Accelerated Engineering of ELP-Based Materials through Hybrid Biomimetic-De Novo Predictive Molecular Design.

Efforts to engineer high-performance protein-based materials inspired by nature have mostly focused on altering naturally occurring sequences to confer the desired functionalities, whereas de novo design lags significantly behind and calls for unconventional innovative approaches. Here, using partially disordered elastin-like polypeptides (ELPs) as initial building blocks this work shows that de novo engineering of protein materials can be accelerated through hybrid biomimetic design, which this work achieves by integrating computational modeling, deep neural network, and recombinant DNA technology. This generalizable approach involves incorporating a series of de novo-designed sequences with α-helical conformation and genetically encoding them into biologically inspired intrinsically disordered repeating motifs. The new ELP variants maintain structural conformation and showed tunable supramolecular self-assembly out of thermal equilibrium with phase behavior in vitro. This work illustrates the effective translation of the predicted molecular designs in structural and functional materials. The proposed methodology can be applied to a broad range of partially disordered biomacromolecules and potentially pave the way toward the discovery of novel structural proteins.

Highly Hydrophobic Films of Engineered Silk Proteins by a Simple Deposition Method.

Molecular engineering of protein structures offers a uniquely versatile route for novel functionalities in materials. Here, we describe a method to form highly hydrophobic thin films using genetically engineered spider silk proteins. We used structurally engineered protein variants containing ADF3 and AQ12 spider silk sequences. Wetting properties were studied using static and dynamic contact angle measurements. Solution conditions and the surrounding humidity during film preparation were key parameters to obtain high hydrophobicity, as shown by contact angles in excess of 120°. Although the surface layer was highly hydrophobic, its structure was disrupted by the added water droplets. Crystal-like structures were found at the spots where water droplets had been placed. To understand the mechanism of film formation, different variants of the proteins, the topography of the films, and secondary structures of the protein components were studied. The high contact angle in the films demonstrates that the conformations that silk proteins take in the protein layer very efficiently expose their hydrophobic segments. This work reveals a highly amphiphilic nature of silk proteins and contributes to an understanding of their assembly mechanisms. It will also help in designing diverse technical uses for recombinant silk.

The complex structure of Fomes fomentarius represents an architectural design for high-performance ultralightweight materials.

High strength, hardness, and fracture toughness are mechanical properties that are not commonly associated with the fleshy body of a fungus. Here, we show with detailed structural, chemical, and mechanical characterization that Fomes fomentarius is an exception, and its architectural design is a source of inspiration for an emerging class of ultralightweight high-performance materials. Our findings reveal that F. fomentarius is a functionally graded material with three distinct layers that undergo multiscale hierarchical self-assembly. Mycelium is the primary component in all layers. However, in each layer, mycelium exhibits a very distinct microstructure with unique preferential orientation, aspect ratio, density, and branch length. We also show that an extracellular matrix acts as a reinforcing adhesive that differs in each layer in terms of quantity, polymeric content, and interconnectivity. These findings demonstrate how the synergistic interplay of the aforementioned features results in distinct mechanical properties for each layer.

Protein-Based Biological Materials: Molecular Design and Artificial Production.

Polymeric materials produced from fossil fuels have been intimately linked to the development of industrial activities in the 20th century and, consequently, to the transformation of our way of living. While this has brought many benefits, the fabrication and disposal of these materials is bringing enormous sustainable challenges. Thus, materials that are produced in a more sustainable fashion and whose degradation products are harmless to the environment are urgently needed. Natural biopolymers─which can compete with and sometimes surpass the performance of synthetic polymers─provide a great source of inspiration. They are made of natural chemicals, under benign environmental conditions, and their degradation products are harmless. Before these materials can be synthetically replicated, it is essential to elucidate their chemical design and biofabrication. For protein-based materials, this means obtaining the complete sequences of the proteinaceous building blocks, a task that historically took decades of research. Thus, we start this review with a historical perspective on early efforts to obtain the primary sequences of load-bearing proteins, followed by the latest developments in sequencing and proteomic technologies that have greatly accelerated sequencing of extracellular proteins. Next, four main classes of protein materials are presented, namely fibrous materials, bioelastomers exhibiting high reversible deformability, hard bulk materials, and biological adhesives. In each class, we focus on the design at the primary and secondary structure levels and discuss their interplays with the mechanical response. We finally discuss earlier and the latest research to artificially produce protein-based materials using biotechnology and synthetic biology, including current developments by start-up companies to scale-up the production of proteinaceous materials in an economically viable manner.

Mesanteric ischemia in a COVID-19 patient with no risk factors: A case report.

BACKGROUND: Acute mesenteric ischemia (AMI) may present in COVID-19 patients without the classic risk factors of AMI, and the symptoms might confuse physicians, leading to delayed diagnosis and increased mortality. In this manuscript, we report a patient with COVID-19 who presented with AMI as its initial presenting symptom. CASE PRESENTATION: A 68-year-old man presented to the emergency room with periumbilical non-radiating pain that had started three days before the admission. He had no defecation or gas passing in the past three days. In the physical exam, the patient had guarding of the abdomen, generalized tenderness, and rebound tenderness. C-reactive protein, Creatine phosphokinase and WBC count were elevate. Abdominal X-ray in the supine position was suggestive of obstruction of the small intestine. Diagnostic laparotomy was performed because of peritonitis and acute abdominal pain. The diagnosis was mesenteric ischemia due to vein thrombosis based on laparotomy and paraclinic findings. In the post-surgery evaluations COVID-19 infection was confirmed based on PCR and chest CT scan. DISCUSSION: Based on paraclinical, clinical, and diagnostic laparotomy findings, the patient was diagnosed with small bowel obstruction and gangrene due to acute mesenteric ischemia (AMI). But as described, this patient didn't have any history of medical diseases that cause a hypercoagulable state, in the past. While AF was ruled out because his ECG was normal. We concluded that in this patient AMI was due to his infection with COVID-19. CONCLUSION: Patients with COVID-19 may not have the typical risk factors for AMI, and AMI diagnosis should be considered in patients presenting with acute abdominal pain, even without the routine risk factors.

Correction: ß-1,3-Glucan synthesis, novel supramolecular self-assembly, characterization and application.

Correction for 'ß-1,3-Glucan synthesis, novel supramolecular self-assembly, characterization and application' by Robert Pylkkänen et al., Nanoscale, 2022, https://doi.org/10.1039/D2NR02731C.

ß-1,3-Glucan synthesis, novel supramolecular self-assembly, characterization and application.

ß-1,3-Glucans are ubiquitously observed in various biological systems with diverse physio-ecological functions, yet their underlying assembly mechanism and multiscale complexation in vitro remains poorly understood. Here, we provide for the first-time evidence of unidentified ß-1,3-glucan supramolecular complexation into intricate hierarchical architectures over several length scales. We mediated these unique assemblies using a recombinantly produced ß-1,3-glucan phosphorylase (Ta1,3BGP) by fine-tuning solution conditions during particle nucleation and growth. We report a synthesis of interconnected parallel hexagonal lamellae composed of 8 nm thick sheets of highly expanded paracrystals. The architecture consists of ß-1,3-glucan triple-helices with considerable inter-intra hydrogen bonding within, as well as in between adjacent triple-helices. The results extend our understanding of ß-1,3-glucan molecular organization and shed light on different aspects of the crystallization processes of biomolecules into structures unseen by nature. The presented versatile synthesis yields new materials for diverse medical and industrial applications.

Bioinspired Functionally Graded Composite Assembled Using Cellulose Nanocrystals and Genetically Engineered Proteins with Controlled Biomineralization.

Nature provides unique insights into design strategies evolved by living organisms to construct robust materials with a combination of mechanical properties that are challenging to replicate synthetically. Hereby, inspired by the impact-resistant dactyl club of the stomatopod, a mineralized biocomposite is rationally designed and produced in the complex shapes of dental implant crowns exhibiting high strength, stiffness, and fracture toughness. This material consists of an expanded helicoidal organization of cellulose nanocrystals (CNCs) mixed with genetically engineered proteins that regulate both binding to CNCs and in situ growth of reinforcing apatite crystals. Critically, the structural properties emerge from controlled self-assembly across multiple length scales regulated by rational engineering and phase separation of the protein components. This work replicates multiscale biomanufacturing of a model biological material and also offers an innovative platform to synthesize multifunctional biocomposites whose properties can be finely regulated by colloidal self-assembly and engineering of its constitutive protein building blocks.

Interfacial Crystallization and Supramolecular Self-Assembly of Spider Silk Inspired Protein at the Water-Air Interface.

Macromolecular assembly into complex morphologies and architectural shapes is an area of fundamental research and technological innovation. In this work, we investigate the self-assembly process of recombinantly produced protein inspired by spider silk (spidroin). To elucidate the first steps of the assembly process, we examined highly concentrated and viscous pendant droplets of this protein in air. We show how the protein self-assembles and crystallizes at the water-air interface into a relatively thick and highly elastic skin. Using time-resolved in situ synchrotron x-ray scattering measurements during the drying process, we showed that the skin evolved to contain a high ß-sheet amount over time. We also found that ß-sheet formation strongly depended on protein concentration and relative humidity. These had a strong influence not only on the amount, but also on the ordering of these structures during the ß-sheet formation process. We also showed how the skin around pendant droplets can serve as a reservoir for attaining liquid-liquid phase separation and coacervation from the dilute protein solution. Essentially, this study shows a new assembly route which could be optimized for the synthesis of new materials from a dilute protein solution and determine the properties of the final products.

Self-Assembly of Silk-like Protein into Nanoscale Bicontinuous Networks under Phase-Separation Conditions.

Liquid-liquid phase separation of biomacromolecules is crucial in various inter- and extracellular biological functions. This includes formation of condensates to control, e.g., biochemical reactions and structural assembly. The same phenomenon is also found to be critically important in protein-based high-performance biological materials. Here, we use a well-characterized model triblock protein system to demonstrate the molecular level formation mechanism and structure of its condensate. Large-scale molecular modeling supported by analytical ultracentrifuge characterization combined with our earlier high magnification precision cryo-SEM microscopy imaging leads to deducing that the condensate has a bicontinuous network structure. The bicontinuous network rises from the proteins having a combination of sites with stronger mutual attraction and multiple weakly attractive regions connected by flexible, multiconfigurational linker regions. These attractive sites and regions behave as stickers of varying adhesion strength. For the examined model triblock protein construct, the ß-sheet-rich end units are the stronger stickers, while additional weaker stickers, contributing to the condensation affinity, rise from spring-like connections in the flexible middle region of the protein. The combination of stronger and weaker sticker-like connections and the flexible regions between the stickers result in a versatile, liquid-like, self-healing structure. This structure also explains the high flexibility, easy deformability, and diffusion of the proteins, decreasing only 10-100 times in the bicontinuous network formed in the condensate phase in comparison to dilute protein solution. The here demonstrated structure and condensation mechanism of a model triblock protein construct via a combination of the stronger binding regions and the weaker, flexible sacrificial-bond-like network as well as its generalizability via polymer sticker models provide means to not only understand intracellular organization, regulation, and cellular function but also to identify direct control factors for and to enable engineering improved protein and polymer constructs to enhance control of advanced fiber materials, smart liquid biointerfaces, or self-healing matrices for pharmaceutics or bioengineering materials.

Luminescent Gold Nanocluster-Methylcellulose Composite Optical Fibers with Low Attenuation Coefficient and High Photostability.

Because of their lightweight structure, flexibility, and immunity to electromagnetic interference, polymer optical fibers (POFs) are used in numerous short-distance applications. Notably, the incorporation of luminescent nanomaterials in POFs offers optical amplification and sensing for advanced nanophotonics. However, conventional POFs suffer from nonsustainable components and processes. Furthermore, the traditionally used luminescent nanomaterials undergo photobleaching, oxidation, and they can be cytotoxic. Therefore, biopolymer-based optical fibers containing nontoxic luminescent nanomaterials are needed, with efficient and environmentally acceptable extrusion methods. Here, such an approach for fibers wet-spun from aqueous methylcellulose (MC) dispersions under ambient conditions is demonstrated. Further, the addition of either luminescent gold nanoclusters, rod-like cellulose nanocrystals or gold nanocluster-cellulose nanocrystal hybrids into the MC matrix furnishes strong and ductile composite fibers. Using cutback attenuation measurement, it is shown that the resulting fibers can act as short-distance optical fibers with a propagation loss as low as 1.47 dB cm-1 . The optical performance is on par with or even better than some of the previously reported biopolymeric optical fibers. The combination of excellent mechanical properties (Young's modulus and maximum strain values up to 8.4 GPa and 52%, respectively), low attenuation coefficient, and high photostability makes the MC-based composite fibers excellent candidates for multifunctional optical fibers and sensors.

Structure of Nora virus at 2.7 Å resolution and implications for receptor binding, capsid stability and taxonomy.

Nora virus, a virus of Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 Å resolution cryoEM map showed Nora virus to have T = 1 symmetry with the characteristic capsid protein ß-barrels found in all the viruses in the Picornavirales order. Strikingly, α-helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.

In Vitro Synthesis and Self-Assembly of Cellulose II Nanofibrils Catalyzed by the Reverse Reaction of Clostridium thermocellum Cellodextrin Phosphorylase.

In nature, various organisms produce cellulose as microfibrils, which are processed into their nano- and microfibrillar and/or crystalline components by humans in order to obtain desired material properties. Interestingly, the natural synthesis machinery can be circumvented by enzymatically synthesizing cellulose from precursor molecules in vitro. This approach is appealing for producing tailor-made cellulosic particles and materials because it enables optimization of the reaction conditions for cellulose synthesis in order to generate particles with a desired morphology in their pure form. Here, we present enzymatic cellulose synthesis catalyzed by the reverse reaction of Clostridium thermocellum cellodextrin phosphorylase in vitro. We were able to produce cellulose II nanofibril networks in all conditions tested, using varying concentrations of the glycosyl acceptors d-glucose or d-cellobiose (0.5, 5, and 50 mM). We show that shorter cellulose chains assemble into flat ribbon-like fibrils with greater diameter, while longer chains assemble into cylindrical fibrils with smaller diameter.

Modulating the Mechanical Performance of Macroscale Fibers through Shear-Induced Alignment and Assembly of Protein Nanofibrils.

Protein-based fibers are used by nature as high-performance materials in a wide range of applications, including providing structural support, creating thermal insulation, and generating underwater adhesives. Such fibers are commonly generated through a hierarchical self-assembly process, where the molecular building blocks are geometrically confined and aligned along the fiber axis to provide a high level of structural robustness. Here, this approach is mimicked by using a microfluidic spinning method to enable precise control over multiscale order during the assembly process of nanoscale protein nanofibrils into micro- and macroscale fibers. By varying the flow rates on chip, the degree of nanofibril alignment can be tuned, leading to an orientation index comparable to that of native silk. It is found that the Young's modulus of the resulting fibers increases with an increasing level of nanoscale alignment of the building blocks, suggesting that the mechanical properties of macroscopic fibers can be controlled through varying the level of ordering of the nanoscale building blocks. Capitalizing on strategies evolved by nature, the fabrication method allows for the controlled formation of macroscopic fibers and offers the potential to be applied for the generation of further novel bioinspired materials.

Controllable coacervation of recombinantly produced spider silk protein using kosmotropic salts.

Recent developments suggest that the phase transition of natural and synthetic biomacromolecules represents an important and ubiquitous mechanism underlying structural assemblies toward the fabrication of high-performance materials. Such a transition results in the formation of condensed liquid droplets, described as condensates or coacervates. Being able to effectively control the assembly of such entities is essential for tuning the quality and their functionality. Here we describe how self-coacervation of genetically engineered spidroin-inspired proteins can be preceded by a wide range of kosmotropic salts. We studied the kinetics and mechanisms of coacervation in different conditions, from direct observation of initial phase separation to the early stage of nucleation/growth and fusion into large fluid assemblies. We found that coacervation induced by kosmotropic salts follows the classical nucleation theory and critically relies on precursor clusters of few weak-interacting protein monomers. Depending on solution conditions and the strength of the supramolecular interaction as a function of time, coacervates with a continuum of physiochemical properties were observed. We observed similar characteristics in other protein-based coacervates, which include having a spherical-ellipsoid shape in solution, an interconnected bicontinuous network, surface adhesion, and wetting properties. Finally, we demonstrated the use of salt-induced self-coacervates of spidroin-inspired protein as a cellulosic binder in dried condition.

Biomimetic composites with enhanced toughening using silk-inspired triblock proteins and aligned nanocellulose reinforcements.

Silk and cellulose are biopolymers that show strong potential as future sustainable materials. They also have complementary properties, suitable for combination in composite materials where cellulose would form the reinforcing component and silk the tough matrix. A major challenge concerns balancing structure and functional properties in the assembly process. We used recombinant proteins with triblock architecture, combining structurally modified spider silk with terminal cellulose affinity modules. Flow alignment of cellulose nanofibrils and triblock protein allowed continuous fiber production. Protein assembly involved phase separation into concentrated coacervates, with subsequent conformational switching from disordered structures into ß sheets. This process gave the matrix a tough adhesiveness, forming a new composite material with high strength and stiffness combined with increased toughness. We show that versatile design possibilities in protein engineering enable new fully biological materials and emphasize the key role of controlled assembly at multiple length scales for realization.

Silica-gentamicin nanohybrids: combating antibiotic resistance, bacterial biofilms, and in vivo toxicity.

INTRODUCTION: Antibiotic resistance is a growing concern in health care. Methicillin-resistant Staphylococcus aureus (MRSA), forming biofilms, is a common cause of resistant orthopedic implant infections. Gentamicin is a crucial antibiotic preventing orthopedic infections. Silica-gentamicin (SiO2-G) delivery systems have attracted significant interest in preventing the formation of biofilms. However, compelling scientific evidence addressing their efficacy against planktonic MRSA and MRSA biofilms is still lacking, and their safety has not extensively been studied. MATERIALS AND METHODS: In this work, we have investigated the effects of SiO2-G nanohybrids against planktonic MRSA as well as MRSA and Escherichia coli biofilms and then evaluated their toxicity in zebrafish embryos, which are an excellent model for assessing the toxicity of nanotherapeutics. RESULTS: SiO2-G nanohybrids inhibited the growth and killed planktonic MRSA at a minimum concentration of 500 µg/mL. SiO2-G nanohybrids entirely eradicated E. coli cells in biofilms at a minimum concentration of 250 µg/mL and utterly deformed their ultrastructure through the deterioration of bacterial shapes and wrinkling of their cell walls. Zebrafish embryos exposed to SiO2-G nanohybrids (500 and 1,000 µg/mL) showed a nonsignificant increase in mortality rates, 13.4±9.4 and 15%±7.1%, respectively, mainly detected 24 hours post fertilization (hpf). Frequencies of malformations were significantly different from the control group only 24 hpf at the higher exposure concentration. CONCLUSION: Collectively, this work provides the first comprehensive in vivo assessment of SiO2-G nanohybrids as a biocompatible drug delivery system and describes the efficacy of SiO2-G nanohybrids in combating planktonic MRSA cells and eradicating E. coli biofilms.

Phase transitions as intermediate steps in the formation of molecularly engineered protein fibers.

A central concept in molecular bioscience is how structure formation at different length scales is achieved. Here we use spider silk protein as a model to design new recombinant proteins that assemble into fibers. We made proteins with a three-block architecture with folded globular domains at each terminus of a truncated repetitive silk sequence. Aqueous solutions of these engineered proteins undergo liquid-liquid phase separation as an essential pre-assembly step before fibers can form by drawing in air. We show that two different forms of phase separation occur depending on solution conditions, but only one form leads to fiber assembly. Structural variants with one-block or two-block architectures do not lead to fibers. Fibers show strong adhesion to surfaces and self-fusing properties when placed into contact with each other. Our results show a link between protein architecture and phase separation behavior suggesting a general approach for understanding protein assembly from dilute solutions into functional structures.

Self-Coacervation of a Silk-Like Protein and Its Use As an Adhesive for Cellulosic Materials.

Liquid-liquid phase separation of biomacromolecules plays a critical role in many of their functions, both as cellular components and in structural assembly. Phase separation is also a key mechanism in the assembly of engineered recombinant proteins for the general aim to build new materials with unique structures and properties. Here the phase separation process of an engineered protein with a block-architecture was studied. As a central block, we used a modified spider silk sequence, predicted to be unstructured. In each terminus, folded globular blocks were used. We studied the kinetics and mechanisms of phase formation and analyzed the evolving structures and their viscoelastic properties. Individual droplets were studied with a micropipette technique, showing both how properties vary between individual drops and explaining overall bulk rheological properties. A very low surface energy allowed easy deformation of droplets and led to efficient infiltration into cellulosic fiber networks. Based on these findings, we demonstrated an efficient use of the phase-separated material as an adhesive for cellulose. We also conclude that the condensed state is metastable, showing an ensemble of properties in individual droplets and that an understanding of protein phase behavior will lead to developing a wider use of proteins as structural polymers.

Coacervation of resilin fusion proteins containing terminal functionalities.

Liquid-liquid phase transition known as coacervation of resilin-like-peptide fusion proteins containing different terminal domains were investigated. Two different modular proteins were designed and produced and their behavior were compared to a resilin-like-peptide without terminal domains. The size of the particle-like coacervates was modulated by the protein concentration, pH and temperature. The morphology and three-dimensional (3D) structural details of the coacervate particles were investigated by cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET) reconstruction. Selective adhesion of the coacervates on cellulose and graphene surfaces was demonstrated.