Self-assembly Of Proteins Research Articles

Conformational switch and multiple supramolecular structures of a newly identified self-assembling protein-mimetic peptide from Pseudomonas aeruginosa YeaZ protein.

Protein-mimetic peptides (PMPs) are shorter sequences of self-assembling proteins, that represent remarkable building blocks for the generation of bioinspired functional supramolecular structures with multiple applications. The identification of novel aminoacidic sequences that permit the access to valuable biocompatible materials is an attractive area of research. In this work, in silico analysis of the Pseudomonas aeruginosa YeaZ protein (PaYeaZ) led to the identification of a tetradecapeptide that represents the shortest sequence responsible for the YeaZ-YeaZ dimer formation. Based on its sequence, an innovative 20-meric peptide, called PMP-2, was designed, synthesized, and characterized in terms of secondary structure and self-assembly properties. PMP-2 conserves a helical character and self-assembles into helical nanofibers in non-polar solvents (DMSO and trifluoroethanol), as well as in dilute (0.5mM) aqueous solutions. In contrast, at higher concentrations (>2mM) in water, a conformational transition from α-helix to β-sheet occurs, which is accompanied by the Protein-mimetic peptide aggregation into 2D-sheets and formation supramolecular gel in aqueous environment. Our findings reveal a newly identified Protein-mimetic peptide that could turn as a promising candidate for future material applications.

Self-Assembly of Amyloid-Beta (Aβ) Peptides from Solution to Near In Vivo Conditions.

Understanding the atomistic resolution changes during the self-assembly of amyloid peptides or proteins is important to develop compounds or conditions to alter the aggregation pathways and suppress the toxicity and potentially aid in the development of drugs. However, the complexity of protein aggregation and the transient order/disorder of oligomers along the pathways to fibril are very challenging. In this Perspective, we discuss computational studies of amyloid polypeptides carried out under various conditions, including conditions closely mimicking in vivo and point out the challenges in obtaining physiologically relevant results, focusing mainly on the amyloid-beta Aβ peptides.

Fabrication and characterization of self-assembled whey protein isolate/short linear glucan core–shell nanoparticles for sustained release of curcumin

The aim of this research was to prepare a bistratal nanocomplex with a high loading capacity (LC) and harsh environment stability for controlled release of curcumin (Cur) in gastrointestinal conditions. Whey protein isolate (WPI)/short linear glucan (SLG) core–shell nanoparticles were fabricated by self-assembly for the delivery of Cur. The results showed that Cur@WPI@SLG nanoparticles had a relatively high LC (12.89 %) and small particle size (89.4 nm). The nanocomplex remained relatively stable in extreme pH conditions (2–4 and 8–10), high temperatures (60–70 °C), and ionic strength (<400 mM). Core–shell nanostructures facilitated the sustained release of Cur in simulated gastrointestinal conditions. In addition, the nanocomplex had little cytotoxicity at high concentrations, yet significantly enhanced the DPPH scavenging activity and reducing power of Cur. This delivery system will significantly improve the sustained release effect of Cur and broaden the application of hydrophobic nutrients in foods.

Antigenicity and immunogenicity of chikungunya virus-like particles from mosquito cells.

The spread of chikungunya virus (CHIKV) is reaching pandemic levels, and vaccines and antivirals to control CHIKV infection have yet to be approved. Virus-like particles (VLPs), a self-assembled native multi-subunit protein structure, could potentially be used as an antigen for serological detection and vaccine development. In the current study, we describe the production of novel CHIKV VLPs from mosquitoes using a Baculovirus/Mosquito (BacMos) system in a simple Biosafety Level-2 laboratory. Substantial envelope and capsid protein secretions were detected in culture medium. Co-fractionation of CHIKV E2, E1, and capsid proteins via sucrose gradient ultracentrifugation provided evidence of VLP formation. Transmission electron microscopy and dynamic light scattering analysis revealed the formation of VLPs in the form of spherical particles with a diameter of roughly 40nm in transduced cells and culture medium. VLP-based IgM capture ELISA in CHIKV patient sera revealed native epitopes on the VLPs. These non-purified VLPs were shown to act as an antigen in CHIKV-specific IgM capture ELISA. The immunization of CHIKV-VLPs alone in mice induced a balance CHIKV-specific IgG2a/IgG1 antibodies and neutralized antibody responses. The study provides support for the hypothesis that mosquito cell-derived CHIKV VLPs could serve as a novel antigen for serological detection and the development of vaccines against CHIKV infection. KEY POINTS: • CHIKV VLPs secreted from BacMos-CHIKV 26S-transduced mosquito cell. • This CHIKV VLPs potentially serve as an alternative capture antigen for MAC-ELISA. • Unadjuvanted CHIK VLPs induce CHIKV-specific IgG and NT responses in mice.

Organizing Enzymes on Self-Assembled Protein Cages for Cascade Reactions.

Cells use self-assembled biomaterials such as lipid membranes or proteinaceous shells to coordinate thousands of reactions that simultaneously take place within crowded spaces. However, mimicking such spatial organization for synthetic applications in engineered systems remains a challenge, resulting in inferior catalytic efficiency. In this work, we show that protein cages as an ideal scaffold to organize enzymes to enhance cascade reactions both in vitro and in living cells. We demonstrate that not only enzyme-enzyme distance but also the improved Km value contribute to the enhanced reaction rate of cascade reactions. Three sequential enzymes for lycopene biosynthesis have been co-localized on the exterior of the engineered protein cages in Escherichia coli, leading to an 8.5-fold increase of lycopene production by streamlining metabolic flux towards its biosynthesis. This versatile system offers a powerful tool to achieve enzyme spatial organization for broad applications in biocatalysis.

Conserved GTPase mechanism in bacterial FtsZ and archaeal tubulin filaments.

Self-assembling protein filaments are at the heart of cell function. Among them, tubulin-like proteins are essential for cell division, DNA segregation and cytoskeletal functions across the domains of life. FtsZ and tubulin share their core structures, a characteristic nucleotide-binding pocket and similar protofilament architecture. GTP hydrolysis between consecutive subunits drives their assembly dynamics. Two recent studies provide previously missing, filament atomic structures of bacterial FtsZ and a recently discovered archaeal tubulin in their nucleotide triphosphate-bound states. Both filament structures reveal strikingly conserved interfacial GTPase active sites, with Mg2+ and K+ /Na+ cations and an NxDxxD/E triad of catalytic residues, probably inherited from the common ancestor of FtsZsand tubulins. Moreover, both proteins exhibit nucleotide-regulated subunit association mediated by interfacial water bridges, as well as polymerization-induced structural changes, likely enabling related dynamic assembly mechanisms.

Vaccination Strategies Based on Bacterial Self-Assembling Proteins as Antigen Delivery Nanoscaffolds.

Vaccination has saved billions of human lives and has considerably reduced the economic burden associated with pandemic and endemic infectious diseases. Notwithstanding major advancements in recent decades, multitude diseases remain with no available effective vaccine. While subunit-based vaccines have shown great potential to address the safety concerns of live-attenuated vaccines, their limited immunogenicity remains a major drawback that still needs to be addressed for their use fighting infectious illnesses, autoimmune disorders, and/or cancer. Among the adjuvants and delivery systems for antigens, bacterial proteinaceous supramolecular structures have recently received considerable attention. The use of bacterial proteins with self-assembling properties to deliver antigens offers several advantages, including biocompatibility, stability, molecular specificity, symmetrical organization, and multivalency. Bacterial protein nanoassemblies closely simulate most invading pathogens, acting as an alarm signal for the immune system to mount an effective adaptive immune response. Their nanoscale architecture can be precisely controlled at the atomic level to produce a variety of nanostructures, allowing for infinite possibilities of organized antigen display. For the bottom-up design of the proteinaceous antigen delivery scaffolds, it is essential to understand how the structural and physicochemical properties of the nanoassemblies modulate the strength and polarization of the immune responses. The present review first describes the relationships between structure and the generated immune responses, before discussing potential and current clinical applications.

The gel mechanism and carrier quality of fibrous and granular whey protein self-assembly

In this study, we induced a 6% protein concentration of whey protein (WP) to form fibrous and granular whey protein self-assembly (FWS and GWS respectively) by a pH-temperature gradient. The fibrous and granular whey protein self-assembly were used as raw material to explore the mechanism of gel formation and their potential a new delivery system loaded with Epigallocatechin gallate (EGCG) was evaluated. Compared with native WP, the α-helix decreased, and the β-sheet increased in FWS, while the opposite was found in GWS. The hydrophobic interaction played a crucial role in the formation process of FWS and GWS, while disulfide bonds played a slightly more vital role in the formation process of GWS. The gels prepared by FWS and GWS had denser microstructure and stronger mechanical strength than native WP gel. Both the water holding capacity and hardness of the gels prepared by FWS and GWS were significantly higher than those prepared by native WP. The water holding capacity of the gel prepared by FWS was higher than that of the gel prepared by GWS at the same protein concentration, but the hardness results were the opposite. Hydrophobic interaction and hydrogen bonds played a dominant role during the gel formation process of FWS and GWS, while disulfide bonds only played a role in the gel formation process of GWS. In addition, the gel formed by FWS and GWS had higher epigallocatechin gallate encapsulation efficiency (94.40% and 95.09%, respectively) and a slow control effect during digestion. Therefore, gel prepared by FWS and GWS can provide essential ideas for building new protein gel technology and is expected to be an ideal carrier for bioactive substances.

Self-Assembled Protein–Surfactant Nanoaggregates for Tunable Peptide Bond Hydrolysis by Polyoxometalate Nanoclusters

Self-Assembled Protein–Surfactant Nanoaggregates for Tunable Peptide Bond Hydrolysis by Polyoxometalate Nanoclusters

Therapeutic and Diagnostic Approaches by using Nanotechnology in SARS-CoV-2 Infections

Severe Acute Respiratory Syndrome Corona Virus-2 infection is a universal threat in recent days, hence early diagnosis and treatment play a pivotal role in controlling the spread thereby preventing them to become endemic. A newer promising approach by Nanotechnology plays an essential role in targeting the specific pathogens for therapeutic and diagnosis of Viral infection. Certain Nano platforms like Microneedle array delivered Virus S1 subunit vaccines, spike protein nanoparticles, Lumazine synthase Nanoparticles, Silver Nanoparticles, Self-Assembling Protein Nanoparticles against Viral therapy are the upcoming applications as a therapeutic approach. Nucleic acid amplification techniques and Surface-enhanced Raman Spectroscopy shows a high specificity with the immunoassay strategy. In recent days, Colloidal Gold – Nanoparticles and silicon nanoparticles have been widely used as a point of care for quick detection of IgG and IgM antibodies obtained from the virus as a diagnostic approach. Additionally, the Nanoparticles serve as a significant improvement in Personal Protective Equipment and protect against exposure to the virus. As a result of repurposing as well as for the development of the drug, apparently, Nanoparticles themselves and their concomitant therapy or their carriers will be advantageous in making a therapeutic and diagnostic approach against Severe Acute Respiratory Syndrome Corona Virus-2 infections.

An Additive-Free Model for Tau Self-Assembly.

Tau is a natively unfolded protein that contributes to the stability of microtubules. Under pathological conditions such as Alzheimer's disease (AD), tau protein misfolds and self-assembles to form paired helical filaments (PHFs) and straight filaments (SFs). Full-length tau protein assembles poorly and its self-assembly is enhanced with polyanions such as heparin and RNA in vitro, but a role for heparin or other polyanions in vivo remains unclear. Recently, a truncated form of tau (297-391) has been shown to self-assemble in the absence of additives which provides an alternative in vitro PHF model system. Here we describe methods to prepare in vitro PHFs and SFs from tau (297-391) named dGAE. We also discuss the range of biophysical/biochemical techniques used to monitor tau filament assembly and structure.

Neuronal Puncta/Aggregate Formation by WT and Mutant UBQLN2.

Protein aggregates are a common feature of nearly all neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Here we describe a method to quickly and accurately measure protein aggregation in cells expressing a fluorescently tagged aggregation-prone protein. This unbiased method obviates the need for manual scoring and facilitates the identification of factors governing protein self-assembly and its downstream consequences for cell heath.

Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH.

Hepatoviruses are atypical hepatotropic picornaviruses that are released from infected cells without lysis in small membranous vesicles. These exosome-like, quasi-enveloped virions (eHAV) are infectious and the only form of hepatitis A virus (HAV) found circulating in blood during acute infection. eHAV is released through multivesicular endosomes in a process dependent on endosomal sorting complexes required for transport (ESCRT). Capsid protein interactions with the ESCRT-associated Bro1 domain proteins, ALG-2-interacting protein X (ALIX) and His domain-containing protein tyrosine phosphatase (HD-PTP), which are both recruited to the pX domain of 1D (VP1pX), are critical for this process. Previous proteomics studies suggest pX also binds the HECT domain, NEDD4 family E3 ubiquitin ligase, ITCH. Here, we confirm this interaction and show ITCH binds directly to the carboxy-terminal half of pX from both human and bat hepatoviruses independently of ALIX. A small chemical compound (compound 5) designed to disrupt interactions between WW domains of NEDD4 ligases and substrate molecules blocked ITCH binding to pX and demonstrated substantial antiviral activity against HAV. CRISPR deletion or small interfering RNA (siRNA) knockdown of ITCH expression inhibited the release of a self-assembling nanocage protein fused to pX and also impaired the release of eHAV from infected cells. The release could be rescued by overexpression of wild-type ITCH, but not a catalytically inactive ITCH mutant. Despite this, we found no evidence that ITCH ubiquitylates pX or that eHAV release is strongly dependent upon Lys residues in pX. These data indicate ITCH plays an important role in the ESCRT-dependent release of quasi-enveloped hepatovirus, although the substrate molecule targeted for ubiquitylation remains to be determined. IMPORTANCE Mechanisms underlying the cellular release of quasi-enveloped hepatoviruses are only partially understood, yet play a crucial role in the pathogenesis of this common agent of viral hepatitis. Multiple NEDD4 family E3 ubiquitin ligases, including ITCH, have been reported to promote the budding of conventional enveloped viruses but are not known to function in the release of HAV or other picornaviruses from infected cells. Here, we show that the unique C-terminal pX extension of the VP1 capsid protein of HAV interacts directly with ITCH and that ITCH promotes eHAV release in a manner analogous to its role in budding of some conventional enveloped viruses. The catalytic activity of ITCH is required for efficient eHAV release and may potentially function to ubiquitylate the viral capsid or activate ESCRT components.

Betv1-displaying elastin-like polypeptide nanoparticles induce a strong humoral and weak CD4+ T-cell response against Betv1 in a murine immunogenicity model.

There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.

Identification and Characterization of Domains Responsible for Cell Wall Binding, Self-Assembly, and Adhesion of S-layer Protein from Lactobacillus acidophilus CICC 6074.

Lactobacillus S-layer protein (SLP) is a biologically active protein on the cell surface. To further elucidate the structures and functions of SLP in Lactobacillus acidophilus CICC 6074, this study was conducted to identify the functional domains of SLP which is responsible for cell wall anchoring, self-assembly, and adhesion. The gene (slpA) of L. acidophilus CICC 6074 SLP was amplified by polymerase chain reaction and speculated functional domains. Fusion proteins of C-terminal truncations from SLP were exogenously expressed in Escherichia coli BL21 (DE3). FITC-labeling N-terminal truncations of SLP were synthesized. The C-terminal domain was more likely to be the binding region, and the cell wall-anchored receptor of SLP was teichoic acid. Furthermore, N-terminal truncations could self-assemble to milk fat globule membrane polar lipid liposomes observed using a fluorescence microscope. Notably, SAN1 (region 32-55) of N-terminal truncations was mainly responsible for the adhesion of SLP to HT-29 cells. These results showed that SLP played a crucial role in the functions of L. acidophilus CICC 6074, which might be of significant reference value for future studies.

Recording of cellular physiological histories along optically readable self-assembling protein chains

Recording of cellular physiological histories along optically readable self-assembling protein chains

Protein Self-Assembly in Crystals and Films

Protein Self-Assembly in Crystals and Films

A self-assembled trimeric protein vaccine induces protective immunity against Omicron variant

The recently emerged Omicron (B.1.1.529) variant has rapidly surpassed Delta to become the predominant circulating SARS-CoV-2 variant, given the higher transmissibility rate and immune escape ability, resulting in breakthrough infections in vaccinated individuals. A new generation of SARS-CoV-2 vaccines targeting the Omicron variant are urgently needed. Here, we developed a subunit vaccine named RBD-HR/trimer by directly linking the sequence of RBD derived from the Delta variant (containing L452R and T478K) and HR1 and HR2 in SARS-CoV-2 S2 subunit in a tandem manner, which can self-assemble into a trimer. In multiple animal models, vaccination of RBD-HR/trimer formulated with MF59-like oil-in-water adjuvant elicited sustained humoral immune response with high levels of broad-spectrum neutralizing antibodies against Omicron variants, also inducing a strong T cell immune response in vivo. In addition, our RBD-HR/trimer vaccine showed a strong boosting effect against Omicron variants after two doses of mRNA vaccines, featuring its capacity to be used in a prime-boost regimen. In mice and non-human primates, RBD-HR/trimer vaccination could confer a complete protection against live virus challenge of Omicron and Delta variants. The results qualified RBD-HR/trimer vaccine as a promising next-generation vaccine candidate for prevention of SARS-CoV-2, which deserved further evaluation in clinical trials.

Self-assembling protein nanoparticles and virus like particles correctly display β-barrel from meningococcal factor H-binding protein through genetic fusion.

Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies. In this work we demonstrated the successful repetitive antigen display of the C-terminal β-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the β-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima, encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.

Supertough and highly stretchable silk protein-based films with controlled biodegradability

Naturally derived protein-based biopolymers are considered potential biomaterials in biomedical applications and eco-friendly materials for replacing current petroleum-based polymers due to their good biocompatibility, low environmental impact, and tunable degradability. However, current strategies for fabricating protein-based materials with superior properties and tailored functionality in a scalable manner are still lacking. Here, we demonstrate an aqueous-based scalable approach for fabricating silk protein-based films through controlled molecular self-assembly (CMS) of silk proteins with plasticizers and salt ions. The films fabricated using this method can achieve a toughness of up to 64±5 MJ/m3 with a stretchability of up to 574±31%. We also demonstrate the tunable enzymatic degradability, low in vitro cytotoxicity, and good in vivo biocompatibility of the films. Furthermore, the films can be patterned with predesigned complex structures through laser cutting and functionalized with bioactive components. The functional silk protein-based films show great potential in various applications, including flexible electronics, bioelectronics, tissue engineering, and bioplastic packaging. Statement of significanceInspired by the naturally optimized multi-scale self-assembly of silk proteins in natural silks, we develop an aqueous-based approach for scalable production of superior protein-based films through controlled molecular self-assembly (CMS) of silk proteins with glycerol and calcium ions. The prepared silk films present outstanding mechanical properties, controlled enzymatic biodegradability, low in vitro cytotoxicity, and good in vivo biocompatibility. Notably, the films fabricated using this method can achieve a high toughness of 64±5 MJ/m3 with a stretchability of 594±31%. The approach introduced in this work provides a facile route toward making silk-based materials with superior properties. It also paves new avenues for developing functional protein-based materials with precisely controlled structures and properties for various applications.