Native Conformation Research Articles

CryoETGAN: Cryo-Electron Tomography Image Synthesis via Unpaired Image Translation.

Cryo-electron tomography (Cryo-ET) has been regarded as a revolution in structural biology and can reveal molecular sociology. Its unprecedented quality enables it to visualize cellular organelles and macromolecular complexes at nanometer resolution with native conformations. Motivated by developments in nanotechnology and machine learning, establishing machine learning approaches such as classification, detection and averaging for Cryo-ET image analysis has inspired broad interest. Yet, deep learning-based methods for biomedical imaging typically require large labeled datasets for good results, which can be a great challenge due to the expense of obtaining and labeling training data. To deal with this problem, we propose a generative model to simulate Cryo-ET images efficiently and reliably: CryoETGAN. This cycle-consistent and Wasserstein generative adversarial network (GAN) is able to generate images with an appearance similar to the original experimental data. Quantitative and visual grading results on generated images are provided to show that the results of our proposed method achieve better performance compared to the previous state-of-the-art simulation methods. Moreover, CryoETGAN is stable to train and capable of generating plausibly diverse image samples.

Structure-Guided Designing Pre-Organization in Bivalent Aptamers.

Multivalent interaction is often used in molecular design and leads to engineered multivalent ligands with increased binding avidities toward target molecules. The resulting binding avidity relies critically on the rigid scaffold that joins multiple ligands as the scaffold controls the relative spatial positions and orientations toward target molecules. Currently, no general design rules exist to construct a simple and rigid DNA scaffold for properly joining multiple ligands. Herein, we report a crystal structure-guided strategy for the rational design of a rigid bivalent aptamer with precise control over spatial separation and orientation. Such a pre-organization allows the two aptamer moieties simultaneously to bind to the target protein at their native conformations. The bivalent aptamer binding has been extensively characterized, and an enhanced binding has been clearly observed. This strategy, we believe, could potentially be generally applicable to design multivalent aptamers.

Curious Case of MAD2 Protein: Diverse Folding Intermediates Leading to Alternate Native States.

Anfinsen's dogma postulates that for one sequence there will be only one unique structure that is necessary for the functioning of the protein. However, over the years there have been a number of departures from this postulate. As far as function is considered, there are growing examples of proteins that "moonlight", perform multiple unrelated functions. With the discovery of intrinsically disordered proteins, morpheeins, chameleonic sequences, and metamorphic proteins that can switch folds, we have acquired a more nuanced understanding of protein folding and dynamics. Appearing to apparently contradict the classical folding paradigm, metamorphic proteins are considered exotic species. In this work, we have explored the free energy landscape and folding pathways of the metamorphic protein MAD2 which is an important component of the spindle checkpoint. It coexists in two alternate states: the inactive open state and the active closed state. Using a dual-basin structure-based model approach we have shown that a variety of intermediates and multiple pathways are available to MAD2 to fold into its alternate forms. This approach involves performing molecular dynamics simulations of coarse-grained models of MAD2 where the structural information regarding both of its native conformations is explicitly included in terms of their native contacts in the force field used. Detailed analyses have indicated that some of the contacts within the protein play a key role in determining which folding pathway will be selected and point to a probable long-range communication between the N and the C termini of the protein that seems to control its folding. Finally, our work also provides a rationale for the experimentally observed preference of the ΔC10 variant of MAD2 to exist in the open state.

A comparative assessment of collagen type 1 from silver carp (fresh water) and milk shark(marine) fish waste.

In this study, a comparative structural and bioactive analysis of collagen extracted from the skin of bony and cartilaginous fishes. The acid-soluble method was followed to extract the collagen from Hypophthalmichthys molitrix (silver carp) andRhizoprionodon acutus (milk shark) followed by purification using Ion exchange chromatography. A higher yield of collagen was obtained from the skin of SCsk (69.45%) as compared to SHsk (55.29%). SDS PAGE displayed the characteristic α, β bands of the collagen type1. The native conformation and secondary structure stability of collagen were confirmed by FTIR, XRD and CD studies. The SEM micrographs exhibited the layered and fibrillar nature of the collagen from SHsk and SCsk, respectively. Relative solubility and thermal denaturation analysis showed SCsk to be more stable, but SHsk could withstand higher temperatures. 53.65% of antioxidant activity was observed in SCsk collagen as compared to SHsk (45.9%). Haemocompatibility, cell viability and adhesion results also displayed silvercarp skin to be a better source than Shark skin collagen. The results establish the potential of silver carp collagen as a biomaterial that can have many commercial applications in tissue engineering, cosmetics and food industries.

Structure and receptor recognition by the Lassa virus spike complex.

Lassa virus (LASV) is a human pathogen, causing substantial morbidity and mortality1,2. Similar to other Arenaviridae, it presents a class-I spike complex on its surface that facilitates cell entry. The virus's cellular receptor is matriglycan, a linear carbohydrate that is present on α-dystroglycan3,4, but the molecular mechanism that LASV uses to recognize this glycan is unknown. In addition, LASV and other arenaviruses have a unique signal peptide that forms an integral and functionally important part of the mature spike5-8; yet the structure, function and topology of the signal peptide in the membrane remain uncertain9-11. Here we solve the structure of a complete native LASV spike complex, finding that the signal peptide crosses the membrane once and that its amino terminus is located in the extracellular region. Together with a double-sided domain-switching mechanism, the signal peptide helps to stabilize the spike complex in its native conformation. This structure reveals that the LASV spike complex is preloaded with matriglycan, suggesting the mechanism of binding and rationalizing receptor recognition by α-dystroglycan-tropic arenaviruses. This discovery further informs us about the mechanism of viral egress and may facilitate the rational design of novel therapeutics that exploit this binding site.

Protein Mutations and Stability, a Link with Disease: The Case Study of Frataxin.

Protein mutations may lead to pathologies by causing protein misfunction or propensity to degradation. For this reason, several studies have been performed over the years to determine the capability of proteins to retain their native conformation under stress condition as well as factors to explain protein stabilization and the mechanisms behind unfolding. In this review, we explore the paradigmatic example of frataxin, an iron binding protein involved in Fe–S cluster biogenesis, and whose impairment causes a neurodegenerative disease called Friedreich’s Ataxia (FRDA). We summarize what is known about most common point mutations identified so far in heterozygous FRDA patients, their effects on frataxin structure and function and the consequences of its binding with partners.

Optimization, Production, Purification and Characterization of HIV-1 GAG-Based Virus-like Particles Functionalized with SARS-CoV-2.

Virus-like particles (VLPs) constitute a promising approach to recombinant vaccine development. They are robust, safe, versatile and highly immunogenic supra-molecular structures that closely mimic the native conformation of viruses without carrying their genetic material. HIV-1 Gag VLPs share similar characteristics with wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, making them a suitable platform for the expression of its spike membrane protein to generate a potential vaccine candidate for COVID-19. This work proposes a methodology for the generation of SARS-CoV-2 VLPs by their co-expression with HIV-1 Gag protein. We achieved VLP functionalization with coronavirus spike protein, optimized its expression using a design of experiments (DoE). We also performed the bioprocess at a bioreactor scale followed by a scalable downstream purification process consisting of two clarifications, an ion exchange and size-exclusion chromatography. The whole production process is conceived to enhance its transferability at current good manufacturing practice (cGMP) industrial scale manufacturing. Moreover, the approach proposed could be expanded to produce additional Gag-based VLPs against different diseases or COVID-19 variants.

Proteomic analysis of <i>Pseudomonas chlororaphis</i> subsp. <i>aurantiacа</i> strains capable of phenasine compounds overproduction

Proteomic analysis is a highly effective method for bacteria identification and the elucidation of protein's content in prokaryotic cells at different growth conditions. To our knowledge this approach is hardly ever used for characterization of producers of biologically active substances. The understanding of the changes in protein profile in mutant strains capable of biologically active substances overproduction helps to recognize the biochemical and molecular basis of metabolic changes which lead to overproduction. So that, proteomic analysis could be especially useful for optimization the producer's creation techniques.The purpose of current research was to carry out proteomic profiling of bacteria P. chlororaphis subsp. aurantiaca mutant strains capable of overproduction of phenazine antibiotics. Microbiological and biochemical methods were used for these aims.In current research a proteomic analysis of strains of P. chlororaphis subsp. aurantiaca producing phenazines was carried out. An early (during log-phase) onset of expression of individual genes of phz-operon which codes enzymes for phenazines synthesis was demonstrated. It was also found that the wild type strain has the highest level of PhzO protein. The gene encoding this protein is located outside the phz-operon. We weren't able to establish the correlation among PhzO protein content and concentration of the derivatives for which appearance PhzO is responsible. A general tendency of producer strains towards the accumulation of enzymes and proteins of the antioxidant defense system was revealed. Producer strains also demonstrate a significant increase in the concentration of proteins involved in DNA repair as well as chaperones involved in the native protein conformation maintenance.

Protein Aggregation and Self Assembly in Health and Disease

: Self-attachment of proteins leading to the formation of highly insoluble protein oligomers and aggregates has become an important focus of research owing to its diverse implications in pathophysiology and diseases. This has become a more frequent phenomenon in most neurological and neurodegenerative diseases as well as in dementia. In recent years such event of protein aggregation has linked to other disease conditions, disorders or adverse health conditions. Interestingly, aggregation of protein also plays role in development, growth or metabolism. Most often physiological proteins are initially bio-synthesised in native or nascent geometrical forms or conformations but later they undergo specific folding pattern and thereby acquire a stable configuration that is biologically relevant and active. It is highly important that these proteins remain in their biologically active configuration in order to exert their functional properties. Any alteration or change to this structural configuration can be detrimental to their specific functions and may cause pathological consequences leading to the onset of diseases or disorders. Several factors such as the action of chaperones, binding partners, physiological metal ions, pH level, temperature, ionic strength, interfacial exposure (solid-liquid, liquid-liquid, gas-liquid), mutation and post translational modification, chemical changes, interaction with small molecules such as lipids, hormones, etc. and solvent environment have been either identified or proposed as important factors in conferring the ultimate status of protein structure and configuration. Among many misfolding protein conformations, self-assembly or aggregation is the most significant. It leads to the formation of highly oligomeric self-aggregates that precipitate and interfere with many biochemical processes with serious pathological consequences. The most common implication of protein aggregation leading to the formation of deposits / plaques of various morphological types is the onset of neurological and neurodegenerative diseases that include Alzheimer’s, Parkinson’s, Huntington, ALS (Amyotrophic Lateral Sclerosis), CJD (Creutzfeldt Jakob Dementia), Prion diseases, Amyloidosis and other forms of dementia. However increasingly studies revealed that protein aggregation may also be associated with other diseases such as cancer, type 2 diabetes, renal, corneal and cardiovascular diseases. Protein aggregation diseases are now considered as part of “Proteinopathy” which refers to conditions where proteins become structurally abnormal or fail to fold into stable normal configurations. In this review, we reflect on various aspects of protein self-aggregation, potential underlying causes, mechanism, role of secondary structures, pathological consequences and possible intervention strategies as reported in published literatures.

A Potential MDM2 Inhibitor Formed by Restoring the Native Conformation of the p53 α-Helical Peptide on Gold Nanoparticles.

Many efforts have been made to develop inhibitors of MDM2 as potential drugs for cancer therapy. In this work, we use our previous developed conformational engineering technique to stabilize the binding conformation of the p53 transcription activation domain (TAD) peptide on gold nanoparticles (AuNPs), and create an AuNP-based anti-MDM2 artificial antibody, denoted as anti-MDM2 Goldbody, that specifically binds MDM2. Though the free TAD peptide is unstructured, circular dichroism (CD) spectra confirm that its α-helical conformation in the original p53 protein is restored on the anti-MDM2 Goldbody, and surface plasmon resonance (SPR) experiments confirm that there is strong specific interaction between the anti-MDM2 Goldbody and MDM2, demonstrating the anti-MDM2 Goldbody as a potential inhibitor of MDM2. This work demonstrates that the conformational engineering technique is not limited to the antigen-antibody systems, but can also be applied more widely in other protein-protein interfaces to create increasingly more artificial proteins for various biomedical applications.

Structural and functional adaptation in extremophilic microbial α-amylases.

Maintaining stable native conformation of a protein under a given ecological condition is the prerequisite for survival of organisms. Extremophilic bacteria and archaea have evolved to adapt under extreme conditions of temperature, pH, salt, and pressure. Molecular adaptations of proteins under these conditions are essential for their survival. These organisms have the capability to maintain stable, native conformations of proteins under extreme conditions. The enzymes produced by the extremophiles are also known as extremozyme, which are used in several industries. Stability and functionality of extremozymes under varying temperature, pH, and solvent conditions are the most desirable requirement of industry. α-Amylase is one of the most important enzymes used in food, pharmaceutical, textile, and detergent industries. This enzyme is produced by diverse microorganisms including various extremophiles. Therefore, understanding its stability is important from fundamental as well as an applied point of view. Each class of extremophiles has a distinctive set of dominant non-covalent interactions which are important for their stability. Static information obtained by comparative analysis of amino acid sequence and atomic resolution structure provides information on the prevalence of particular amino acids or a group of non-covalent interactions. Protein folding studies give the information about thermodynamic and kinetic stability in order to understand dynamic aspect of molecular adaptations. In this review, we have summarized information on amino acid sequence, structure, stability, and adaptability of α-amylases from different classes of extremophiles.

Investigation of protective effect of ethanol on the natural structure of protein with infrared spectroscopy

The stability of biological drugs with protein as an active substance depends heavily on the retention of natural protein structure during freeze-drying. Stabilizers have become important substances in the process of protein freeze-drying. In order to further understand the mechanism of the interaction between protein and stabilizers, human serum albumin (HSA) and simple hydroxyl compound ethanol were used as models. Infrared (IR) spectroscopy combined with chemometrics was implemented to investigate the changes of secondary structure and hydration of HSA when different concentrations of ethanol were considered as interference. Through the analysis of the protein secondary structure and hydrated layer, we found that the addition of ethanol-d6 increased the α-helix of HSA and reduced the disordered structure. The hydrogen bond structure around HSA was enhanced and intermolecular aggregation was reduced through the action of the water molecules. The hypothesis was verified by circular dichroism (CD) and transmission electron microscopy (TEM) observation by adding different concentrations of ethanol-d6. It was found that a small amount of ethanol could protect the native conformation of HSA. In conclusion, this study revealed the mechanism of ethanol as a protein protector, provided a new idea for protein purification process and a theoretical basis for biomolecular interaction.

In vitro cytotoxicity, antibacterial activity and HSA and ct-DNA interaction studies of chlorogenic acid loaded on γ-Fe2O3@SiO2 as new nanoparticles

In this study, nanoparticles with both anticancer and antibacterial features were synthesized through loading chlorogenic acid (CGA) of essential oils on magnetic nanoparticles (MNPs). Characterization of γ-Fe2O3@SiO2-CGA MNPs was performed using Fourier transform infrared (FT-IR) spectroscopy and transmission electron microscopy (TEM) that show effective coating of the MNPs with SiO2 and CGA ligand and spherical shape of the nanoparticles with a mean diameter of 16 nm, respectively. The cytotoxicity study demonstrated that γ-Fe2O3@SiO2-CGA MNPs had fewer toxic effects on normal cells (Huvec) than on cancerous cells (U-87 MG, A-2780 and A-549), and could be a new potential candidate for use in biological and pharmaceutical applications. The interaction of calf thymus deoxyribonucleic acid (ct-DNA) with γ-Fe2O3@SiO2-CGA MNPs indicated that the anticancer activity might be associated with the DNA binding properties of γ-Fe2O3@SiO2-CGA MNPs. Moreover, the interaction of γ-Fe2O3@SiO2-CGA MNPs with human serum albumin (HSA) suggests that the native conformation of HSA was preserved at the level of secondary structure, indicating that the γ-Fe2O3@SiO2-CGA MNPs do not show any cytotoxicity effect when they are injected into the blood. Antibacterial tests were performed and represented γ-Fe2O3@SiO2-CGA MNPs attained better antibacterial function than CGA as free.

Inhibition of Polyglutamine Misfolding with D-Enantiomeric Peptides Identified by Mirror Image Phage Display Selection.

Nine heritable diseases are known that are caused by unphysiologically elongated polyglutamine tracts in human proteins leading to misfolding, aggregation and neurodegeneration. Current therapeutic strategies include efforts to inhibit the expression of the respective gene coding for the polyglutamine-containing proteins. There are, however, concerns that this may interfere with the physiological function of the respective protein. We aim to stabilize the protein’s native conformation by D-enantiomeric peptide ligands to prevent misfolding and aggregation, shift the equilibrium between aggregates and monomers towards monomers and dissolve already existing aggregates into non-toxic and functional monomers. Here, we performed a mirror image phage display selection on the polyglutamine containing a fragment of the androgen receptor. An elongated polyglutamine tract in the androgen receptor causes spinal and bulbar muscular atrophy (SBMA). The selected D-enantiomeric peptides were tested for their ability to inhibit polyglutamine-induced androgen receptor aggregation. We identified D-enantiomeric peptide QF2D-2 (sqsqwstpqGkwshwprrr) as the most promising candidate. It binds to an androgen receptor fragment with 46 consecutive glutamine residues and decelerates its aggregation, even in seeded experiments. Therefore, QF2D-2 may be a promising drug candidate for SBMA treatment or even for all nine heritable polyglutamine diseases, since its aggregation-inhibiting property was shown also for a more general polyglutamine target.

Stabilization and structure determination of integral membrane proteins by termini restraining.

Integral membrane proteins isolated from cellular environment often lose activity and native conformation required for functional analyses and structural studies. Even in their native state, they lack sufficient surfaces to form crystal contacts. Furthermore, most of them are too small for cryogenic electron microscopy detection and too big for solution NMR. To overcome these difficulties, we recently developed a strategy to stabilize the folded state of membrane proteins by restraining their two termini with a self-assembling protein coupler. The termini-restrained membrane proteins from distinct functional families retain their activities and show increased stability and yield. This strategy enables their structure determination at near-atomic resolution by facilitating the entire pipeline from crystallization, crystal identification, diffraction enhancement and phase determination, to electron density improvement. Furthermore, stabilization of membrane proteins enables their biochemical and biophysical characterization. Here we present the protocol of membrane protein engineering (2 weeks), quality assessment (1-2 weeks), protein production (1-6 weeks), crystallization (1-2 weeks), diffraction improvement (1-3 months) and crystallographic data analysis (1 week). This protocol is intended not only for structural biologists, but also for biochemists, biophysicists and pharmaceutical scientists whose research focuses on membrane proteins.

The design of TOPK inhibitors using structure-based pharmacophore modeling and molecular docking based on an MD-refined homology model.

The TOPK enzyme (also known as PBK) is a serine-threonine protein kinase that is rarely detected in normal tissues yet is found to be overexpressed and activated in a variety of cancers such as lung, colorectal, breast, and esophageal cancer. Its prevalence in cancerous cells is associated with their poor prognosis and responsiveness to treatment. This enzyme plays a vital role in cell division, specifically in regulating cytokinesis. Unlike drugs targeting early phases in mitosis, inhibition of cytokinesis by targeting biomolecules that are unique to multiplying cells poses no threat to the normal function of non-multiplying cells. Studies have shown that inhibition of cytokinesis is promising in suppressing the growth of proliferating cancerous cells as exemplified by the complete tumor regression seen with the suppression of TOPK. Herein, we report the identification of potent TOPK inhibitors with anticancer potential using a structure-based drug design approach. The only available crystal structure of TOPK corresponds to a double mutant (T9E and T198E) dimer with a distorted N-lobe conformation, thus 3D homology modeling was implemented to rebuild the enzyme's native conformation. The resulting refined model was used to generate 3D pharmacophore models for the virtual screening of small molecules databases. Retrieved hits were filtered, docked into the ATP binding site of the enzyme, rescored, and the binding free energies for the top consensually scoring hits were calculated. Consequently, 45 compounds were selected and their in vitro inhibitory activity against TOPK was tested revealing four potential hits with the most active compound having an IC50 of 3.85µM. This compound will be chosen as a lead compound to synthesize analogs aiming to enhance potency and drug-like properties and to enrich the SAR data.

Expression, Purification, and Refolding of Chikungunya Virus Full-Length Envelope E2 Protein along with B-Cell and T-Cell Epitope Analyses Using Immuno-Informatics Approaches.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which causes severe illness in humans and is responsible for epidemic outbreaks in Africa, Asia, North and South America, and Europe. Despite its increased global prevalence, no licensed vaccines are available to date for treating or preventing CHIKV infection. The envelope E2 protein is one of the promising subunit vaccine candidates against CHIKV. In this study, we describe successful cloning, expression, and purification of CHIKV E2 full-length (E2-FL) and truncated (E2-ΔC and E2-ΔNC) proteins in the Escherichia coli expression system. The recombinant E2 proteins were purified from inclusion bodies using Ni-NTA chromatography. Further, we describe a detailed refolding procedure for obtaining the CHIKV E2-FL protein in native conformation, which was confirmed using circular dichroism and Fourier transform infrared spectroscopy. BALB/c mice immunized with the three different E2 proteins exhibited increased E2-specific antibody titers compared to sham-immunized controls, suggesting induction of strong humoral immune response. On analyzing the E2-specific antibody response generated in immunized mice, the CHIKV E2-FL protein was observed to be the most immunogenic among the three different CHIKV E2 antigens used in the study. Our B-cell and T-cell epitope mapping results indicate that the presence of specific immunogenic peptides located in the N-terminal and C-terminal regions of the CHIKV E2-FL protein may contribute to its increased immunogenicity, compared to truncated CHIKV E2 proteins. In summary, our study provides a detailed protocol for expressing, purifying, and refolding of the CHIKV E2-FL protein and provides an understanding of its immunogenic epitopes, which can be exploited for the development of novel multiepitope-based anti-CHIKV vaccine strategies.

Immobilization of β-Galactosidase by Encapsulation of Enzyme-Conjugated Polymer Nanoparticles Inside Hydrogel Microparticles.

Increasing the shelf life of enzymes and making them reusable is a prominent topic in biotechnology. The encapsulation inside hydrogel microparticles (HMPs) can enhance the enzyme’s stability by preserving its native conformation and facilitating continuous biocatalytic processes and enzyme recovery. In this study, we present a method to immobilize β-galactosidase by, first, conjugating the enzyme onto the surface of polymer nanoparticles, and then encapsulating these enzyme-conjugated nanoparticles (ENPs) inside HMPs using microfluidic device paired with UV-LEDs. Polymer nanoparticles act as anchors for enzyme molecules, potentially preventing their leaching through the hydrogel network especially during swelling. The affinity binding (through streptavidin-biotin interaction) was used as an immobilization technique of β-galactosidase on the surface of polymer nanoparticles. The hydrogel microparticles of roughly 400 μm in size (swollen state) containing unbound enzyme and ENPs were produced. The effects of encapsulation and storage in different conditions were evaluated. It was discovered that the encapsulation in acrylamide (AcAm) microparticles caused an almost complete loss of enzymatic activity. Encapsulation in poly(ethylene glycol) (PEG)-diacrylate microparticles, on the other hand, showed a residual activity of 15–25%, presumably due to a protective effect of PEG during polymerization. One of the major factors that affected the enzyme activity was presence of photoinitiator exposed to UV-irradiation. Storage studies were carried out at room temperature, in the fridge and in the freezer throughout 1, 7 and 28 days. The polymer nanoparticles showcased excellent immobilization properties and preserved the activity of the conjugated enzyme at room temperature (115% residual activity after 28 days), while a slight decrease was observed for the unbound enzyme (94% after 28 days). Similar trends were observed for encapsulated ENPs and unbound enzyme. Nevertheless, storage at −26°C resulted in an almost complete loss of enzymatic activity for all samples.

Is Protein Folding a Thermodynamically Unfavorable, Active, Energy-Dependent Process?

The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy G. We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that ΔG of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process.

Amyloid and Hydrogel Formation of a Peptide Sequence from a Coronavirus Spike Protein

We demonstrate that a conserved coronavirus spike protein peptide forms amyloid structures, differing from the native helical conformation and not predicted by amyloid aggregation algorithms. We investigate the conformation and aggregation of peptide RSAIEDLLFDKV, which is a sequence common to many animal and human coronavirus spike proteins. This sequence is part of a native α-helical S2 glycoprotein domain, close to and partly spanning the fusion sequence. This peptide aggregates into β-sheet amyloid nanotape structures close to the calculated pI = 4.2, but forms disordered monomers at high and low pH. The β-sheet conformation revealed by FTIR and circular dichroism (CD) spectroscopy leads to peptide nanotape structures, imaged using transmission electron microscopy (TEM) and probed by small-angle X-ray scattering (SAXS). The nanotapes comprise arginine-coated bilayers. A Congo red dye UV–vis assay is used to probe the aggregation of the peptide into amyloid structures, which enabled the determination of a critical aggregation concentration (CAC). This peptide also forms hydrogels under precisely defined conditions of pH and concentration, the rheological properties of which were probed. The observation of amyloid formation by a coronavirus spike has relevance to the stability of the spike protein conformation (or its destabilization via pH change), and the peptide may have potential utility as a functional material. Hydrogels formed by coronavirus peptides may also be of future interest in the development of slow-release systems, among other applications.