that appropriate methodologies are used to inform technical decision-making during biopharmaceutical development. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1240-1245, 2015 [ABSTRACT FROM AUTHOR]

The two major storage proteins identified in Brassica napus (canola) were isolated and studied for their molecular composition, structural characteristics and the responses of structural features to the changes in pH and temperature. Cruciferin, a complex of six monomers, has a predominantly β-sheet-containing secondary structure. This protein showed low pH unstable tertiary structure, and distinctly different solubility behaviour with pH when intact in the seed cellular matrix. Cruciferin structure unfolds at pH 3 even at ambient temperature. Temperature-induced structure unfolding was observed above the maximum denaturation temperature of cruciferin. Napin was soluble in a wider pH range than cruciferin and has α-helices dominating secondary structure. Structural features of napin showed less sensitivity to the changes in medium pH and temperature. The surface hydrophobicity (S0) and intrinsic fluorescence of tryptophan residue appear to be good indicators of cruciferin unfolding, however they were not the best to demonstrate structural changes of napin. These two storage proteins of B. napus have distinct molecular characteristics, therefore properties and functionalities they provide are contrasting rather than complementary.

Halophilic enzymes have been manifested for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation in presence of high temperature, pH, presence of organic solvents and chaotropic agents. The present study aims at understanding the stability and activity of a halophilic Bacillus sp. EMB9 protease in organic solvents. The protease was uniquely stable in polar solvents. A clear correlation was evident between the protease function and conformational transitions, validated by CD and fluorescence spectral studies. The study affirms that preservation of protein structure, possibly due to charge screening of the protein surface by Ca and Na ions provides stability against organic solvents and averts denaturation. Salt was also found to exert a protective effect on dialyzed protease against chaotropism of solvents. Presence of 1 % (w/v) NaCl restored the activity in the dialyzed protease and prevented denaturation in methanol, toluene and n-decane. The work will have further implication on discerning protein folding in saline as well as non-aqueous environments. [ABSTRACT FROM AUTHOR]

Immobilization procedures are a fundamental step in the technological use of enzymes. Among the different immobilization procedures sol-gel technique is widely recognised as a valuable approach to obtain very high quality catalytic supports. In this paper different optical techniques have been used and compared to investigate structural and dynamic properties of glucose oxidase (GOD) prior and after sol-gel immobilization process. In particular, using Fourier Transform infrared micro-spectroscopy and time-resolved fluorescence the secondary structure of GOD and the flavin adenine dinucleotide (FAD) conformational changes have been respectively investigated. Infrared spectroscopy measurements have confirmed that enzymatic activity is preserved and a predominant β-sheet subcomponent is retained by immobilized GOD. By time-resolved FAD fluorescence a three-exponential decaying behaviour has been observed for both free and immobilized enzymes with three different lifetimes, each being characteristic of a peculiar conformational state of the FAD structure. The comparison between lifetime values for free and immobilized GOD has not shown significant differences, while the fractional steady-state intensities of the single exponential components have been changed by immobilization procedure. All results reported and discussed in this paper have confirmed once again the efficacy of the adopted sol-gel immobilization procedure for enzymes and proteins. In addition, the joint use of different optical spectroscopic techniques has shown to be a very valuable tool for getting a better insight into structural and dynamic properties of immobilized enzymes. [ABSTRACT FROM AUTHOR]