Silk-like Proteins Research Articles

HcN57, A Novel Unusual Acidic Silk-Like Matrix Protein from Hyriopsis cumingii, Participates in Framework Construction and Nacre Nucleation During Nacreous Layer Formation.

In the classic molecular model of nacreous layer formation, unusual acidic matrix proteins rich in aspartic acid (Asp) residues are essential for nacre nucleation due to their great affinity for binding calcium. However, the acidic matrix proteins discovered in the nacreous layer so far have been weakly acidic with a high proportion of glutamate. In the present study, several silk-like matrix proteins, including the novel matrix protein HcN57, were identified in the ethylenediaminetetraacetic acid-soluble extracts of the nacreous layer of Hyriopsis cumingii. HcN57 is a highly repetitive protein that consists of a high proportion of alanine (Ala, 34.4%), glycine (Gly, 22.5%), and serine (Ser, 11.4%). It forms poly Ala blocks, GlynX repeats, an Ala-Gly repeat, and a Ser-Ala-rich region, exhibiting significant similarity to silk proteins found in spider species. The expression of HcN57 was specifically located in the dorsal epithelial cells of the mantle pallium and mantle center. Notably, expression of HcN57 was relatively high during nacreous layer regeneration and pearl nacre deposition, suggesting HcN57 is a silk matrix protein in the nacreous layer. Importantly, HcN57 also contains a certain content of Asp residues, making it an unusual acidic matrix protein present in the nacreous layer. These Asp residues are mainly distributed in three large hydrophilic acidic regions, which showed inhibitory activity against aragonite deposition and morphological regulation of calcite in vitro. Moreover, HcN57-dsRNA injection resulted in failure of nacre nucleation in vivo. Taken together, our results show that HcN57 is a bifunctional silk protein with poly Ala blocks and Gly-rich regions that serve as space fillers within the chitinous framework to prevent crystallization at unnecessary nucleation sites and Asp-rich regions that create a calcium ion supersaturated microenvironment for nucleation in the center of nacre tablets. These observations contribute to a better understanding of the mechanism by which silk proteins regulate framework construction and nacre nucleation during nacreous layer formation.

Spidroins under the Influence of Alcohol: Effect of Ethanol on Secondary Structure and Molecular Level Solvation of Silk-Like Proteins.

Future sustainable materials based on designer biomolecules require control of the solution assembly, but also interfacial interactions. Alcohol treatments of protein materials are an accessible means to this, making understanding of the process at the molecular level of seminal importance. We focus here on the influence of ethanol on spidroins, the main proteins of silk. By large-scale atomistically detailed molecular dynamics (MD) simulations and interconnected experiments, we characterize the protein aggregation, secondary structure changes, molecular level origins of them, and solvation environment changes for the proteins, as induced by ethanol as a solvation additive. The MD and circular dichoroism (CD) findings jointly show that ethanol promotes ordered structure in the protein molecules, leading to an increase of helix content and turns but also increased aggregation, as revealed by dynamic light scattering (DLS) and light microscopy. The structural changes correlate at the molecular level with increased intramolecular hydrogen bonding. The simulations reveal that polar amino acids, such as glutamine and serine, are most influenced by ethanol, whereas glycine residues are most prone to be involved in the ethanol-induced secondary structure changes. Furthermore, ethanol engages in interactions with the hydrophobic alanine-rich regions of the spidroin, significantly decreasing the hydrophobic interactions of the protein with itself and its surroundings. The protein solutes also change the microstructure of water/ethanol mixtures, essentially decreasing the level of larger local clustering. Overall, the work presents a systematic characterization of ethanol effects on a widely used, common protein type, spidroins, and generalizes the findings to other intrinsically disordered proteins by pinpointing the general features of the response. The results can aid in designing effective alcohol treatments for proteins, but also enable design and tuning of protein material properties by a relatively controllable solvation handle, the addition of ethanol.

Liquid-Liquid Phase Separation and Assembly of Silk-like Proteins is Dependent on the Polymer Length.

Phase transitions have an essential role in the assembly of nature’s protein-based materials into hierarchically organized structures, yet many of the underlying mechanisms and interactions remain to be resolved. A central question for designing proteins for materials is how the protein architecture and sequence affects the nature of the phase transitions and resulting assembly. In this work, we produced 82 kDa (1×), 143 kDa (2×), and 204 kDa (3×) silk-mimicking proteins by taking advantage of protein ligation by SpyCatcher/Tag protein-peptide pair. We show that the three silk proteins all undergo a phase transition from homogeneous solution to assembly formation. In the assembly phase, a length- and concentration-dependent transition between two distinct assembly morphologies, one forming aggregates and another coacervates, exists. The coacervates showed properties that were dependent on the protein size. Computational modeling of the proteins by a bead-spring model supports the experimental results and provides us a possible mechanistic origin for the assembly transitions based on architectures and interactions.

Inhibitory Effects of Bacterial Silk-like Biopolymer on Herpes Simplex Virus Type 1, Adenovirus Type 7 and Hepatitis C Virus Infection.

Bacterial polymeric silk is produced by Bacillus sp. strain NE and is composed of two proteins, called fibroin and sericin, with several biomedical and biotechnological applications. In the current study and for the first time, the whole bacterial silk proteins were found capable of exerting antiviral effects against herpes simplex virus type-1 (HSV-1), adenovirus type 7 (AD7), and hepatitis C virus (HCV). The direct interaction between bacterial silk-like proteins and both HSV-1 and AD7 showed potent inhibitory activity against viral entry with IC50 values determined to be 4.1 and 46.4 μg/mL of protein, respectively. The adsorption inhibitory activity of the bacterial silk proteins showed a blocking activity against HSV-1 and AD7 with IC50 values determined to be 12.5 and 222.4 ± 1.0 μg/mL, respectively. However, the bacterial silk proteins exhibited an inhibitory effect on HSV-1 and AD7 replication inside infected cells with IC50 values of 9.8 and 109.3 μg/mL, respectively. All these results were confirmed by the ability of the bacterial silk proteins to inhibit viral polymerases of HSV-1 and AD7 with IC50 values of 164.1 and 11.8 μg/mL, respectively. Similarly, the inhibitory effect on HCV replication in peripheral blood monocytes (PBMCs) was determined to be 66.2% at concentrations of 100 μg/mL of the bacterial silk proteins. This antiviral activity against HCV was confirmed by the ability of the bacterial silk proteins to reduce the ROS generation inside the infected cells to be 50.6% instead of 87.9% inside untreated cells. The unique characteristics of the bacterial silk proteins such as production in large quantities via large-scale biofermenters, low costs of production, and sustainability of bacterial source offer insight into its use as a promising agent in fighting viral infection and combating viral outbreaks.

From reductionism to synthesis: The case of hagfish slime.

Reductionist strategies aim to understand the mechanisms of complex systems by studying individual parts and their interactions. In this review, we discuss how reductionist approaches have shed light on the structure, function, and production of a complex biomaterial - hagfish defensive slime. Hagfish slime is an extremely dilute hydrogel-like material composed of seawater, mucus, and silk-like proteins that can deploy rapidly. Despite being composed almost entirely of water, hagfish slime has remarkable physical properties, including high strength and toughness. While hagfish slime has a promising future in biomimetics, including the development of eco-friendly high-performance fibers, recreating hagfish slime in the lab has been a difficult challenge. Over the past two decades, reductionist experiments have provided a wealth of information about the individual components of hagfish slime. However, a reductionist approach provides a limited understanding because hagfish defensive slime, like most biological phenomena, is more than just the sum of its parts. We end by providing some thoughts about how the knowledge generated in the last few decades might be synthesized into a working model that can explain hagfish slime structure and function.

Bioengineered elastin- and silk-biomaterials for drug and gene delivery.

Advances in medical science have led to diverse new therapeutic modalities, as well as enhanced understanding of the progression of various disease states. These findings facilitate the design and development of more customized and exquisite drug delivery systems that aim to improve therapeutic indices of drugs to treat a variety of conditions. Synthetic polymer-based drug carriers have often been the focus of such research. However, these structures suffer from challenges with heterogeneity of the starting material, limited chemical features, complex functionalization methods, and in some cases a lack of biocompatibility. Consequently, protein-based polymers have garnered much attention in recent years due to their monodisperse features, ease of production and functionalization, and biocompatibility. Genetic engineering techniques enable the advancement of protein-based drug delivery systems with finely tuned physicochemical properties, and thus an expanded level of customization unavailable with synthetic polymers. Of these genetically engineered proteins, elastin-like proteins (ELP), silk-like proteins (SLP), and silk-elastin-like proteins (SELP) provide a unique set of alternatives for designing drug delivery systems due to their inherent chemical and physical properties and ease of engineering afforded by recombinant DNA technologies. In this review we examine the advantages of genetically engineered drug delivery systems with emphasis on ELP and SLP constructions. Methods for fabrication and relevant biomedical applications will also be discussed.

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 β 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.

Silk-Like Protein with Persistent Radicals Identified in Oyster Adhesive by Solid-State NMR.

The cement produced by the Eastern oyster, Crassostrea virginica, may provide blueprints for waterproof biocompatible adhesives synthesized under benign conditions. The composition of this organic-inorganic composite, and of an organic extract, was characterized by 13C and 1H solid-state NMR and also compared withC. virginica shell and its organic extract. Quantification of the organic fraction by 13C and 1H NMR spectroscopy consistently showed 3 wt % organics in cement, which was higher than the 1.2 wt % in the shell. According to 13C NMR with spectral editing, the organic fraction of cement consisted of 73% protein, 25% polysaccharide, and 2% lipid. The organic acid-insoluble extract from the cement was mostly made up of protein remarkably rich in alanine and glycine. The unusual amino acid content matched the composition of silk-like proteins in the C. virginica or C. gigas genomes, including spidron-1-like and shelk2 previously found to be upregulated at the mantle edge. The corresponding extract from the shell contained 32% glycine and was also enriched in serine but not alanine, which was consistent with a previous wet-chemistry study. The 13C and 1H NMR spin-lattice relaxation in the organic component of cement and the acid-insoluble extract was 4-40 times faster than in the shell and showedpronounced nonexponentiality, indicating a high concentration of persistent radicals in the organic components of cement, in agreement with a prior EPR study. The presence of radicals in the acid-insoluble cement fraction was confirmed by observation of a paramagnetic shift anisotropy. 13C NMR corroborated prior observations that the calcium carbonate in the shell and pseudonacre was mostly calcite, whereas cement had an enhanced aragonite fraction. Surprisingly, 1H-13C NMR indicated that aragonite in cement was more distant from the organic fraction than wascalcite. These results help advance our understanding of how oysters achieve adhesion within their wet environment.

Biomimetic Chitin–Silk Hybrids: An Optically Transparent Structural Platform for Wearable Devices and Advanced Electronics

AbstractThe cuticles of insects and marine crustaceans are fascinating models for man‐made advanced functional composites. The excellent mechanical properties of these biological structures rest on the exquisite self‐assembly of natural ingredients, such as biominerals, polysaccharides, and proteins. Among them, the two commonly found building blocks in the model biocomposites are chitin nanofibers and silk‐like proteins with β‐sheet structure. Despite being wholly organic, the chitinous protein complex plays a key role for the biocomposites by contributing to the overall mechanical robustness and structural integrity. Moreover, the chitinous protein complex alone without biominerals is optically transparent (e.g., dragonfly wings), thereby making it a brilliant model material system for engineering applications where optical transparency is essentially required. Here, inspired by the chitinous protein complex of arthropods cuticles, an optically transparent biomimetic composite that hybridizes chitin nanofibers and silk fibroin (β‐sheet) is introduced, and its potential as a biocompatible structural platform for emerging wearable devices (e.g., smart contact lenses) and advanced displays (e.g., transparent plastic cover window) is demonstrated.

Polyelectrolyte Fiber Assembly of Plant-Derived Spider Silk-like Proteins

Spider dragline silk is a proteinaceous material that combines superior toughness and biocompatibility, which makes it a promising biomaterial. The distinct protein structure and the fiber formation process contribute to the superior toughness of dragline silk. Previously, we have produced recombinant spider silk-like proteins in transgenic tobacco that are readily purified from plant extracts. The plant-derived spidroin-like proteins consisted of native major ampullate spidroin 1 or spidroin 2 N- and C-termini flanking 8, 16, or 32 copies of their respective consensus block repeats (mini-spidroins). Here, we present the generation of fibers from mini-spidroins (rMaSp1R8 and rMaSp2R8) by polyelectrolyte complex formation using an anionic polyelectrolyte, gellan gum. Mini-spidroins, when treated with acetic acid and cross-linked by glutaraldehyde, formed a thin film at the interface when overlaid with a gellan gum solution. Immediate pulling of the film resulted in autofluorescent fibrous materials from either mini-spidroin alone or a combination of rMaSp1R8 and rMaSp2R8 (70:30). Addition of chitosan to the mini-spidroin solutions permitted continuous fiber production until the spinning dope supply was exhausted. When air-dried as-spun fibers were rehydrated and stretched in water, the fiber diameter decreased and the overall toughness improved. This study showed that spider silk-like fibers can be produced in large quantities through charge attraction that assembles chitosan, mini-spidroins, and gellan gum into fibrous complexes. We speculate that the spider silk self-assembly process in the duct may involve attraction of variously charged chitinous polymers, spidroins, and glycoproteins.

The Widespread Prevalence and Functional Significance of Silk-Like Structural Proteins in Metazoan Biological Materials.

In nature, numerous mechanisms have evolved by which organisms fabricate biological structures with an impressive array of physical characteristics. Some examples of metazoan biological materials include the highly elastic byssal threads by which bivalves attach themselves to rocks, biomineralized structures that form the skeletons of various animals, and spider silks that are renowned for their exceptional strength and elasticity. The remarkable properties of silks, which are perhaps the best studied biological materials, are the result of the highly repetitive, modular, and biased amino acid composition of the proteins that compose them. Interestingly, similar levels of modularity/repetitiveness and similar bias in amino acid compositions have been reported in proteins that are components of structural materials in other organisms, however the exact nature and extent of this similarity, and its functional and evolutionary relevance, is unknown. Here, we investigate this similarity and use sequence features common to silks and other known structural proteins to develop a bioinformatics-based method to identify similar proteins from large-scale transcriptome and whole-genome datasets. We show that a large number of proteins identified using this method have roles in biological material formation throughout the animal kingdom. Despite the similarity in sequence characteristics, most of the silk-like structural proteins (SLSPs) identified in this study appear to have evolved independently and are restricted to a particular animal lineage. Although the exact function of many of these SLSPs is unknown, the apparent independent evolution of proteins with similar sequence characteristics in divergent lineages suggests that these features are important for the assembly of biological materials. The identification of these characteristics enable the generation of testable hypotheses regarding the mechanisms by which these proteins assemble and direct the construction of biological materials with diverse morphologies. The SilkSlider predictor software developed here is available at https://github.com/wwood/SilkSlider.

Spider silk-like proteins derived from transgenic Nicotiana tabacum.

The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids.

The Potential of Silk and Silk-Like Proteins as Natural Mucoadhesive Biopolymers for Controlled Drug Delivery.

Drug delivery across mucus membranes is a particularly effective route of administration due to the large surface area. However, the unique environment present at the mucosa necessitates altered drug formulations designed to (1) deliver sensitive biologic molecules, (2) promote intimate contact between the mucosa and the drug, and (3) prolong the drug's local residence time. Thus, the pharmaceutical industry has an interest in drug delivery systems formulated around the use of mucoadhesive polymers. Mucoadhesive polymers, both synthetic and biological, have a history of use in local drug delivery. Prominently featured in the literature are chitosan, alginate, and cellulose derivatives. More recently, silk and silk-like derivatives have been explored for their potential as mucoadhesive polymers. Both silkworms and spiders produce sticky silk-like glue substances, sericin and aggregate silk respectively, that may prove an effective, natural matrix for drug delivery to the mucosa. This mini review will explore the potential of silk and silk-like derivatives as a biocompatible mucoadhesive polymer matrix for local controlled drug delivery.

Effect of Fe3+ on the Silk Fibroin Regulated Direct Growth of Nacre-like Aragonite Hybrids

Silk fibroin (SF), which has been used as an analogue of the silk-like proteins in studying the biomineralization process of calcium carbonate in vitro, shows a high preference for the formation of aragonite polymorph. In this study, Fe3+, which has been found with relatively high levels in the shells of marine mollusks, was introduced into the SF-regulated biomineralization system. The addition of Fe3+ into the SF aqueous solutions induced a conformational transition of SF from random coil to β-sheet. The promoted β-sheet structures provided Ca2+-binding sites for SF and, accordingly, enabled the formation of an amorphous calcium carbonate precursor. Such a precursor has an aligned parallel structure and acts as a template for the direct growth of aragonite. The corresponding aragonite products showed similar organic–inorganic hierarchical structure to nacre building in vivo as well as higher thermal stability compared with that of the other Fe3+-free aragonite products in terms of the polymorph. It is i...

Biomimetic Production of Silk-Like Recombinant Squid Sucker Ring Teeth Proteins

The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called "suckerins", which assemble into nanoconfined β-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombinant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to β-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in β-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications.

Purification and cytotoxicity of tag‐free bioengineered spider silk proteins

Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels, and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue, two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in Escherichia coli. The proteins were free of tag sequence and were purified using two methods based on (1) thermal and (2) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and as the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications.

Expression of redesigned mussel silk-like protein in Escherichia coli

Silks have been used widely for human beings due to their several extraordinary properties. Until now, the studies on silk proteins have mainly focused on spiders and silkworms. Because silk properties are organism-dependent, novel silk protein types can be found and developed through investigation of new silk-bearing organisms. We noticed that marine mussel has silk-like domains containing many repeats with abundance of glycine and alanine. In the present work, we redesigned mussel-derived silk-like gene sequence which contains alternating repeated and nonrepeated regions with optimized codons for Escherichia coli. For successful expression of recombinant mussel silk-like protein in E. coli cells, we employed several experimental strategies, including use of strong promoter, cold shock expression, and genetic fusions. We observed significant repression on cell growths by even low expression levels of soluble mussel silk-like proteins in cold shock- and glutathione s-transferase (GST) fusion-based systems. Thus, we finally used baculoviral polyhedrin protein as a fusion partner and successfully expressed insoluble mussel silk-like protein with relatively high expression level and without cell growth repression in E. coli.

Synthesis and Characterization of Novel Silk-Like Proteins Using Genetic Engineering Methods

Using genetic engineering methods, we attempted to produce novel silk-like proteins with new function by combining several functional sequences selected from fibroin of Bombyx mori (B.mori), Samia Cynthia ricini (S.c.ricini) and spider silks or by inducing cell adhesive sequence or calcium binding sequence into silk proteins. The secondary structure of these silk-like proteins was characterized with solid state NMR. Cell adhesion assay indicated that silk-like proteins have higher cell activity. Mineralization of fibroin protein was improved with induction of calcium binding sequence. Nanofiber formation of silk-like proteins was achieved using electrospinning. Fiber was formed from silk-like proteins. These silk-like proteins might be candidates to meet requirement in the field of biomaterials.

Harnessing disorder: onychophorans use highly unstructured proteins, not silks, for prey capture

Onychophora are ancient, carnivorous soft-bodied invertebrates which capture their prey in slime that originates from dedicated glands located on either side of the head. While the biochemical composition of the slime is known, its unusual nature and the mechanism of ensnaring thread formation have remained elusive. We have examined gene expression in the slime gland from an Australian onychophoran, Euperipatoides rowelli, and matched expressed sequence tags to separated proteins from the slime. The analysis revealed three categories of protein present: unique high-molecular-weight proline-rich proteins, and smaller concentrations of lectins and small peptides, the latter two likely to act as protease inhibitors and antimicrobial agents. The predominant proline-rich proteins (200 kDa+) are composed of tandem repeated motifs and distinguished by an unusually high proline and charged residue content. Unlike the highly structured proteins such as silks used for prey capture by spiders and insects, these proteins lack ordered secondary structure over their entire length. We propose that on expulsion of slime from the gland onto prey, evaporative water loss triggers a glass transition change in the protein solution, resulting in adhesive and enmeshing thread formation, assisted by cross-linking of complementary charged and hydrophobic regions of the protein. Euperipatoides rowelli has developed an entirely new method of capturing prey by harnessing disordered proteins rather than structured, silk-like proteins.

The role of salt and shear on the storage and assembly of spider silk proteins

Major ampullate silk fibers of orb web-weaving spiders have impressive mechanical properties due to the fact that the underlying proteins partially fold into helical/amorphous structures, yielding relatively elastic matrices that are toughened by anisotropic nanoparticulate inclusions (formed from stacks of beta-sheets of the same proteins). In vivo the transition from soluble protein to solid fibers involves a combination of chemical and mechanical stimuli (such as ion exchange, extraction of water and shear forces). Here we elucidate the effects of such stimuli on the in vitro aggregation of engineered and recombinantly produced major ampullate silk-like proteins (focusing on structure-function relationships with respect to their primary structures), and discuss their relevance to the storage and assembly of spider silk proteins in vivo.