Rearrangement Of Secondary Structure Research Articles

Impact of NaCl perturbation on physicochemical and structural properties of preheat–treated egg white protein modulating foaming property

This study aimed to investigate the mechanism of NaCl perturbed preheat–treated egg white proteins' (EWPs) physicochemical and structural properties to modulate the foaming property (FP). The results revealed that NaCl regulated the salinolysis (5 mM) – salt precipitation (50 mM) – gradual or complete coverage with hydrated Na+ of the hydration layer (100–300 mM) – enhanced Cl− hydration repulsion (500 mM) of EWP, showing a gradual decrease in aggregates particle size, and reversibility of structural freedom, including moleculer flexibility and surface hydrophobicity. Whereas preheating temperature affected the secondary structure rearrangement and tertiary conformation exposure, and excessive temperature reduced foaming capacity while enhanced foam stability, with a tight correlation between NaCl–mediated EWPs’ FP and the extent of Na+ covering the hydration layer. The findings provide a theoretical basis for processing factors to modulate the protein hydration layer to influence the functional properties.

Exploring the mechanism and inhibitory effect of robinin on xanthine oxidase using multi-spectroscopy and molecular dynamics simulation methods

Xanthine oxidase (XOD) is a key enzyme in purine metabolism that is tightly associated with hyperuricemia (HUA). Natural flavonoids can inhibit XOD activity. In this study, the inhibitory activity and mechanism of the novel flavonoid robinin on XOD were analyzed using enzyme inhibition kinetics, multispectral techniques, molecular docking, and molecular dynamics (MD) simulations. Robinin reversibly inhibited the activity of XOD in a mixed competitive manner, with an IC50 value of 31.79 ± 0.15 μg/mL. Moreover, robinin spontaneously binds to a specific site on XOD and effectively quenches its intrinsic fluorescence via a single static quenching mechanism. The combination of XOD and robinin induced secondary structure rearrangement and conformational changes in XOD and prevented the entry of substrates, thus producing a strong inhibitory effect. Molecular docking and MD simulation revealed that robinin mainly binds to XOD through hydrogen bonding and hydrophobic interactions to form more stable complexes with a binding energy of −7.4 kcal/mol. Overall, this study established a theoretical reference for robinin as a promising natural XOD inhibitor for relieving HUA.

Allergenicity and Conformational Diversity of Allergens

Allergens are substances that cause abnormal immune responses and can originate from various sources. IgE-mediated allergies are one of the most common and severe types of allergies, affecting more than 20% of the population in Western countries. Allergens can be subdivided into a limited number of families based on their structure, but this does not necessarily indicate the origin or the route of administration of the allergen, nor is the molecular basis of allergenicity clearly understood. This review examines how understanding the allergenicity of proteins involves their structural characterization and elucidates the study of conformational diversity by nuclear magnetic resonance spectroscopy. This article also discusses allergen cross-reactivity and the mechanisms by which IgE antibodies recognize and bind to allergens based on their conformational and linear epitopes. In addition, we outline how the pH, the proteolytic susceptibility and the endosomal degradation affect the outcome of allergic reactions, and how this is correlated with conformational changes and secondary structure rearrangement events. We want to emphasize the importance of considering structural diversity and dynamics, proteolytic susceptibility and pH-dependent factors to fully comprehend allergenicity.

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.

RNA excited states by NMR and their impact on function.

Many functions of RNA depend on rearrangements in secondary structure that are triggered by external factors, such as protein or small molecule binding. These transitions can feature on one hand localized structural changes in base-pairs or can be presented by a change in chemical identity of e.g. a nucleo-base tautomer [1].

Demineralisation and recovery of whey proteins from commercial full-fat salty Cheddar whey using size-exclusion chromatography

Extraction of the whey proteins from salty whey would add value to milk waste streams. Size-exclusion chromatography (SEC) is a non-invasive method to screen proteins, based on molecular size. Here, compositional analysis of commercial salty whey showed total solids (TS), ash, and protein contents were 12.3 ± 0.1 %, 8.92 ± 0.01 %, and 0.9 ± 0.1 %, respectively. Prior to SEC, salty whey pH (5.5) was adjusted to pH 4.6, 7.0 and 9.0. This adjustment was associated with protein losses through aggregation. Following SEC, a substantial amount of whey protein (82–100 %) and salt (>99 %) was recovered/removed. SEC samples exhibited similar ranges of conductivity (1.8 × 102 – 2.4 × 102 μS.cm−1) and NaCl (0.005–0.009 %). SEC protein and salt fractions were characterised for particle size, and zeta potential, and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy. Generally, demineralisation resulted in decreased conductivity and particle size, whereas zeta potential was pH-dependent. FTIR analysis showed modifications and/or rearrangement of whey protein secondary structure after SEC. This work suggests that SEC can be used to demineralise and recover WPs from salty whey.

Binding of heparan sulfate to human cystatin C modulates inhibition of cathepsin L: Putative consequences in mucopolysaccharidosis

Mucopolysaccharidoses (MPS) are a group of rare lysosomal storage diseases characterized by glycosaminoglycan (GAG) accumulation causing progressive multi-organs dysfunction and ultimately severe cardio-respiratory damages. Human cystatin C (hCC), a potent inhibitor of cysteine cathepsins, plays an important role in respiratory diseases. However, its regulation remained unknown in MPS. Herein, elevated hCC levels were measured in respiratory specimens from MPS-I, -II, and -III patients and were significantly correlated with severe respiratory symptoms (rs = 0.7173). Heparan sulfate (HS), a prominent GAG, dampened its inhibitory activity toward cathepsin L in a dose-dependent manner. HS and HS-oligosaccharides bound tightly hCC, in combination with a secondary structure rearrangement. Molecular modeling studies identified three HS binding regions in hCC, including the N-terminus, which is crucial in the inhibition of cathepsins. Impairment of inhibitory potential of hCC may reflect abnormal regulation of proteolytic activity of cathepsin L in lung, ultimately contributing to the severity of MPS.

Improvement of the solubility and emulsification of rice protein isolate by the pH shift treatment

SummaryIn the current research, we have adopted a pH shift treatment method that can significantly improve the solubility and emulsification of rice protein isolate (RPI) under neutral conditions. Our results showed that the RPI reached a quasi‐equilibrium state at 1 h after alkali shift treatment, and the solubility and emulsifying properties of RPI were significantly improved compared with the control. In addition, the particle size of RPI decreased considerably under the alkali shift treatment. The results of electron microscopy showed that the RPI aggregates were depolymerised and the particle size distribution was more uniform. The pH shift treatment induced the rearrangement of protein secondary structure, and the exposure of hydrophobic groups increase the surface hydrophobicity of RPI. The change in endogenous fluorescence spectrum, UV and the disulfide bond content all showed that the conformation of RPI changed significantly through pH shift treatment. In addition, it was found that the RPI treated under pH 10–11 shift possessed the best property to stabilise the emulsion. This study showed that under the condition of pH10 and pH11 shift treatment, the structural and functional properties of RPI were vastly improved, and the application of RPI in food was greatly ameliorated.

Aggregation and conformational change of mushroom (Agaricus bisporus) polyphenol oxidase subjected to atmospheric cold plasma treatment

Atmospheric cold plasma (ACP) is a novel nonthermal technology with potential applications in maintaining and improving food quality. The effect of ACP on the activity and structure of mushroom (Agaricus bisporus) polyphenol oxidase (PPO) was evaluated. Results demonstrated that the dielectric barrier discharge (DBD) based plasma technology could inactivate PPO (up to 69%) at 50 kV with the increased concentrations of H2O2 and NOx. An obvious enhancement of surface hydrophobicity was observed, whereas a gradual reduction of total sulfhydryl content was recorded with the increasing exposure time. Data from circular dichroism, atomic force microscopy, particle size distribution and fluorescence spectra displayed the rearrangement of secondary structure and disruption of the tertiary structure. Red shifts of fluorescence spectra showed positive correlations with the inactivation rate of PPO. Therefore, ACP treatment could be served as an alternative approach to inactivate undesirable enzymes to minimize the loss of food nutrition and quality.

Liquid metal-tailored gluten network for protein-based e-skin

Designing electronic skin (e-skin) with proteins is a critical way to endow e-skin with biocompatibility, but engineering protein structures to achieve controllable mechanical properties and self-healing ability remains a challenge. Here, we develop a hybrid gluten network through the incorporation of a eutectic gallium indium alloy (EGaIn) to design a self-healable e-skin with improved mechanical properties. The intrinsic reversible disulfide bond/sulfhydryl group reconfiguration of gluten networks is explored as a driving force to introduce EGaIn as a chemical cross-linker, thus inducing secondary structure rearrangement of gluten to form additional β-sheets as physical cross-linkers. Remarkably, the obtained gluten-based material is self-healing, achieves synthetic material-like stretchability (>1600%) and possesses the ability to promote skin cell proliferation. The final e-skin is biocompatible and biodegradable and can sense strain changes from human motions of different scales. The protein network microregulation method paves the way for future skin-like protein-based e-skin.

Structural basis of adenylyl cyclase 9 activation

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

Inhibition of porphyra polysaccharide on xanthine oxidase activity and its inhibition mechanism

Xanthine oxidase (XO) is a purine catabolic enzyme related to hyperuricemia and gout. Porphyra polysaccharide (PP) is a kind of sulfated polysaccharide with potent biological activity. Herein, the interaction mechanism between PP and XO was studied by enzyme kinetics and multi-spectroscopy methods for the first time. Inhibition kinetics assay showed that PP reversibly inhibited XO activity in a mixed competitive manner with an IC50 of 10.53 ± 0.69 mg/ml. Fluorescence titration studies and thermodynamic parameter calculations revealed that PP could spontaneously bind to XO through hydrophobic interactions, with a class of binding site. Circular dichroism analysis demonstrated that PP induced secondary structure rearrangement and conformational change of XO. Molecular docking further revealed that PP inserted into the hydrophobic cavity of XO, occupying the catalytic center, leading to the inhibition of XO activity. This study may provide new insights into the inhibitory mechanism of PP as a promising XO inhibitor.

Effects of ionic strength and (−)-epigallocatechin gallate on physicochemical characteristics of soybean 11S and 7S proteins

Polyphenols can interact with proteins to improve their physicochemical and functional properties. This study was aimed to determine the interactions of EGCG with 11S and 7S at different NaCl concentrations. Results of turbidity indicated that the binding affinity of EGCG with 11S and 7S were strongest at 0.3 M NaCl concentration. Zeta potential results confirmed that 11S-EGCG and 7S-EGCG complexes had stronger stability in salt solutions, but the stability decreased with the increase of NaCl concentration to 0.6 M. Fourier transform infrared spectra showed that NaCl could affect hydrogen bonds between soybean proteins and EGCG. Based on Raman spectroscopy, the microenvironment of the tryptophan and tyrosine residues and intermolecular hydrogen of 11S, 7S, 11S-EGCG and 7S-EGCG were influenced by NaCl concentration. The NaCl induced a decrease in α-helix and an increase in β-sheet of 11S protein, while the secondary structure of 7S was not sensitive to NaCl concentration. The bindings of EGCG to 11S and 7S resulted in the secondary structure rearrangement of two proteins. The NaCl addition led the transformation of the α-helix to the β-sheet in 11S-EGCG and 7S-EGCG complexes. The 11S-EGCG and 7S-EGCG complexes had more compact microstructure with a smooth surface than 11S and 7S proteins, while the microstructure of the complexes became undesirable with the increase of NaCl concentration. These results will lay the foundation for the development 11S-EGCG and 7 S-EGCG complexes as a new food material in the food industry.

Conformational Structural Alteration of NCS1 Protein Through Pb2+and Mn2+ Association

NCS1 (Frequenin) is a member of neuronal calcium sensor (NCS) family. This globular protein contains four EF-hand motifs and an amino-terminal myristylation sequence. Pb2+has been widely viewed as a health concern for its anthropogenic toxicity. It has been observed that Pb2+ modulates interactions between calcium-binding protein calmodulin (CaM) and its binding partners through binding in the EF-hands of CaM and it may play a significant role in Pb2+-induced neurotoxicity. Mn is a highly abundant metal in nature which has been found in human brain and associated with the several diseases like multiple sclerosis, Alzheimer's and Parkinson's disease and is considered as potential toxic element. Previous studies from our group showed Pb2+ and Cd2+ binding to DREAM protein that can contribute to Pb2+/Cd2+ induced neurotoxicity. Here we showed that Pb2+ binds to EF-hands in apo-NCS1with a high affinity (Kd = 7.0±1.5 nM) whereas Cd2+ did not show any binding with NCS1. For the first time we investigated the Mn2+ association to NCS1 protein. This cation binds with an apparent equilibrium dissociation constant of 340±67 µM. Pb2+ and Mn2+binding trigger secondary and tertiary structural rearrangements of NCS1 that are analogous to those observed for Ca2+ association. In addition, Pb2+ as well as Mn2+ promote NCS1 interaction with hydrophobic molecule 1,8-ANS. Interestingly,Ca2+NCS1:1,8-ANS complex exhibits a co-operative binding of Pb2+ with Kd = 103±23 µM whereas Mn2+ showed apparent Kd value 924±185 µM with Ca2+NCS1:1,8-ANS complex. These results suggest allosteric interactions between the 1,8-ANS binding site and Pb2+ binding EF-hands. Data provided here points towards ability of EF-hands in NCS-1 to interact with various metals and a possible role of NCS1 in Pb2+/Mn2+ neurotoxicity.

Comprehensive Sequence-to-Function Mapping of Ligand-Dependent RNA Catalysis

Massively-parallel, quantitative measurements of biochemical activity have the potential to revolutionize our understanding of biomolecular sequence-structure-function relationships. We report the development of a novel RNA array assay which enables such measurements to be performed on self-cleaving ribozymes, allowing determination of real-time cleavage kinetics of tens of thousands of sequence variants in a single experiment. We applied this methodology to the glmS ribozyme riboswitch, which performs self-cleavage in a ligand-dependent manner in order to regulate bacterial cell wall synthesis. We determined the cleavage rates of all single and double mutants of this RNA under a range of regulatory ligand concentrations, enabling the calculation of kcat and KM for active variants. This trove of quantitative kinetic parameters across the mutational landscape enabled a number of further analyses. Comparing single-mutant activities with naturally-occurring sequence variation at each position, we found that conservation is driven by cleavage rate, and not by ligand binding affinity. Comprehensive double-mutant measurements permitted a global analysis of rescue interactions, which revealed the functional relevance of several known tertiary contacts, as well as providing evidence for a number of compensatory secondary and tertiary structural rearrangements. Double-mutant kinetics and rescue interactions also provided a combined structural and functional mapping of the ribozyme which successfully identified the full secondary structure at nucleotide resolution, while additionally revealing relative structural and catalytic contributions of individual residues. We believe that our approach will be broadly applicable and extensible, enabling a deeper understanding of sequence-structure-function relationships across a variety of functional RNAs.

Novel Bovine Serum Albumin Protein Backbone Reassembly Study: Strongly Twisted β-Sheet Structure Promotion upon Interaction with GO-PAMAM.

This study investigates the formation of a graphene oxide-polyamidoamine dendrimer complex (GO-PAMAM) and its association and interaction with bovine serum albumin (BSA). Fourier-transform infrared spectrometry and X-ray photoelectron spectrometry indicated the formation of covalent linkage between the GO surface and PAMAM with 7.22% nitrogen content in the GO-PAMAM sample, and various interactions between BSA and GO-PAMAM, including π-π* interactions at 291.5 eV for the binding energy value. Thermogravimetric analysis highlighted the increasing thermal stability throughout the modification process, from 151 to 192 °C for the 10% weight loss temperature. Raman spectrometry and X-ray diffraction analysis were used in order to examine the complexes’ assembly, showing a prominent (0 0 2) lattice in GO-PAMAM. Dynamic light scattering tests proved the formation of stable graphenic and graphenic-protein aggregates. The secondary structure rearrangement of BSA after interaction with GO-PAMAM was investigated using circular dichroism spectroscopy. We have observed a shift from 10.9% β-sheet composition in native BSA to 64.9% β-sheet composition after the interaction with GO-PAMAM. This interaction promoted the rearrangement of the protein backbone, leading to strongly twisted β-sheet secondary structure architecture.

Effects of catechins on the polymerisation behaviour, conformation and viscoelasticity of wheat gluten

SummaryThe effects of catechins on the structural properties of wheat gluten were investigated in terms of their polymerisation behaviour, conformation and viscoelasticity. Depolymerisation of glutenin macropolymers was analysed by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) of gluten with catechins, revealing major variation in sulfhydryl (SH) content. Compared with controls, the SH content of catechin‐enhanced gluten was significantly increased due to breakage of disulphide (SS) bonds. Reversed‐phase HPLC revealed that catechins had minimal effect on subunit distribution of gliadin and glutenin. Fourier transform infrared (FTIR) spectroscopy showed that catechins induced the transformation of α‐helices to β‐sheets, β‐turns and antiparallel β‐sheets. Rearrangement of secondary structure when supplemented with catechins might be a consequence of altered non‐covalent interactions. Catechins increased tan δ, indicating enhanced gluten fluid properties. These results imply that catechins might weaken the network structure of gluten and change the rheological properties by inhibiting SS cross‐linkage formation and influencing non‐covalent bonds.

Marine Structural Protein Stability Induced by Hofmeister Salt Annealing and Enzymatic Cross-Linking.

The Humboldt squid is one of the fiercest marine predators thanks in part to its sucker ring teeth that are biopolymer blends of a protein isoform family called suckerin with compression strength that rivals silkworm silk. Here, we focus on the popular suckerin-12 isoform to understand what makes the secondary structure of this biopolymer different in water and the potential role of diverse physical and chemical cross-linkings. By choosing a salt post-treatment, in accordance with the Hofmeister series, we achieved film stability with salt annealing that is comparable to chemical cross-links. By correlating the film morphology with the protein secondary structure changes, suckerin-12 films were shown to contract upon treatment with kosmotropic salts and exhibited increased stability in water. These changes are related to the rearrangement of suckerin-12 secondary structure from random coils and helices to β-sheets. Overall, understanding secondary structure changes caused by aqueous and ionic environments can be instructive for the tuning of the suckerin film sclerotization, its conversion to a tough biological material, and to ultimately produce the natural squid sucker ring teeth.

Improvement of postharvest quality, enzymes activity and polyphenoloxidase structure of postharvest Agaricus bisporus in response to high voltage electric field

Abstract High voltage electric field (HVEF) is an innovative technology used in the non-thermal food processing. To explore effects of HVEF on the postharvest quality and its possible mechanisms of Agaricus. bispourus (mushroom), Agaricus. bispourus were stored in the HVEF at 4 °C for 12 days. The results revealed that HVEF could reduce the loss of firmness and maintain the whiteness index for A. bisporus samples after 12 days of storage. Furthermore, HVEF treatment significantly prevented the accumulation of malondialdehyde (MDA), delayed loss of total phenolic, enhanced the superoxide dismutase (SOD) and catalase (CAT) activity, and had the better microstructure compared with that in the control group. In addition, HVEF treatment induced the rearrangement of secondary structure and irreversibly disrupted the tertiary structure of the polyphenol oxidase (PPO) to bury the active site, resulting in inactivation of PPO. These results indicated that HVEF treatment is a potential approach to prolong the shelf life of A. bisporus, maintaining its postharvest quality and consumer acceptance during storage.

Humanin induces conformational changes in the apoptosis regulator BAX and sequesters it into fibers, preventing mitochondrial outer-membrane permeabilization

The mitochondrial, or intrinsic, apoptosis pathway is regulated mainly by members of the B-cell lymphoma 2 (BCL-2) protein family. BCL-2-associated X apoptosis regulator (BAX) plays a pivotal role in the initiation of mitochondria-mediated apoptosis as one of the factors causing mitochondrial outer-membrane permeabilization (MOMP). Of current interest are endogenous BAX ligands that inhibit its MOMP activity. Mitochondrial-derived peptides (MDPs) are a recently identified class of mitochondrial retrograde signaling molecules and are reported to be potent apoptosis inhibitors. Among them, humanin (HN) has been shown to suppress apoptosis by inhibiting BAX translocation to the mitochondrial outer membrane, but the molecular mechanism of this interaction is unknown. Here, using recombinant protein expression, along with light-scattering, CD, and fluorescence spectroscopy, we report that HN and BAX can form fibers together in vitro Results from negative stain EM experiments suggest that BAX undergoes secondary and tertiary structural rearrangements and incorporates into the fibers, and that its membrane-associating C-terminal helix is important for the fibrillation process. Additionally, HN mutations known to alter its anti-apoptotic activity affect fiber morphology. Our findings reveal for the first time a potential mechanism by which BAX can be sequestered by fibril formation, which can prevent it from initiating MOMP and committing the cell to apoptosis.