Changes In Protein Structure Research Articles

Persistent topological Laplacian analysis of SARS-CoV-2 variants.

Topological data analysis (TDA) is an emerging field in mathematics and data science. Its central technique, persistent homology, has had tremendous success in many science and engineering disciplines. However, persistent homology has limitations, including its inability to handle heterogeneous information, such as multiple types of geometric objects; being qualitative rather than quantitative, e.g., counting a 5-member ring the same as a 6-member ring, and a failure to describe non-topological changes, such as homotopic changes in protein-protein binding. Persistent topological Laplacians (PTLs), such as persistent Laplacian and persistent sheaf Laplacian, were proposed to overcome the limitations of persistent homology. In this work, we examine the modeling and analysis power of PTLs in the study of the protein structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain (RBD). First, we employ PTLs to study how the RBD mutation-induced structural changes of RBD-angiotensin-converting enzyme 2 (ACE2) binding complexes are captured in the changes of spectra of the PTLs among SARS-CoV-2 variants. Additionally, we use PTLs to analyze the binding of RBD and ACE2-induced structural changes of various SARS-CoV-2 variants. Finally, we explore the impacts of computationally generated RBD structures on a topological deep learning paradigm and predictions of deep mutational scanning datasets for the SARS-CoV-2 Omicron BA.2 variant. Our results indicate that PTLs have advantages over persistent homology in analyzing protein structural changes and provide a powerful new TDA tool for data science.

Effects of covalent binding of different polyphenols on structure, rheology and functional properties of whey protein isolate

In this paper, conjugated four polyphenols (epigallocatechin gallate [EGCG], procyanidine [PA], apple polyphenol [AP], puerarin [PUE]) with whey protein isolate (WPI) was prepared under alkaline condition. The covalent interaction between polyphenols and WPI was confirmed by the determination of polyphenol binding equivalent. Fluorescence spectroscopy and fourier transform infrared spectra analysis were used to identify the changes in whey protein structure. The scanning electron microscopy data and the particle size distribution demonstrated surface changes and increases in the particle sizes of conjugates. Rheology analysis showed that the initial value of apparent viscosity and curve shape of all the samples have certain differences and exhibited a pseudoplastic behavior. Conjugation significantly improved foaming and emulsifying properties of the WPI, while its surface hydrophobicity was greatly decreased.These results provide a new basis of theory for the development of novel protein ingredient.

Evolution of lean meat tenderness stimulated by coordinated variation of water status, protein structure and tissue histology during cooking of braised pork

Tenderness of lean meat in braised pork is of great importance to the consumer palatability and acceptance. The influence of water status, protein structure and histological changes on lean meat tenderness during cooking was investigated. Results indicated that lean meat began to tenderize mainly after 20 min-cooking. In the early period of cooking, the decrease of total sulfhydryl content caused the protein oxidative cross-linking, leading to the gradual unfolding of the protein structure, thus resulting in a decrease of T22 and an increase of centrifugal loss, which decreased the tenderness of lean meat. However, after cooking for 20 min, the β-sheet decreased and random coil increased, thus generating conversion between P21 and P22. The rupture of perimysium structure was observed. Changes in protein structure, water status, and tissue histology could facilitate the initiation and development of lean meat tenderness.

Evaluating Tyndallisation's potential for composite food products and quality considerations

Tyndallization presents potential as a cost-effective alternative sterilisation method for small-scale producers. This research aimed to assess the effectiveness of tyndallization at various temperatures in sterilising pempek, a composite food product, and its impact on product quality. The study employed the SDS-PAGE technique to investigate protein polymerisation mechanisms and FTIR to evaluate protein structure, colour, pH, and texture changes. The results indicated protein polymerisation occurred, evidenced by a decrease in Molecule High Chain (MHC) and an increase in actin, corresponding to the rising temperature and tyndallization cycles with a sinusoidal pattern. FTIR spectra demonstrated that the β-helix structure dominated secondary protein structures, consistently observed across different levels and temperature variations. This study contributes new insights into applying thermal processes in preparing composite food products. Regarding quality, tyndallization caused a shift towards a more yellow colour in the pempek and a softer texture, although there were no significant pH changes. The alterations in protein patterns resulted from the varying cycles and temperatures experienced during the tyndallization process. The findings suggest that tyndallization can be considered an effective alternative for sterilising composite products like pempek; however, its implementation still requires the appropriate combination of time, temperature, and tyndallization cycles while considering the potential impact on quality changes during the sterilisation process.

Clinical Characteristics and Genetic Analyses of Patients with Idiopathic Hypogonadotropic Hypogonadism

Idiopathic hypogonadotropic hypogonadism (IHH) is classified into two groups-Kalman syndrome and normosmic IHH (nIHH). Half of all cases can be explained by mutations in >50 genes. Targeted gene panel testing with nexrt generation sequencing (NGS) is required for patients without typical phenotypic findings. The aim was to determine the genetic etiologies of patients with IHH using NGS, including 54 IHH-associated genes, and to present protein homology modeling and protein stability analyzes of the detected variations. Clinical and demographic data of 16 patients (eight female), aged between 11.6-17.8 years, from different families were assessed. All patients were followed up for a diagnosis of nIHH, had normal cranial imaging, were without anterior pituitary hormone deficiency other than gonadotropins, had no sex chromosome anomaly, had no additional disease, and underwent genetic analysis with NGS between the years 2008-2021. Rare variants were classified according to the variant interpretation framework of the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology. Changes in protein structure caused by variations were modeled using RoseTTAFold and changes in protein stability resulting from variation were analyzed. Half of the 16 had no detectable variation. Three (18.75%) had a homozygous (pathogenic) variant in the GNRHR gene, one (6.25%) had a compound heterozygous [likely pathogenic-variants of uncertain significance (VUS)] variant in PROK2 and four (25%) each had a heterozygous (VUS) variant in HESX1, FGF8, FLRT3 and DMXL2. Protein models showed that variants interpreted as VUS according to ACMG could account for the clinical IHH. The frequency of variation detection was similar to the literature. Modelling showed that the variant in five different genes, interpreted as VUS according to ACMG, could explain the clinical IHH.

Formation mechanism for stable system of nanoparticle/protein corona and phospholipid membrane

In the physiological environment, nanoparticles (NPs) interact with proteins to form a protein-rich layer on the surface which is called "protein corona". Understanding and analyzing the formation process of protein corona and protein corona-nanoparticles is of great significance for biological related nano research. Many separation techniques have been used to analyze the composition of protein corona, but in situ analysis of protein corona is still absent. With the development of detection technology, sum frequency generation (SFG) is an effective instrument to analyze the surface protein structure and dynamic changes of protein corona in situ. In this work the molecular mechanism and surface structure effect of the interaction between nanoparticles with surface protein corona (S-NPP) and phospholipid membrane were studied. When S-NPP interacts with phospholipid membrane, the bond affinity network formed by the binding water can stabilize S-NPP around the lipid bilayer. In this process, S-NPP can be found wrapped in the hydration shell. This ultimately leads to a more moderate interaction between particles and phospholipid membrane.

Fatigue-driven compliance increase and collagen unravelling in mechanically tested anterior cruciate ligament

Approximately 300,000 anterior cruciate ligament (ACL) tears occur annually in the United States, half of which lead to the onset of knee osteoarthritis within 10 years of injury. Repetitive loading is known to result in fatigue damage of both ligament and tendon in the form of collagen unravelling, which can lead to structural failure. However, the relationship between tissue’s structural, compositional, and mechanical changes are poorly understood. Herein we show that repetitive submaximal loading of cadaver knees causes an increase in co-localised induction of collagen unravelling and tissue compliance, especially in regions of greater mineralisation at the ACL femoral enthesis. Upon 100 cycles of 4× bodyweight knee loading, the ACL exhibited greater unravelled collagen in highly mineralized regions across varying levels of stiffness domains as compared to unloaded controls. A decrease in the total area of the most rigid domain, and an increase in the total area of the most compliant domain was also found. The results highlight fatigue-driven changes in both protein structure and mechanics in the more mineralized regions of the ACL enthesis, a known site of clinical ACL failure. The results provide a starting point for designing studies to limit ligament overuse injury.

Changes in advanced protein structure during dense phase carbon dioxide induced gel formation in golden pompano surimi correlate with gel strength

Changes in protein structure are closely related to gel strength. Dense phase carbon dioxide (DPCD) treatment is an excellent non-thermal food processing method that can be used to induce gel formation in surimi. The sensory, water holding capacity and gel strength of DPCD induced gels are superior to heat-induced gels. Fourier-transform infrared spectroscopy was used to investigate the role of DPCD in the quality of golden pompano surimi gels and changes in protein structure. The intermolecular forces of surimi gels were analyzed in terms of ionic and hydrogen bonds, disulfide covalent and non-disulfide covalent bonds, as well as hydrophobic interactions. Correlation analysis was used to investigate the relationship between the changes in advanced protein structure and gel strength during DPCD-induced gel formation in golden pompano surimi. The results showed that the α-helix and random coil levels of surimi gel were significantly decreased (p < 0.05), while the β-sheet and β-turn content was significantly increased (p < 0.05). The number of ionic and hydrogen bonds in gel proteins decreased significantly (p < 0.05), while the hydrophobic interactions, and disulfide and non-disulfide covalent bonds increased significantly (p < 0.05) after DPCD treatment. Correlation analysis showed that β-sheets, β-turns, hydrophobic interactions, and disulfide and non-disulfide covalent bonds were strongly positively correlated with gel strength, whereas α-helices, random coils, and ionic and hydrogen bonds were strongly negatively correlated with gel strength. Therefore, the α-helix and random coil structures of surimi gels were transformed into β-sheet and β-turn structures after DPCD treatment. Hydrophobic interactions, and disulfide and non-disulfide covalent bonds were the main intermolecular forces during the DPCD-induced gel formation of surimi. Ionic and hydrogen bonds were not the main intermolecular forces. The results provide fundamental data for elucidating the mechanism of DPCD-induced protein gel formation.

High carrier frequency of pathogenic PATL2 gene mutations predicted in population: a bioinformatics-based approach.

Topoisomerase II homologue 2 (PATL2) has been confirmed to be a key gene that contributes to oocyte maturation. However, the allele distribution and carrier frequency of these mutations remain uncharacterized. So a bioinformatics subcategory analysis of PATL2 mutations from outcome data and Single Nucleotide Polymorphism (SNP) databases was conducted. Altogether, the causative PATL2 mutation number detected in patients with oocyte maturation defects in the clinical studies and pathogenic PATL2 mutation sites predicted by software based on the database was approximately 53. The estimated carrier frequency of pathogenic mutation sites was at least 1.14‰ based on the gnomAD and ExAC database, which was approximately 1/877. The highest frequency of mutations detected in the independent patients was c.223-14_223-2del13. The carrier frequency of this mutation in the population was 0.25‰, which may be a potential threat to fertility. Estimated allele and carrier frequency are relatively higher than those predicted previously based on clinical ascertainment. A review of PATL2 mutation lineage identified in 34 patients showed that 53.81%, 9.22% and 14.72% of the oocytes with PATL2 mutations were arrested at the germinal vesicle (GV) stage, metaphase I (MI) stage and first polar body stage, respectively. Oocytes that could develop to the first polar body stage were extremely rare to fertilise, and their ultimate fate was early embryonic arrest. Phenotypic variability is related to the function of the regions and degree of loss of function of PATL2 protein. A 3D protein structure changes predicted by online tools, AlphaFold, showed aberrations at the mutation sites, which may explain partially the function loss. When the mutated and wild-type proteins are not in the same amino acid category, the protein structure will be considerably unstable. The integration of additional mutation sites with phenotypes is helpful in drawing a complete picture of the disease. Bioinformatics analysis of PATL2 mutations will help reveal molecular epidemiological characteristics and provide an important reference for new mutation assessment, genetic counselling and drug research.

From amyloid fibrils to microfiber condensates: Tuning microfluidic coextrusion of food-grade β-lactoglobulin-pectin core-shell fibers by changes of protein structure

Protein-based microfibers have potential applications in bioengineering and food but preserving and utilizing the unique nanomechanical properties of their protein building blocks at the micrometer scale remains a challenge. This study investigates the bottom-up fabrication of core-shell fibers by coaxial microfluidic spinning of pectin and β-lactoglobulin in different conformational states (monomeric, amyloid fibrils, shortened amyloid fibrils in their isotropic/nematic phases), gelled in CaCl2 solution. Fiber diameters ranged between 478 and 855 μm (wet state) and 107–135 μm (dry state). They showed clear core-shell cross-sections, except pectin-β-lactoglobulin monomer fibers where the compact protein is presumably understood to diffuse through the pectin matrix. The molecular orientation of the fiber building blocks was expressed as order parameters representing the alignment of pectin chains and amyloid fibrils parallel to the fiber axis calculated from synchrotron wide-angle X-ray scattering (WAXS) with a spatial resolution of 20 μm. Introduction of amyloid fibrils as the protein core increased the Young's modulus from 3.3 to 6.4 GPa and tensile strength from 117 to 182 MPa compared to pure pectin fibers. Increasing the protein core flow rate from 1 to 2 mL/h, however, caused helical bending of the core jet, a decrease in order, and ultimately worsened mechanical performance. Overall, full length amyloid fibrils proved to be more beneficial to the mechanical properties than shortened amyloid fibrils. By providing insight into the relationship between protein conformation, spinning flow rate, and resulting mechanical properties of core-shell microfibers, these results may contribute to the field of novel fibrous protein-based materials.

Identification of potentially pathogenic variants for autism spectrum disorders using gene-burden analysis.

Gene- burden analyses have lately become a very successful way for the identification of genes carrying risk variants underlying the analysed disease. This approach is also suitable for complex disorders like autism spectrum disorder (ASD). The gene-burden analysis using Testing Rare Variants with Public Data (TRAPD) software was conducted on whole exome sequencing data of Slovenian patients with ASD to determine potentially novel disease risk variants in known ASD-associated genes as well as in others. To choose the right control group for testing, principal component analysis based on the 1000 Genomes and ASD cohort samples was conducted. The subsequent protein structure and ligand binding analysis usingI-TASSER package were performed to detect changes in protein structure and ligand binding to determine a potential pathogenic consequence of observed mutation. The obtained results demonstrate an association of two variants-p.Glu198Lys (PPP2R5D:c.592G>A) and p.Arg253Gln (PPP2R5D:c.758G>A) with the ASD. Substitution p.Glu198Lys (PPP2R5D:c.592G>A) is a variant, previously described as pathogenic in association with ASD combined with intellectual disability, whereas p.Arg253Gln (PPP2R5D:c.758G>A) has not been described as an ASD-associated pathogenic variant yet. The results indicate that the filtering process was suitable and could be used in the future for detection of novel pathogenic variants when analysing groups of ASD patients.

Spy chemistry enables stable protein immobilization on iron oxide nanoparticles with enhanced magnetic properties

Iron oxide nanoparticles (IONPs) modified with functional proteins hold great promise in the biomedical field. However, conventional protein modification strategies, such as adsorption and covalent coupling, are either unstable or nonspecific, or may result in the changes of protein structure and ultimately the loss of protein activity. Modification of active proteins on small-sized IONPs with a particle size of less than 30 nm is especially difficult due to their high surface energy. Herein, we developed a universal modification method based on Spy chemistry for rapid and stable protein immobilization on small-sized IONPs, which only requires the presence of active groups on the surface of nanoparticles that can couple with SpyCatcher. In short, the SpyCatcher peptides were first coated on the surface of IONPs by cross-linking with activated groups, and then the SpyTag peptide fused with a model protein (enhanced green fluorescent protein, EGFP) was engineered (SpyTag-EGFP) and directly coupled to SpyCatcher-modified IONPs by self-assembly, which is spontaneous and robust while avoiding the effect of chemical reactions on functional protein activity. The obtained EGFP-functionalized IONPs exhibited enhanced and stable green fluorescence and improved magnetic properties. In addition, the cell internalization efficiency of EGFP-functionalized IONPs was significantly increased as compared to unmodified IONPs, providing an ideal solution for efficient cell labeling and tracking. In conclusion, here we report a rapid and easy strategy for EGFP immobilization on IONPs based on Spy chemistry, which could be further adapted to other functional proteins in the future. SpyCatcher-modified IONPs and SpyTag-X (arbitrary functional fusion proteins) hold great potential to be applied as a versatile platform for protein immobilization on IONPs and enable its multifunctional application in the future.

Time Evolution of Ultrasmall Gold Nanoparticle-Protein Interactions.

To date, much effort has been devoted toward the study of protein corona formation onto large gold nanoparticles (GNPs). However, the protein corona concept breaks down for GNPs in the ultrasmall size regime (<3 nm), and, as a result, our understanding of ultrasmall GNP (usGNP)-protein interactions remains incomplete. Herein, we used anionic usGNPs and six different proteins as model systems to systematically investigate usGNP-protein interactions, with particular focus on the time evolution and long-term behavior of complex formation. The different proteins comprised chymotrypsin (Cht), trypsin (Try), thrombin (Thr), serum albumin (HSA), cytochrome c (Cyt c), and factor XII (FXII). We used a range of biochemical and biophysical methods to estimate binding affinities, determine the effects of usGNPs on protein structure and function, assess the reversibility of any protein structural and functional changes, and evaluate usGNP-protein complex stability. Among the main findings, we observed that prolonged (24 h)─but not short-term (10 min)─interactions between proteins and usGNPs permanently altered protein function, including enzyme activities (Try, Thr, and FXIIa), peroxidase-like activity (Cyt c), and ligand-binding properties (HSA). Remarkably, this occurred without any large-scale loss of the native global conformation, implying time-dependent effects of usGNPs on local protein conformation or dynamics. We also found that both short-(10 min) and long-term (24 h) interactions between proteins and usGNPs yielded short-lived complexes, i.e., there was no time-dependent "hardening" of the interactions at the binding interface as usually seen with large GNPs. The present study increases our fundamental understanding of nano-bio interactions in the ultrasmall size regime, which may assist the safe and effective translation of usGNPs into the clinic.

Tracking the disulfide rearrangement of heated lactoglobulin by matrix-assisted laser desorption/ionization-in-source decay top-down analysis.

Disulfide bond rearrangement is a common occurrence during protein analysis or treatment. A convenient and rapid method has been developed to investigate heat-induced disulfide rearrangement of lactoglobulin using matrix-assisted laser desorption/ionization-in-source decay (MALDI-ISD) technology. By analyzing heated lactoglobulin in reflectron and linear mode, we demonstrated that cysteines C66 and C160 exist as free residues other than linked ones in some protein isomers. This method provides a straightforward and expeditious way to assess the cysteine status and structural changes of proteins under heat stress.

Effects of soluble aggregates sizes on rheological properties of soybean protein isolate under high temperature

The rheological properties of five concentrated soluble soybean protein isolates (SPI, 600 g/kg) with different average particle sizes, sulfhydryl content, and surface hydrophobicity were investigated at two different temperatures, i.e., 60 °C and 140 °C. The dynamic rheological results at 60 °C exhibited protein aggregation as the most significant factor affecting the storage and loss modulus. Time-sweep test at a high temperature (140 °C) and the temperature sweep test (30 °C–150 °C) exhibited that the SPI aggregates with large size need more energy to enter the flow state, and the protein interactions among these aggregates may also be hindered in the thermo-mechanical process. Furthermore, in order to exploring the protein interactions, N-Ethylmaleimide (NEM), sodium dodecyl sulfate (SDS), or urea were added to five soluble SPI samples during time-sweep experiments at 140 °C. The large particle sizes of pre-aggregated soy proteins may be an obstacle to further interactions (disulfide bonds, hydrophobic interactions, and hydrogen bonds) between protein molecules in the thermo-mechanical process. Proteins easily formed thermally irreversible aggregates via hydrophobic interactions or hydrogen bonds when disulfide bonds were hindered, and hydrogen bonds were indispensable forces for maintaining the viscoelasticity of the soy protein system during the extrusion-like condition (140 °C, 80% strain, 10 Hz frequency). The reaction behavior of soy protein under extrusion-like conditions can be used to understand the protein structural changes during the extrusion processes and produce consumer-popular meat analogs.

Computational modeling and prediction of deletion mutants

In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially due to the lack of comprehensive datasets including a structural readout. In addition, the recent breakthrough in structure prediction through deep learning demands an update of computational deletion mutation prediction. In this study, we deleted individually every residue of a small α-helical sterile alpha motif domain and investigated the structural and thermodynamic changes using 2D NMR spectroscopy and differential scanning fluorimetry. Then, we tested computational protocols to model and classify observed deletion mutants. We show a method using AlphaFold2 followed by RosettaRelax performs the best overall. In addition, a metric containing pLDDT values and Rosetta ΔΔG is most reliable in classifying tolerated deletion mutations. We further test this method on other datasets and show they hold for proteins known to harbor disease-causing deletion mutations.

Lutein fortification of wheat bread with marigold powder: Impact on rheology, water dynamics, and structure.

Demands for foods conducive to eye health have been on the increase in the global healthcare sector. Marigold powder as a major source of lutein was utilized to produce lutein-fortified breads for ocular health. The physicochemical characteristics of the doughs and breads were investigated in terms of rheology, water mobility, and protein secondary structures. The incorporation of marigold powder decreased the water absorption of doughs without significantly altering thermo-mechanical properties. With a range of fortification levels (1 ~ 3%), marigold powder led to decreased storage and loss modulus of doughs by weakening their gluten network, which was supported by their T2 relaxation times. The resistances of the doughs weakened with increasing levels of marigold powder, while their extensibilities significantly incremented. FTIR spectral deconvolution revealed the changes of wheat protein structures upon marigold powder incorporation, in which the proportion of β-turn increased at the expense of β-sheet ratio. The breads with marigold powder displayed increased specific volume from 4.034 to 4.368 mL/g, accompanied by softer textures. The baking process led to heat-induced losses in lutein concentration of less than 10% within the crumbs and approximately 30% in the crusts. The use of marigold powder induced changes in protein secondary structure and extensional features of doughs, contributing to increased loaf volume and softer texture. Overall, this study provides fundamental information about the rheological and structural effects of marigold powder in a wheat bread system, consequently encouraging the food industry to utilize marigold power as a functional food ingredient. This article is protected by copyright. All rights reserved.

Synergistic effect of high-intensity ultrasound and pH-shifting on the functionalities of chicken wooden breast myofibrillar protein: Reveal the mechanism of protein structure change

Due to the deterioration of wooden breast myofibrillar protein (WBMP) functional properties (such as gel and emulsification performance) caused by oxidative stress, making it difficult to use as normal MP in the processing and utilization of protein gel/emulsion products. The functional properties of chicken deteriorate, affecting its processing and utilization. High-intensity ultrasound (HIU) was introduced in the unfolding‒refolding process of pH-shifting treatment to improve functional properties on WBMP. The results showed that the WUH-R group (HIU applied during the WBMP unfolding stage) had the higher solubility, emulsification, and rheology. The zeta potential and particle size indicated that HIU significantly disrupted the protein structures (P < 0.05). The fluorescence spectrum showed that protein hydrophobicity was significantly (P < 0.05) reduced from 69.60 ± 2.03 (WBMP) to 43.39 ± 1.33 (WUH-R). Similarly, circular dichroism and Fourier transform infrared spectroscopy indicated that HIU facilitated the conversion of α-helix (25.12 ± 0.78% to 46.37 ± 2.14%) to β-sheet (27.61 ± 2.47% to 7.28 ± 0.89%). Our results demonstrate that HIU can promote the complete unfolding of WBMP. Nonetheless, in the WU-RH group (HIU applied during the WBMP refolding stage), HIU inhibited the assembly of proteins. In the WU-RH group, the particle size became larger, while the solubility and emulsibility became worse. In general, HIU is only added in the unfolding phase of protein pH-shifting to improve its functional properties and facilitate the reuse of WBMP.

Antibacterial and Antibiofilm Efficacy and Mechanism of Ginger (Zingiber officinale) Essential Oil against Shewanella putrefaciens.

Ginger (Zingiber officinale) has unique medicinal value and can be used to treat colds and cold-related diseases. The chemical composition and antibacterial activity of ginger essential oil (GEO) against Shewanella putrefaciens were determined in the present study. Zingiberene, α-curcumene, and zingerone were the main active compounds of GEO. GEO displayed significant antibacterial activity against S. putrefaciens, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 2.0 and 4.0 μL/mL, respectively. Changes in intracellular ATP content, nucleic acid and protein structure, exopolysaccharides (EPS) content, and extracellular protease production indicated that GEO disrupted the membrane integrity of S. putrescens. At the same time, changes in biofilm metabolic activity content and the growth curve of biofilm showed that GEO could destroy the biofilm. Both scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) observations confirmed that GEO destroyed the cell membrane and lead to the leakage of the constituents. The above results indicate that GEO entered the cells via contact with bacterial membranes, and then inhibited the growth of S. putrefaciens and its biofilms by increasing membrane permeability and inhibiting various virulence factors such as EPS. The findings showed that GEO could destroy the structure of cell membrane and biofilm of tested S. putrefaciens, indicating its potential as a natural food preservative.

Chiral and Structural Polymorphism of Fibril Architectures of Homologous Lysozymes.

Amyloid fibrils are fascinating and complex structures with the multilayered chiral organization. Using the multimodal methodology, including VCD, ECD, cryo-EM, and TEM, we characterized in detail different levels of organization (secondary structure/protofilament/mesoscopic structure) of amyloid fibrils prepared from proteins highly homologous in the structure (hen egg white and human lysozymes). Our results demonstrate that small changes in the native protein structure or preparation conditions translate into significant differences in the handedness and architecture of the formed fibrils at various levels of their complexity. In particular, fibrils of hen egg white and human lysozymes obtained in vitro at the same preparation conditions, possess different secondary structure, protofilament twist and ultrastructure. Yet, formed fibrils adopted a relatively similar mesoscopic structure, as observed in high-resolution 3D cryo-EM, scarcely used up to now for fibrils obtained in vitro in denaturing condition. Our results add to other puzzling experiments implicating the indeterministic nature of fibril formation.