Structural Stability Of Protein Research Articles

Amelioration of ovalbumin gel properties by EGCG via protein aggregation, hydrogen, and van der Waals force.

The mechanism of epigallocatechin gallate (EGCG)-modified ovalbumin gel (EMOG) was investigated. Results indicated that, with the increase of EGCG concentration from 0% to 0.05%, the opacity, hardness, and cohesiveness of EMOG increased significantly from 0.058 to 0.133, 321.0g to 377.6g, and 0.879 to 0.951, respectively, while the soluble protein, surface hydrophobicity, and free sulfhydryl decreased significantly by 41.74%, 28.26%, and 39.36%, respectively. Moreover, EGCG promoted the formation of dense and stable microstructures of EMOG, changed the expansion rate, and improved the stability of EMOG. Moreover, the results of silico simulation showed that EGCG would insert into ovalbumin and interact with the amino acids through van der Waals force and hydrogen bonds, leading to a compact and stable protein structure. In this paper, the mechanism of modification of ovalbumin by EGCG was elucidated at the macro and micro levels, providing insights into the action of polyphenols and proteins.

Effects of static magnetic field (SMF) and alternating magnetic field (AMF) assisted freezing on the microstructure and protein properties of channel catfish (Ictalurus punctatus) fillet

The effect of static and alternating magnetic fields assisted freezing with intensity of 1, 2, and 3 mT on the microstructure and protein properties of channel catfish fillet were investigated. The results showed that the magnetic field treatment shortened the phase transition time of freezing, and significantly reduced the size of the formed ice crystals. The changes of trichloroacetic acid-soluble peptide, Ca2+-ATPase activity, particle size, and Zeta potential, which represented solubility, denaturation and aggregation of protein, indicated that magnetic field treatment could improve the protein stability. The chemical force analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Fourier transform infrared spectroscopy (FTIR) results proved that the magnetic field could change the structure of protein. Furthermore, these changes had effects on the thermal stability of catfish meat protein which reflected by increasing of the transition temperature and enthalpy. However, the waveform and intensity of magnetic field affected the stability of protein structure.

Nitrogen regulates the synthesis of hydrophobic amino acids to improve protein structural and gel properties in common buckwheat

Nitrogen (N) fertilizer impacts the grain quality of common buckwheat, but the effects and regulatory mechanisms of N on various protein parameters of buckwheat are not fully understood. The purpose of this study was to investigate the particle morphology, structural and gel properties, and regulation mechanism of buckwheat protein under four N levels. The bulk density, surface hydrophobicity, particle size, and thermal properties of the buckwheat protein were maximized through the optimal N application (180 kg N/ha), further enhancing the thermal stability of the protein. N application increased the β-sheet content and reduced the random coil content. Appropriate N fertilizer input enhanced the tertiary structure stability and gel elasticity of buckwheat protein by promoting hydrophobic interactions, disulfide bonds, ionic bonds, storage modulus and loss modulus. The differentially expressed proteins induced by N are primarily enriched in small ribosomal subunit and ribosome, improving protein quality mainly by promoting the synthesis of hydrophobic amino acids. Future agriculture should pay attention to the hydrophobic amino acid content of buckwheat to effectively improve protein quality. This study further advances the application of buckwheat protein in the field of food processing and provides a theoretical basis for the extensive development and utilization of buckwheat protein.

Role of hydrogen-bond networks on the donor side of photosynthetic reaction centers from purple bacteria.

For the last decades, significant progress has been made in studying the biological functions of H-bond networks in membrane proteins, proton transporters, receptors, and photosynthetic reaction centers. Increasing availability of the X-ray crystal and cryo-electron microscopy structures of photosynthetic complexes resolved with high atomic resolution provides a platform for their comparative analysis. It allows identifying structural factors that are ensuring the high quantum yield of the photochemical reactions and are responsible for the stability of the membrane complexes. The H-bond networks are known to be responsible for proton transport associated with electron transfer from the primary to the secondary quinone as well as in the processes of water oxidation in photosystem II. Participation of such networks in reactions proceeding on the periplasmic side of bacterial photosynthetic reaction centers is less studied. This review summarizes the current understanding of the role of H-bond networks on the donor side of photosynthetic reaction centers from purple bacteria. It is discussed that the networks may be involved in providing close association with mobile electron carriers, in light-induced proton transport, in regulation of the redox properties of bacteriochlorophyll cofactors, and in stabilization of the membrane protein structure at the interface of membrane and soluble phases.

Development of noodles with high highland barley flour incorporation level with proteins and pre‐gelatinized flour/starch

SummaryIn this study, pre‐gelatinized highland barley flour (PHBF), pre‐gelatinized sweet potato starch (PSPS), wheat gluten (WG), and egg white protein (EWP) were used to develop highland barley noodles. Their effects on noodle quality were analysed, and then noodle formula optimization was carried out based on them. Changes in quality characteristics and intermolecular structure were analysed to explore the underlying mechanism. PHBF, PSPS, WG, and EWP influence the quality characteristics of HB noodles from diverse angles. After formula optimization, noodles with improved cooking and textural properties were obtained, in which HBF accounts for 93.0% of the composite flour and can be categorised as low expected glycemic index (eGI) foods. The viscosity and elasticity of the dough both improved; a more stable protein structure and enhanced starch structure were formed; and increased intermolecular forces were observed, which would contribute to the enhanced protein cross‐linking, structural order, and textural properties of noodles.

Effect of Titanium dioxide nanoparticles in Enzymatic Activity of Epigeic Earthworm species “Eudrilus eugenia”

Titanium dioxide nanoparticles (TiO2 NPs) are commonly used in different industries because of their physico-chemical properties. They are widely used and their environmental occurrence has raised concerns about the potential toxicity as the release of nanoparticles to the soil directly or indirectly through air and water is gradually increasing. The soil is contaminated with nanoparticles in the long term and soil microorganisms can adversely be affected by these accumulated nanoparticles. Studies show that nanoparticles can have a lethal effect on soil microorganisms by causing the production of reactive oxygen species, by damaging the membrane permeability, cell signaling processes and the stability of enzymes and protein structures. In this study, Eudrilus eugenia earthworms were exposed to varying concentrations of TiO2 nanoparticles for a specified duration. Enzymatic activity assays were performed to evaluate the impact of nanoparticle exposure on key enzymes involved in antioxidant defense systems such as glutathione peroxidase (GPx) superoxide dismutase (SOD), glutathione reductase (GR) detoxification enzymes and glutathione S-transferases (GSTs). The activity of acetylcholinesterase (AChE), an enzyme involved in neurotransmission, was also assessed. The results demonstrated the exposure to TiO2 nanoparticles significantly altered the enzymatic activity of Eudrilus eugenia earthworms. The activity of GPx and GR enzymes exhibited a concentration-dependent decrease (0, 5, 50 and 500 mg/kg), indicating a disruption in antioxidant defense mechanisms. This disruption suggests a potential imbalance between reactive oxygen species (ROS) and antioxidant capacity, leading to oxidative stress. The superoxide dismutase (SOD) activity and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio significantly decreased under the 500 mg/kg nTiO2 treatment. Also, the activity of AChE was found to be significantly affected by TiO2 nanoparticle exposure, suggesting potential disruption in neurotransmission processes.

HSP110 aggravates ischemia-reperfusion injury after liver transplantation by promoting NF-κB pathway

BackgroundIschemia-reperfusion injury (IRI) poses a significant challenge to liver transplantation (LT). The underlying mechanism primarily involves overactivation of the immune system. Heat shock protein 110 (HSP110) functions as a molecular chaperone that helps stabilize protein structures. MethodsAn IRI model was established by performing LT on Sprague-Dawley rats, and HSP110 was silenced using siRNA. Hematoxylin-eosin staining, TUNEL, immunohistochemistry, ELISA and liver enzyme analysis were performed to assess IRI following LT. Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes. ResultsOur findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT (P < 0.05). However, when rats were injected with siRNA-HSP110, IRI subsequent to LT was notably reduced (P < 0.05). Additionally, the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced (P < 0.05). Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver (P < 0.05). ConclusionsHSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells. Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI.

Molecular dynamics simulations identify the topological weak spots of a protease CN2S8A

Thermophilic enzymes are highly desired in industrial applications due to their efficient catalytic activity at high temperature. However, most enzymes exhibit inferior thermostability and it remains challenging to identify the optimal sites for designing mutations to improve protein stability. To tackle this issue, we integrated topological analysis and all-atom molecular dynamics simulations to efficiently pinpoint the thermally-unstable regions in protein structures. Using a protease CN2S8A as the model, we analyzed the intramolecular hydrogen bonding interactions between adjacent secondary structure elements, and then identified the topological weak spots of CN2S8A where weak hydrogen bonding interactions were formed. To examine the role of these sites in protein structural stability, we designed three virtual mutations at different weak spots and characterized the effects of these mutations on the structural properties of CN2S8A. The results showed that all three mutations increased the protein structural stability. In conclusion, these findings provide a novel method to identify the topological weak spots of proteins, with implications in the rational design of biocatalysts with superior thermostability.

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4–0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design.

Clinical feature and genetic analysis of a patient with Idiopathic hypogonadotropic hypogonadism due to a novel variant of CHD7 gene

To explore the clinical feature and genetic etiology of a patient with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) due to variant of CHD7 gene. A patient who had presented at Anhui Provincial Children's Hospital in October 2022 was selected as the study subject. Clinical data of the patient was collected. The patient and his parents were subjected to trio-whole exome sequencing. Candidate variant was verified by Sanger sequencing and bioinformatic analysis. The patient had featured delayed development of secondary sexual characteristics but normal olfactory function. Genetic testing revealed that he has harbored a c.3052C>T (p.Pro1018Ser) missense variant of the CHD7 gene, for which both of his parents were of the wild type. The variant has not been recorded in the PubMed and HGMD databases. Analysis of amino acid sequences suggested that the variant site is highly conserved, and the variant may affect the stability of protein structure. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.3032C>T variant was classified as a likely pathogenic (PS2+PM2_Supporting+PP2+PP3+PP4). The delayed development of secondary sexual characteristics of the patient may be attributed to the c.3052C>T (p.Pro1018Ser) variant of the CHD7 gene. Above finding has expanded the variation spectrum of the CHD7 gene.

Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine.

Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential. The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (kcat /Km ) toward 1-acetylnaphthalene and the stability (half-life t1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value>99.5%) within 10h under 25% DMSO. The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

C-Terminal β8-α9 Interaction Modulates Thermal Stability and Enzymatic Activity Differently in Hyperthermophilic Esterase EstE1 and Mesophilic Esterase rPPE.

Hydrophobic interactions and hydrogen bonds are 2 types of noncovalent interactions that play distinct roles in the folding and structural stability of proteins. However, the specific roles of these interactions in hydrophobic or hydrophilic environments in α/β-hydrolases are not fully understood. A hyperthermophilic esterase EstE1 in a dimer maintains the C-terminal β8-α9 strand-helix via hydrophobic interactions (Phe276 and Leu299), constituting a closed dimer interface. Moreover, a mesophilic esterase rPPE in a monomer maintains the same strand-helix via a hydrogen bond (Tyr281 and Gln306). Unpaired polar residues (F276Y in EstE1 and Y281A/F and Q306A in rPPE) or reduced hydrophobic interactions (F276A/L299A in EstE1) between the β8-α9 strand-helix decrease thermal stability. EstE1 (F276Y/L299Q) and rPPE WT, both with the β8-α9 hydrogen bond, showed the same thermal stability as EstE1 WT and rPPE (Y281F/Q306L), which possess hydrophobic interactions instead. However, EstE1 (F276Y/L299Q) and rPPE WT exhibited higher enzymatic activity than EstE1 WT and rPPE (Y281F/Q306L), respectively. This suggests that α/β-hydrolases favor the β8-α9 hydrogen bond for catalytic activity in monomers or oligomers. Overall, these findings demonstrate how α/β-hydrolases modulate hydrophobic interactions and hydrogen bonds to adapt to different environments. Both types of interactions contribute equally to thermal stability, but the hydrogen bond is preferred for catalytic activity. IMPORTANCE Esterases hydrolyze short to medium-chain monoesters and contain a catalytic His on a loop between the C-terminal β8-strand and α9-helix. This study explores how hyperthermophilic esterase EstE1 and mesophilic esterase rPPE adapt to different temperatures by utilizing the β8-α9 hydrogen bonds or hydrophobic interactions differently. EstE1 forms a hydrophobic dimer interface, while rPPE forms a monomer stabilized by a hydrogen bond. The study demonstrates that these enzymes stabilize β8-α9 strand-helix differently but achieve similar thermal stability. While the β8-α9 hydrogen bond or hydrophobic interactions contribute equally to thermal stability, the hydrogen bond provides higher activity due to increased catalytic His loop flexibility in both EstE1 and rPPE. These findings reveal how enzymes adapt to extreme environments while maintaining their functions and have implications for engineering enzymes with desired activities and stabilities.

Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain.

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur.

Characterization and Mechanism of Gel Deterioration of Egg Yolk Powder during Storage.

Egg yolk forms have several health and industrial applications, but their storage characteristics and gel mechanisms have not been thoroughly studied. In order to investigate the relationship between the changes in structure and properties of egg yolk gel and egg yolk powder during storage, in this paper, egg yolk powder was stored at 37 °C for 0, 1, 3, and 6 months in an accelerated storage experiment, and the influence of storage time on the gel properties of egg yolk powder was analyzed. The results showed that the contents of protein carbonylation and sulfhydryl in the yolk decreased gradually with the extension of storage time. Circular dichroism and fluorescence spectra showed that the ordered structure and structural stability of egg yolk proteins decreased gradually. Oxidation led to the formation of intermolecular crosslinking in the egg yolk proteins and oxidized aggregates, resulting in a decrease in surface hydrophobicity, which affected the gel properties of the egg yolk powder after rehydration, resulting in the phenomenon of lipid migration and gel degradation. The results provide a theoretical basis for improving egg yolk powder's overall quality and storage stability.

Differential glycosylation in mutant vitamin D-binding protein decimates the binding stability of vitamin D

Vitamin D (VD) is produced by the skin upon exposure to sunlight or is obtained from dietary sources. Several risk factors are associated with VD deficiency including mutations and post-translational modifications in its transport protein known as vitamin D binding protein (VDBP) or GC-globulin. The two common single nucleotide polymorphisms rs7041 and rs4588 create three major isoforms of VDBP, including GC-1F also called wild type, GC1S, and GC-2. The 3D models for both GC-1F and GC-2 were constructed in their glycosylated states to decipher the effect of these mutations on the overall conformational changes and VD-binding affinity. The binding affinities were estimated using the Molecular Mechanics Poison-Boltzmann surface area (MM-PBSA) method and conformational changes were investigated after free energy landscapes estimations. Total free energies suggest that GC-1F exhibits stronger affinity (ΔE = −116.09 kJ/mol) than GC-2 (ΔE = −95 kJ/mol) variant with VD. The GC-1F isoforms had more streamlined motion compared to GC-2 isoforms, predicting a trade-off between cross-talk residues that significantly impacts protein structural stability. The data suggest that glycation at Thr418 plays a vital role in the overall VDBP-VD affinity by stabilizing the N-T loop that holds the domain I (VD-pocket) and domain III intact. The loss of glycation in GC-2 has a pivotal role in the inter-domain conformational stability of VDBP, which may ultimately affect VD transportation and maturation. These findings describe a novel mechanism in how mutations distant from the VD-active site change the overall conformational of the VDBP and abrogate the VDBP-VD interaction. Communicated by Ramaswamy H. Sarma

Structure Relaxation Approximation (SRA) for Elucidation of Protein Structures from Ion Mobility Measurements (II). Protein Complexes.

Characterizing structures of protein complexes and their disease-related aberrations is essential to understanding molecular mechanisms of many biological processes. Electrospray ionization coupled with hybrid ion mobility/mass spectrometry (ESI-IM/MS) methods offer sufficient sensitivity, sample throughput, and dynamic range to enable systematic structural characterization of proteomes. However, because ESI-IM/MS characterizes ionized protein systems in the gas phase, it generally remains unclear to what extent the protein ions characterized by IM/MS have retained their solution structures. Here, we discuss the first application of our computational structure relaxation approximation [Bleiholder, C.; et al. J. Phys. Chem. B 2019, 123 (13), 2756-2769] to assign structures of protein complexes in the range from ∼16 to ∼60 kDa from their "native" IM/MS spectra. Our analysis shows that the computed IM/MS spectra agree with the experimental spectra within the errors of the methods. The structure relaxation approximation (SRA) indicates that native backbone contacts appear largely retained in the absence of solvent for the investigated protein complexes and charge states. Native contacts between polypeptide chains of the protein complex appear to be retained to a comparable extent as contacts within a folded polypeptide chain. Our computations also indicate that the hallmark "compaction" often observed for protein systems in native IM/MS measurements appears to be a poor indicator of the extent to which native residue-residue interactions are lost in the absence of solvent. Further, the SRA indicates that structural reorganization of the protein systems in IM/MS measurements appears driven largely by remodeling of the protein surface that increases its hydrophobic content by approximately 10%. For the systems studied here, this remodeling of the protein surface appears to occur mainly by structural reorganization of surface-associated hydrophilic amino acid residues not associated with β-strand secondary structure elements. Properties related to the internal protein structure, as assessed by void volume or packing density, appear unaffected by remodeling of the surface. Taken together, the structural reorganization of the protein surface appears to be generic in nature and to sufficiently stabilize protein structures to render them metastable on the time scale of IM/MS measurements.

Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges. Here, we report the combination of protein-denaturation principles with quantitative cross-linking mass spectrometry using isobaric quantitative protein interaction reporter technologies. This method enables the evaluation of ligand-induced protein engagement through analysis of cross-link relative ratios across chemical denaturation. As a proof of concept, we found ligand-stabilized cross-linked lysine pairs in well-studied bovine serum albumin and ligand bilirubin. These links map to the known binding sites Sudlow Site I and subdomain IB. We propose that protein denaturation and qXL-MS can be combined with similar peptide-level quantification approaches, like SPROX, to increase the coverage information profiled for facilitating protein-ligand engagement efforts.

Cryoprotective Effects of Carrageenan Oligosaccharides on Crayfish (Procambarus clarkii) during Superchilling.

Cryoprotectants are widely used to protect muscle tissue from ice crystal damage during the aquatic products freezing process, but traditional phosphate cryoprotectants may cause an imbalance in the calcium-to-phosphorus ratio for the human body. This study evaluated the effects of carrageenan oligosaccharides (CRGO) on quality deterioration and protein hydrolysis of crayfish (Procambarus clarkii) during superchilling. The physical-chemical analyses showed that CRGO treatments could significantly (p < 0.05) inhibit the increase of pH values, TVB-N, total viable counts, and thawing loss, and improve the water holding capacity and the proportion of immobilized water, which indicated that CRGO treatment effectively delayed the quality deterioration of crayfish. The myofibrillar protein structural results demonstrated that the increase of the disulfide bond, carbonyl content, S0-ANS, and the decrease of total sulfhydryl content were suppressed significantly (p < 0.05) in CRGO treatment groups. Furthermore, SDS-PAGE results showed that the band intensity of myosin heavy chain and actin in CRGO treatment groups were stronger than in the control. Overall, the application of CRGO to crayfish might maintain better quality and stable protein structure during the superchilling process, and CRGO has the potential to replace phosphate as a novel cryoprotectant for aquatic products.

Measurement of charges and chemical bonding in a cryo-EM structure

Hydrogen bonding, bond polarity, and charges in protein molecules play critical roles in the stabilization of protein structures, as well as affecting their functions such as enzymatic catalysis, electron transfer, and ligand binding. These effects can potentially be measured in Coulomb potentials using cryogenic electron microscopy (cryo-EM). We here present charges and bond properties of hydrogen in a sub-1.2 Å resolution structure of a protein complex, apoferritin, by single-particle cryo-EM. A weighted difference map reveals positive densities for most hydrogen atoms in the core region of the complex, while negative densities around acidic amino-acid side chains are likely related to negative charges. The former positive densities identify the amino- and oxo-termini of asparagine and glutamine side chains. The latter observations were verified by spatial-resolution selection and a dose-dependent frame series. The average position of the hydrogen densities depends on the parent bonded-atom type, and this is validated by the estimated level of the standard uncertainties in the bond lengths.

Investigating the Activation Mechanism Differences between Human and Mouse cGAS by Molecular Dynamics Simulations.

Cyclic GMP-AMP synthase (cGAS) has been widely investigated as a drug target for its crucial role in innate immunity. However, the inhibitors designed using mouse model were often shown to be ineffective for humans. This outcome indicates that the activation mechanisms of human and mouse cGAS (mcGAS) are different. The cGAS activation is achieved by dimerization via binding to DNA, the detailed mechanism of which, however, is not entirely clear. To investigate these mechanisms, molecular dynamics (MD) simulations were performed on several states of four types of cGAS, namely, the mcGAS, the wild-type and A- and C-type mutations of human cGAS (hcGAS). We find that sequence differences between hcGAS and mcGAS can directly affect the protein structure stability, especially that of the siteB domain. The sequence and structural differences also contribute to DNA-binding differences. In addition, the conformational fluctuations of cGAS are found to correlate with the regulation of catalytic capacity. More importantly, we illustrate that dimerization enhances the correlation among distant residues and significantly reinforces the allosteric signal transmission among the DNA-binding interfaces and the catalytic pocket, which facilitates rapid immune response to cytosolic DNA. We conclude that siteB domain plays a prominent role in mcGAS activation, while siteA domain is key to hcGAS activation.