Intermolecular Β-sheets Research Articles

Cryo-EM structure of a natural prion: chronic wasting disease fibrils from deer

Chronic wasting disease (CWD) is a widely distributed prion disease of cervids with implications for wildlife conservation and also for human and livestock health. The structures of infectious prions that cause CWD and other natural prion diseases of mammalian hosts have been poorly understood. Here we report a 2.8 Å resolution cryogenic electron microscopy-based structure of CWD prion fibrils from the brain of a naturally infected white-tailed deer expressing the most common wild-type PrP sequence. Like recently solved rodent-adapted scrapie prion fibrils, our atomic model of CWD fibrils contains single stacks of PrP molecules forming parallel in-register intermolecular β-sheets and intervening loops comprising major N- and C-terminal lobes within the fibril cross-section. However, CWD fibrils from a natural cervid host differ markedly from the rodent structures in many other features, including a ~ 180° twist in the relative orientation of the lobes. This CWD structure suggests mechanisms underlying the apparent CWD transmission barrier to humans and should facilitate more rational approaches to the development of CWD vaccines and therapeutics.

Thermally reversible emulsion gels and high internal phase emulsions based solely on pea protein for 3D printing

This work compared thermally reversible emulsion gels and high internal phase emulsions (HIPEs) based on pea protein for 3D printing at different oil volume fraction (ϕ) ranging from 0.2 to 0.8. Thermally reversible emulsion gels were obtained with ϕ from 0.2 to 0.65, which melt into liquid emulsion as temperature increased, and recovered into emulsion gels upon cooling as reflected by consistent rheological measurements. The Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed the transition between intermolecular β-sheet at 1630 cm−1 and intramolecular β-sheet at 1618 cm−1 through reversible H-bonds upon changes in temperature, which was responsible for the repeated thermo-reversibility. With ϕ increased to 0.74–0.8, HIPEs were confirmed by Scanning Electron Microscope (SEM) and Confocal Laser Scanning Microscope (CLSM), yet the thermo-reversibility was not maintained. In addition, the thermally reversible emulsion gels exhibited strong shear-thinning and thixotropic recovery through the untangling and reformation of the H-bonded gel network. Whereas deformation and restructuring of the close packed droplets were responsible for such behaviors in HIPEs. 3D printing test has further demonstrated excellent performance of thermally reversible emulsion-filled gel with ϕ 0.4, comparable to HIPE with ϕ 0.74 b y enabling printed objects with high shape fidelity (printability index (Pr) of 0.95–1.08) and shape stability (prints height maintenance (Hm) of 82.2%–87.8%). This research opens up thermally reversible emulsion gels solely from sustainable protein source for 3D printing, with low oil content and no synthetic ingredients needed.

Study of Monoclonal Antibody Aggregation at the Air-Liquid Interface under Flow by ATR-FTIR Spectroscopic Imaging.

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular β-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.

The 75-99 C-Terminal Peptide of URG7 Protein Promotes α-Synuclein Disaggregation.

Up Regulation Gene seven (URG7) is the pseudogene 2 of the transporter ABCC6. The translated URG7 protein is localized with its single transmembrane α-helix in the endoplasmic reticulum (ER) membrane, orienting the N- and C-terminal regions in the lumen and cytoplasm, respectively, and it plays a crucial role in the folding of ER proteins. Previously, the C-terminal region of URG7 (PU, residues 75-99) has been shown to modify the aggregation state of α-synuclein in the lysate of HepG2 cells. PU analogs were synthesized, and their anti-aggregation potential was tested in vitro on α-synuclein obtained using recombinant DNA technology. Circular dichroism (CD), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and microscopic techniques were used to assess the sample's behavior. The results show that the peptides studied by themselves are prone to clathrate-like structure formation of variable stability. Aggregation of α-synuclein is accompanied by desolvation of its peptide chain and an increase in intermolecular β-sheets. The PU analogs all interact with α-synuclein aggregates and those possessing the most stable clathrate-like structures have the highest disaggregating effect. These findings suggest that the C-terminal region of URG7 may have a role in interacting and modulating α-synuclein structures and could be used to generate interesting therapeutic candidates as disaggregators of α-synuclein.

Study on the anti-retrogradation of wheat amylopectin by addition of alkali-soluble glutenin

The retrogradation of wheat amylopectin during cold storage is the main reason for the increasing hardness of flour products such as steamed bread, bread and pastries, etc. Addition of gluten protein components is a green, safe, cheap and efficient method to inhibit the retrogradation of wheat amylopectin. In this paper, as being stored at 4 °C for 7 d, retrogradation rate of wheat amylopectin decreased from 55.02 % to 14.37 % after it was mixed with 20 % alkali-soluble glutenin (ASG) at 30 °C for 90 min, a 73.8 % reduction. The infrared results showed that the intensity of bending vibration of water molecules and intra-molecular β-sheet content of ASG decreased during the interaction between amylopectin and ASG. Meanwhile, intermolecular β-sheet and random coil contents of ASG increased. The results of 13C Solid-state NMR indicated that Qβ, Pγ and Lγ of ASG involved in interaction of wheat amylopectin, ASG and molecule of water. Under the optimal conditions, the interaction of wheat amylopectin and ASG began to form spheres containing disulfide bonds, resulting in the attenuation or disappearance of the diffraction peak at 2θ 19.7°, which may be marked as the criterion for the best mixing time of wheat amylopectin and ASG. The retrogradation kinetic index (n) of wheat amylopectin decreased significantly with the addition of ASG and formation of disulfide bond was the key factor. ASG could be potentially used as an anti-retrogradation agent for amylopectin.

Structural evidence for protein-protein interaction between the non-canonical methyl-CpG-binding domain of SETDB proteins and C11orf46

SETDB1 and SETDB2 mediate trimethylation of histone H3 lysine 9 (H3K9), an epigenetic hallmark of repressive chromatin. They contain a non-canonical methyl-CpG-binding domain (MBD) and bifurcated SET domain, implying interplay between H3K9 trimethylation and DNA methylation in SETDB functions. Here, we report the crystal structure of human SETDB2 MBD bound to the cysteine-rich domain of a zinc-binding protein, C11orf46. SETDB2 MBD comprises the conserved MBD core and a unique N-terminal extension. Although the MBD core has the conserved basic concave surface for DNA binding, it utilizes it for recognition of the cysteine-rich domain of C11orf46. This interaction involves the conserved arginine finger motif and the unique N-terminal extension of SETDB2 MBD, with a contribution from intermolecular β-sheet formation. Thus, the non-canonical MBD of SETDB1/2 seems to have lost methylated DNA-binding ability but gained a protein-protein interaction surface. Our findings provide insight into the molecular assembly of SETDB-associated repression complexes.

Glucose-Triggered Gelation of Supramolecular Peptide Nanocoils with Glucose-Binding Motifs.

Peptide self-assembly is a powerful tool to prepare functional materials at the nanoscale. Often, the resulting materials have high aspect-ratio, with intermolecular β-sheet formation underlying 1D fibrillar structures. Inspired by dynamic structures in nature, peptide self-assembly is increasingly moving toward stimuli-responsive designs wherein assembled structures are formed, altered, or dissipated in response to a specific cue. Here, a peptide bearing a prosthetic glucose-binding phenylboronic acid (PBA) is demonstrated to self-assemble into an uncommon nanocoil morphology. These nanocoils arise from antiparallel β-sheets, with molecules aligned parallel to the long axis of the coil. The binding of glucose to the PBA motif stabilizes and elongates the nanocoil, driving entanglement and gelation at physiological glucose levels. The glucose-dependent gelation of these materials is then explored for the encapsulation and release of a therapeutic agent, glucagon, that corrects low blood glucose levels. Accordingly, the release of glucagon from the nanocoil hydrogels is inversely related to glucose level. When evaluated in a mouse model of severe acute hypoglycemia, glucagon delivered from glucose-stabilized nanocoil hydrogels demonstrates increased protection compared to delivery of the agent alone or within a control nanocoil hydrogel that is not stabilized by glucose.

β-Strand-mediated domain-swapping in the absence of hydrophobic core repacking

Domain swapping is a process wherein a portion of a protein is exchanged with its counterpart in another copy of the molecule, resulting in the formation of homo-oligomers with concomitant repacking of a hydrophobic core. Here, we report domain swapping triggered upon modifying a β-hairpin sequence within a single-layer β-sheet (SLB) of a model protein, OspA that did not involve the formation of a reorganized hydrophobic core. The replacement of two β-hairpin sequences with a Gly-Gly and shorteing of a β-hairpin resulted in a protein that formed two distinct crystal structures under similar conditions: one was monomeric, similar to the parental molecule, whereas the other was a domain-swapped dimer, mediated by an intermolecular β-sheet in the SLB portion. Based on the dimer interface structure, we replaced the Gly-Gly sequence with three-residue sequences that enable the formation of a consecutive intermolecular β-sheet, including the Cys-Thr-Cys sequence that formed a stable disulfide-linked dimer. These results provide new insights into protein folding, evolution, and the designability of protein structure.

Effect of High-Hydrostatic-Pressure Treatment on the Physicochemical Properties of Kafirin.

The kafirin derived from Jin Nuo 3 sorghum underwent a high-hydrostatic-pressure (HHP) treatment of 100, 300, and 600 MPa for 10 min to investigate alterations in its physicochemical attributes. The findings exhibited a reduction in protein solubility, declining from 83% to 62%, consequent to the application of the HHP treatment. However, this treatment did not lead to subunit-specific aggregation. The absorption intensity of UV light diminished, and the peak fluorescence absorption wavelength exhibited a shift from 342 nm to 344 nm, indicating an increased polarity within the amino acid microenvironment. In an aqueous solution, the specific surface area expanded from 294.2 m2/kg to 304.5 m2/kg, while the average particle-size value in a 70% ethanol solution rose to 26.3 nm. Conversely, the zeta-potential value decreased from 3.4 mV to 1.3 mV, suggesting a propensity for aggregation in ethanol solutions. A notable rise in the intermolecular β-sheet content to 21.06% was observed, along with a shift in the peak denaturation temperature from 76.33 °C to 86.33 °C. Additionally, the content of disulfide bonds increased to 14.5 μmol/g. Collectively, the application of the HHP treatment not only enhanced the thermal stability but also induced a more ordered secondary structure within the kafirin.

Ready for the sheet: β-strand folding of phosphorylation clusters guides GPCR binding to arrestin

The molecular dynamics of arrestin binding to G protein-coupled receptors (GPCRs) are still poorly understood. In this issue of Structure, Guillien etal. show that negative charges in GPCR key phosphorylation clusters induce the formation of a transient β-strand that participates in an intermolecular β-sheet in the associated complex.

Influence of pH on Heat-Induced Changes in Skim Milk Containing Various Levels of Micellar Calcium Phosphate.

The present study investigated the effect of micellar calcium phosphate (MCP) content and pH of skim milk on heat-induced changes in skim milk. Four MCP-adjusted samples, ranging from 67 to 113% of the original MCP content, were heated (90 °C for 10 min) at different pH values (6.3, 6.6, 6.9, and 7.2), followed by determining changes in particle size, turbidity, protein distribution, and structure. The results demonstrate a strong effect of MCP level and pH on heat-induced changes in milk, with the MCP67 samples revealing the greatest thermal stability. Specifically, decreasing MCP content by 33% (MCP67) led to a smaller increase in non-sedimentable κ-casein and a lower decrease in αs2-casein concentrations after heating compared to other samples. Lower MCP content resulted in a moderate rise in the average particle size and turbidity, along with lower loading of β-turn structural component after heating at low pH (pH 6.3). Notably, MCP113 exhibited instability upon heating, with increased particle size, turbidity, and a significant decrease in non-sedimentable αs2-casein concentration, along with a slight increase in non-sedimentable κ-casein concentration. The FTIR results also revealed higher loading of intermolecular β-sheet, β-turn, and random coil structures, as well as lower loading of α-helix and β-sheet structures in MCP-enhanced skim milk samples. This suggests significant changes in the secondary structure of milk protein and greater formation of larger aggregates.

Three-Dimensional Printing Parameter Optimization for Salmon Gelatin Gels Using Artificial Neural Networks and Response Surface Methodology: Influence on Physicochemical and Digestibility Properties.

This study aimed to optimize the 3D printing parameters of salmon gelatin gels (SGG) using artificial neural networks with the genetic algorithm (ANN-GA) and response surface methodology (RSM). In addition, the influence of the optimal parameters obtained using the two different methodologies was evaluated for the physicochemical and digestibility properties of the printed SGG (PSGG). The ANN-GA had a better fit (R2 = 99.98%) with the experimental conditions of the 3D printing process than the RSM (R2 = 93.99%). The extrusion speed was the most influential parameter according to both methodologies. The optimal values of the printing parameters for the SGG were 0.70 mm for the nozzle diameter, 0.5 mm for the nozzle height, and 24 mm/s for the extrusion speed. Gel thermal properties showed that the optimal 3D printing conditions affected denaturation temperature and enthalpy, improving digestibility from 46.93% (SGG) to 51.52% (PSGG). The secondary gel structures showed that the β-turn structure was the most resistant to enzymatic hydrolysis, while the intermolecular β-sheet was the most labile. This study validated two optimization methodologies to achieve optimal 3D printing parameters of salmon gelatin gels, with improved physicochemical and digestibility properties for use as transporters to incorporate high value nutrients to the body.

Dissociation process of polyalanine aggregates by free electron laser irradiation.

Polyalanine (polyA) disease-causative proteins with an expansion of alanine repeats can be aggregated. Although curative treatments for polyA diseases have not been explored, the dissociation of polyA aggregates likely reduces the cytotoxicity of polyA. Mid-infrared free electron laser (FEL) successfully dissociated multiple aggregates. However, whether the FEL dissociates polyA aggregates like other aggregates has not been tested. Here, we show that FEL at 6.1 μm experimentally weakened the extent of aggregation of a peptide with 13 alanine repeats (13A), and the irradiated 13A exerted lesser cytotoxicity to neuron-like cells than non-irradiated 13A. Then, we applied molecular dynamics (MD) simulation to follow the dissociation process by FEL. We successfully observed how the intermolecular β-sheet of polyA aggregates was dissociated and separated into monomers with helix structures upon FEL irradiation. After the dissociation by FEL, water molecules inhibited the reformation of polyA aggregates. We recently verified the same dissociation process using FEL-treated amyloid-β aggregates. Thus, a common mechanism underlies the dissociation of different protein aggregates that cause different diseases, polyA disease and Alzheimer's disease. However, MD simulation indicated that polyA aggregates are less easily dissociated than amyloid-β aggregates and require longer laser irradiation due to hydrophobic alanine repeats.

Study on the mechanism of structure modification of amylopectin co-crystalized by sodium chloride to promote disulfide bond formation of alkali-soluble glutenin

Strength and stiffness are the main characteristics of high-quality dough products. As one of the key components to endow those functions to noodles, formation of disulfide bonds in alkali-soluble glutenin (ASG) is very important. Interaction of ASG and co-crystalized wheat amylopectin with NaCl could prompt formation of disulfide bonds of ASG and its mechanism was deduced in the paper. The results showed that the disulfide bonds contents of ASG were increased significantly by interaction with co-crystalized wheat amylopectin. The co-crystalized wheat amylopectin with 2% NaCl for 24 h at 30 °C was distinguished by the Mn distribution of 0.55–0.95 × 106 g/mol, more chains with 18∼24 glucose residues being grafted from chains of 4∼7 glucose residues during the eutectic process, presence of the weak resonance enhancement at 96.5 ppm, molecular aggregate diameter less than 120 μm, and only one of X-diffraction angles 2θ at 20.2°. In the process of interaction, Qβ, Qδ, Yγ, Yε of ASG and C1, C6 of what amylopectin involved in the formation of disulfide bond, the contents of intermolecular β-sheet decreased and those of intra-molecular aggregation extended β-sheet and random coils increased. The peak melting temperature and melting enthalpy of the mixture decreased significantly after co-crystallized wheat amylopectin interacted with the ASG. Co-crystalized wheat amylopectin with NaCl can be used as a reinforcing agent of wheat flour.

Effects of CaCl2 on the structure of high-density lipoprotein and low-density lipoprotein isolated from rapidly salted separated egg yolk

According to previous research, adding CaCl2 to the salting solution improves the quality of salted separated egg yolk. To further understand the improvement mechanism of CaCl2, this paper investigated the effect of CaCl2 on the structure of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) during the salting process. The results indicated that the addition of CaCl2 can affect the composition of HDL and LDL apolipoproteins, improving the orderliness of the HDL structure and the looseness of the LDL structure. It was discovered by atomic force microscopy (AFM) that adding CaCl2 to the salting solution can weaken the aggregation behavior of HDL. Simultaneously, the addition of CaCl2 decreased the relative content of intermolecular β-sheets in the secondary structure of HDL and LDL, influenced their tertiary conformation, and prevented HDL and LDL from participating in the formation of a three-dimensional gel structure by influencing their hydrogen bonds and hydrophobic interactions.

Effect of High-Molecular Weight Glutenin Subunits (HMW-GSs) on Gluten Polymerization during Biscuit Making: Insights from Experimental and Molecular Dynamics Simulation Study.

The effect of high-molecular weight glutenin subunits (HMW-GSs) on gluten polymerization during biscuit making was investigated using a set of HMW-GS deletion lines. Results showed that the deletion of HMW-GSs improved the biscuit quality compared with the wild type (WT), especially in x-type HMW-GS deletion lines. Slight gluten depolymerization was observed during dough mixing, while progressive gluten polymerization occurred during biscuit baking. The deletion of HMW-GSs suppressed the polymerization of glutenin and gliadin compared with the WT during biscuit baking, especially in x-type HMW-GS deletion lines. These actions resulted in less elevation of the intermolecular β-sheet and ordered α-helix and altering the disulfide (SS) conformation to a less stable conformation in HMW-GS deletion lines compared with the WT during baking. Molecular dynamics simulation analysis further demonstrated that x-type HMW-GSs had higher thermal stability compared with y-type HMW-GSs during heating.

Age-Related Fourier-Transform Infrared Spectroscopic Changes in Protein Conformation in an Aging Model of Human Dermal Fibroblasts

The loss of proteostasis, which results in the accumulation of misfolded proteins, is one of the hallmarks of aging and is frequently associated with the aging process. Fibroblasts are a cellular model widely used in the study of aging and can mimic the loss of proteostasis that occurs in the human body. When studying human aging using fibroblasts, two approaches can be used: fibroblasts from the same donor aged in vitro until senescence or fibroblasts from donors of different ages. A previous study by our group showed that the first approach can be used in the study of aging. Thus, this work aimed to study the spectroscopic profile of human dermal fibroblasts from four donors of different ages using Fourier-transform infrared spectroscopy to identify changes in protein conformation and to compare results with those obtained in the previous study. Partial least squares regression analysis and peak intensity analysis suggested that fibroblasts from older donors were characterized by an increase in the levels of antiparallel β-sheets and a decrease in intermolecular β-sheets, in agreement with our previous results.

Impact of Cavitation Jet on the Structural, Emulsifying Features and Interfacial Features of Soluble Soybean Protein Oxidized Aggregates

A cavitation jet can enhance food proteins' functionalities by regulating solvable oxidized soybean protein accumulates (SOSPI). We investigated the impacts of cavitation jet treatment on the emulsifying, structural and interfacial features of soluble soybean protein oxidation accumulate. Findings have shown that radicals in an oxidative environment not only induce proteins to form insoluble oxidative aggregates with a large particle size and high molecular weight, but also attack the protein side chains to form soluble small molecular weight protein aggregates. Emulsion prepared by SOSPI shows worse interface properties than OSPI. A cavitation jet at a short treating time (<6 min) has been shown to break the core aggregation skeleton of soybean protein insoluble aggregates, and insoluble aggregates into soluble aggregates resulting in an increase of emulsion activity (EAI) and constancy (ESI), and a decrease of interfacial tension from 25.15 to 20.19 mN/m. However, a cavitation jet at a long treating time (>6 min) would cause soluble oxidized aggregates to reaggregate through an anti-parallel intermolecular β-sheet, which resulted in lower EAI and ESI, and a higher interfacial tension (22.44 mN/m). The results showed that suitable cavitation jet treatment could adjust the structural and functional features of SOSPI by targeted regulated transformation between the soluble and insoluble components.

Structural basis for membrane attack complex inhibition by CD59

CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.

Phosphorylation at Ser289 Enhances the Oligomerization of Tau Repeat R2.

In tauopathies such as Alzheimer's disease (AD), aberrant phosphorylation causes the dissociation of tau proteins from microtubules. The dissociated tau then aggregates into sequent forms from soluble oligomers to paired helical filaments and insoluble neurofibrillary tangles (NFTs). NFTs is a hallmark of AD, while oligomers are found to be the most toxic form of the tau aggregates. Therefore, understanding tau oligomerization with regard to abnormal phosphorylation is important for the therapeutic development of AD. In this study, we investigated the impact of phosphorylated Ser289, one of the 40 aberrant phosphorylation sites of full-length tau proteins, on monomeric and dimeric structures of tau repeat R2 peptides. We carried out intensive replica exchange molecular dynamics simulation with a total simulation time of up to 0.1 ms. Our result showed that the phosphorylation significantly affected the structures of both the monomer and the dimer. For the monomer, the phosphorylation enhanced ordered-disordered structural transition and intramolecular interaction, leading to more compactness of the phosphorylated R2 compared to the wild-type one. As to the dimer, the phosphorylation increased intermolecular interaction and β-sheet formation, which can accelerate the oligomerization of R2 peptides. This result suggests that the phosphorylation at Ser289 is likely to promote tau aggregation. We also observed a phosphorylated Ser289-Na+-phosphorylated Ser289 bridge in the phosphorylated R2 dimer, suggesting an important role of cation ions in tau aggregation. Our findings suggest that phosphorylation at Ser289 should be taken into account in the inhibitor screening of tau oligomerization.