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Advanced search. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. Subjects Molecular biophysics Proteins Structural biology. Abstract A huge variety of proteins are able to form fibrillar structures 1 , especially at high protein concentrations. Access through your institution. Buy or subscribe. This is a preview of subscription content. Change institution.
Buy article Get time limited or full article access on ReadCube. Figure 2: Assembly and aggregation properties of our spider silk-like proteins. Figure 3: Stability and folding of spider dragline silk constructs. Figure 4: Fibre assembly mechanism of dragline silk proteins. Accession codes Primary accessions Protein Data Bank 2khm Data deposits The resonance assignment obtained was deposited at the BMRB data bank under accession code and the atomic coordinates of the best 20 structures plus a regularized average structure have been deposited at the Protein Data Bank under accession code 2khm.
References 1 Dobson, C. Acknowledgements This paper is in memoriam of R. View author publications. Ethics declarations Competing interests The authors declare no competing financial interests. Supplementary information. PowerPoint slides PowerPoint slide for Fig. PowerPoint slide for Fig. Rights and permissions Reprints and Permissions.
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Ferritin can store iron and has a hollow cavity. Therapeutic agents can be encapsulated within ferritin by reversible self-assembly. There are two kinds of ferritins, heavy H and light L , and they can complement each other. Each ferritin is made up of 24 subunits.
Despite a rigid structure of ferritin, the connection between its 24 subunits is pH-dependent, indicating that it decomposes under acidic conditions and re-assembles itself in a neutral environment [ 83 ]. Unlike other temperature-sensitive proteins, ferritin is thermostable due to hydrogen bonds and salt bridges between subunits [ 84 ]. The binding of ferritin to transferrin receptors allows it to be internalized by tumor tissues, and thus ferritin nanoparticles can be applied to deliver drugs.
In addition, the surface groups of ferritin like amino, carboxyl, and sulfhydryl groups can be connected with ligands chemically, and its cavity can bind with metals with high affinity, making it a multifunctional nanocarrier for effective drug delivery [ 27 , 85 , 86 ].
PEG can be used to modify the surface of ferritin and the PEGylated ferritin can circulate longer in vivo [ 87 ]. Ferritin has a strongly negative charge in its inner cavity, so it can be easily bonded with positively charged metal ions by electrostatic interactions [ 88 ].
Beeman and his coworkers injected cationized ferritin into healthy rats intravenously using ferritin itself as a contrast agent for magnetic resonance imaging to study its biodistribution. The results showed that ferritin was distributed at the kidney, liver, lung, and spleen and could be cleared by the liver after 7 days. Furthermore, ferritin had no toxic effects on the kidney and liver [ 90 ]. A summary and comparison of the aforementioned protein nanoparticles PNs are concluded in Table 1.
The desolvation method is also called the coacervation method and is commonly used in the preparation of protein nanoparticles [ 72 ]. Desolvation refers to adding a reagent that can reduce the solubility of the protein in the protein solution so that the protein can agglomerate and settle. The different solubility of proteins in different solvents is exploited to realize phase separation [ 32 ].
The size of protein nanoparticles can be controlled by multiple variables, such as protein concentrations, ratios of desolvating agent antisolvents to original solvents, and pH values. Higher pH values tend to produce nanoparticles with smaller sizes.
After the formation of protein nanoparticles, cross-linking agents like glutaraldehyde are added [ 91 , 92 ]. The advantages of the desolvation method include relatively mild reaction conditions, no need for surfactants, and a time-saving simple preparation process [ 72 ]. This method is widely used in the drug encapsulation of protein nanoparticles [ 93 ]. For example, Weber et al. They explored the influence of various process variables on the surface amino groups and particle size of nanoparticles.
They found out that desolvation had a great effect on the size of nanoparticles. Matrix density is a vital factor to influence the drug release rate for protein nanoparticles used in drug loading. Rao et al.
They investigated the effect of pH and desolvating agent concentrations on gelatin nanoparticle synthesis in detail and concluded the mechanism. By changing the pH, they could vary the size and matrix density of gelatin nanoparticles easily. Then, the resistance to protease and fluorescein release was compared among gelatin nanoparticles with different matrix densities.
The results indicated the potential of protein nanoparticles synthesized by the desolvation method in regulating the drug release rate [ 94 ]. Emulsification is a method to prepare nanoparticles through the formation of emulsion.
Emulsion is a multi-phase dispersion system formed by two immiscible liquids. The liquid in a droplet form is named the dispersed phase, and the other is named the continuous phase [ 95 ]. Surfactants have an important influence on the process of emulsification to form an emulsion [ 96 ]. In the emulsion constituted by oil and water, the phase with a high surfactant solubility is generally the continuous phase of the emulsion. After emulsification, flocculation, coalescence, and Ostwald ripening usually happen during the aging of emulsion as it is a thermodynamically unstable system [ 95 ].
For the preparation of protein nanoparticles, emulsions usually form by mixing an aqueous phase containing the protein and an organic phase containing surfactants with a miscible solvent of oil and water [ 97 ]. Then the protein nanoparticles will be generated by adding such emulsions to preheated or precooled oil drop by drop. Wu et al.
The liquid PEG with a low molecular weight was used as the organic phase, which was green and safe in the application of delivering drugs. The nanoparticle size could be controlled easily by tuning the concentration of silk protein. It helped the nanoparticles to get an efficient drug loading and great ability to control cargo release.
The emulsion method is also used in combination with other advanced technology platforms to prepare protein nanoparticles. They fabricated the nanofluidic device using a two-step lithographic method for generating the droplets. Nanoemulsions production can be tuned by the relationship between the current speed of the dispersed phase and the continuous phase.
After the collection of protein nanodroplets, a de-emulsified and subsequently re-emulsified process could be done to get the aqueous phase protein nanogel [ 99 ]. Electron microscopy results indicated precise control of emulsion sizes and a narrow distribution with the dispersion Fig.
A Illustration of protein nanogels formation by the production of monodisperse water-in-oil nanodroplets in the nanochannels and subsequent de-emulsification steps. G , H are the size distributions of nanoparticles in E and F , respectively [ 99 ]. Salting-out is a very simple but efficient way to prepare protein-based nanoparticles. The process is a bit like desolvation, but no organic solvents are included. Protein coacervates will form from solutions by adding salt ions with a relatively high concentration [ 67 , ].
Since salt ions are more hydrophilic than protein, they compete with protein micelles and are combined with water, destroying the protein hydration layers. This decreases the solubility of the protein. In addition, the salts for the salting-out method have a strong dissociation effect.
The dissociation of salt can inhibit the dissociation of weak protein electrolytes, which reduces the charge of the protein and makes it easier to aggregate and precipitate [ 72 , ].
The conformational structure of proteins stays the same after the salting-out based strategy. So, the bioactivity and function of proteins can be retained after the nanoparticles are formed.
Silk fibroin nanoparticles with controlling features were prepared by the salting-out process for drug delivery [ ]. The morphology and salting-out efficiency were determined by the pH value as well as the ionic strengths of potassium phosphate solutions.
The drawback of the salting-out method is the wide size distribution of protein nanoparticles by the approach. Spray drying is a liquid atomization technology, an important method for producing a drying powder. It disperses the solution into small droplets through a nozzle, transmits heat and mass using a hot and drying medium, and obtains dry particles through the evaporation of the solvent [ , , ]. Recently, spray drying has been well-established as the preparation strategy for protein nanoparticles [ , ].
Most of the protein nanoparticles produced by the spray drying are spherical, with good fluidity and dispersibility.
Tan et al. The size and morphology of nanoparticles mainly depend on the surfactant concentrations and spray mesh sizes. Spray drying granulation has the advantages of continuous work, convenient operation and control, and is suitable for large-scale production [ , ].
Since protein is a natural amphiphilic polymer, it has the potential to form nanoparticles through self-assembly. More and more protein supramolecular nano-complex systems are formed by self-assembly of naturally or artificially prepared protein subunits through non-covalent bonds [ , ].
Ferritin is a rigid structure under neutral pH conditions. When the pH is dropped to 2, the ferritin nanocage structure is destroyed and disassembled to form a single subunit. When the pH of the system is adjusted to neutral again, the ferritin subunits can re-assemble to form a nearly complete structure [ , ]. This unique property makes self-assembly an ideal strategy for encapsulating bioactive compounds into ferritin nanoparticles. In a recent study, the self-assembly of ferritin nanocages was regulated by introducing His motifs through genetic engineering [ ].
When the solution pH equaled to 7. If the pH was adjusted to This process was reversible as pH changed back to 7. An example of designing and preparing protein nanoparticles by self-assembly.
The resultant subunits were natural ferritin tetramers at pH 7. Such tetramers could self-assemble into reconstituted nanocages at pH Conversely, the nanocages would be disassembled into ferritin tetramers by the stimuli of pH or EDTA [ ].
It is normal to obtain VLPs using self-assembly [ , ]. Hong et al. The model antigen peptide was displayed onto the coat protein CP , which was then self-assembled with the scaffolding protein SP to get the P22 VLP-Antigen Peptide with an icosahedral structure. Further results verified that the VLP was useful to work as a platform to deliver peptide antigens with high efficiency in therapeutic cancer vaccines. A comparison of the advantages and disadvantages of different preparation strategies of protein nanoparticles is summarized in Table 2.
Ideally, the use of nanoparticle platforms for oncological applications should be sufficiently effective to produce a unique function in vivo with minimal toxicity and low risk to the body [ , ]. Noble metal-derived nanoparticles platinum, gold, etc. However, the unknown long-term accumulations in the host body have limited their clinical considerations [ , ].
Inorganic nanoparticles can be easily synthesized and modified, but they may release excess toxic ions when used for cancer treatment [ , ]. For instance, carbon nanomaterials are usually hard to degrade in vivo and have the risk of increased side effects [ , ].
Polymeric nanoparticles possess good biocompatibility and manufacturing advantages, but they have limited tumor targeting ability without additional ligand modifications [ , ]. Compared with other nano-systems for cancer therapy and imaging, protein-based nanoparticles have several unique advantages: 1 Proteins can be produced through bioengineering strategies, avoiding the use of chemical synthesis and toxic solvents [ 67 , ]. Especially, a genetic engineering strategy can be used to display antigen epitopes or other functional groups on the protein nanoparticles for cancer nanomedicine [ , , , ].
A lot of protein nanoparticles have been developed as ideal drug cargo loading agents for cancer therapy to reduce drug toxicity and improve therapeutic effects. Compared with normal drug delivery systems such as inorganic nanoparticles, protein nanoparticles have incomparable advantages such as biocompatibility and biodegradability [ 32 ]. The albumin's affinity with hydrophobic drugs is reversible, and the complex is allowed to be transported in the blood and released on the specific cell surface [ , ].
Also, researchers constructed hyaluronic acid HA coated albumin nanoparticles and used the composite nanoparticles to load both hydrophobic and hydrophilic drugs due to the hydrophobic binding sites of albumin as well as the hydrophilic binding sites of HA [ 93 ]. The binding strength between HSA and drug is significant because it determines when the drug is released in vivo.
Qi et al. The non-immunogenicity, low cytotoxicity, and multiple drug binding sites give HSA nanoparticles a big advantage over other drug loading systems. HSA-based nanoparticles can also avoid unnecessary interactions with sera in addition to increasing the solubility of hydrophobic molecules in the blood [ ]. In addition, protein nanoparticles can use their hollow structures to load drugs.
Auoxo3, a gold compound, is a kind of cytotoxic anti-cancer drug. Ferraro et al. The results of cell experiments showed that compared to non-tumorigenic cells, the nanocarriers were much more toxic to aggressive human cancer cells. Also, Huang et al. Although inorganic porous nanomaterials can also be used to deliver drugs, some of them even have a high drug loading rate.
However, without modification, they cannot avoid drug leakage during transportation [ ]. The unique cavity structure and pH-dependent self-assembly and decomposition of ferritin give it unique superiority in drug delivery. The combination of disulfiram and copper is believed to have anti-tumor properties. Xu et al. Together with disulfiram, the Fn-HSA-Cu complex showed a great antitumor ability both in cell and animal experiments.
Both bombyx mori silk fibroin SF and sericin SS have a negatively charged surface and a high encapsulation efficacy. Therefore, the silk proteins were used as biotemplates to direct the self-assembly of silica nanoparticles and nanofibers.
Analogously, Shuai et al. Collagen is a kind of fibrous protein available in the extracellular matrix of different tissues, including tumors [ , ].
As a nature-derived biopolymer, collagen is widely used as the matrix material for drug cargo loading [ ]. Protein-based compositions can enhance the solubility of the whole nano-systems. The fibrous structural characteristics of collagen can increase the mechanical stability of nanoparticles [ , ]. Jiang et al. In another study, collagen was combined with nanohydroxyapatite and fucoidan to prepare nanomaterials for drug delivery [ ].
The physico-chemical properties of the materials were improved with better crosslinking and non-toxicity to overcome the original disadvantages. A cell migration assay indicated that the composite material is a promising drug delivery platform for killing cancer cells. In the past, many tumor-targeting drugs relied on passive targeting based on the enhanced permeability and retention EPR effect [ ]. Nanodrugs or nanoparticles can target tumors actively through the affinity with highly expressed tumor-specific receptors in tumor cells or tumor microenvironment such as tumor blood vessels and fibroblasts [ ].
SPARC and gp60 are highly expressed in glioma and tumor vascular endothelial cells. They can bind specifically to albumin. Lin et al. The composite nanoparticles could target in situ and subcutaneous glioma effectively. The cell-penetrating peptide CPP LMWP modified on the nanoparticles enhanced the interaction between the nanoparticles and cancer cells, leading to better targeted delivery and intensive tumor accumulation.
Li et al. ZnF 16 Pc is a kind of near-infrared photosensitizer. They then attached an affinity single-chain variable fragment scFv of fibroblast activation protein FAP to the surface of nanoparticles. In addition, they found that photodynamic therapy increased particle aggregation at the tumor site. In addition to the binding with specific and highly expressed receptors in the tumor microenvironment, protein nanoparticles can actively target tumors by grafting targeting peptides on the surface as well.
Integrin is overexpressed on many tumor cells. Bari et al. The targeting peptide significantly increased the particle internalization into human urinary bladder ECV cancer cells. Shan et al. VLPs inherit the advantages of proteins and can be genetically modified with targeting peptides, whereas inorganic nanoparticles are generally modified with the targeting ligands through direct chemical coupling or through the use of organic polymer linkers [ ].
In addition, studies have shown that the targeting ability of VLPs will not be adversely affected by protein corona [ ].
However, when silica was modified with transferrin, the protein corona formed on the surface of silica would shield transferrin, making it lose the ability to bind with transferrin receptors.
They were highly expressed in many tumors, thus losing the ability to target tumors [ ]. Protein nanoparticles can deliver drugs responding to internal stimuli such as glutathione GSH , pH value, and enzyme concentration or external stimuli like temperature through engineering [ ].
Seib et al. The produced silk nanoparticles were negatively charged, and hence they could be loaded with positively-charged DOX through electrostatic attraction. The research indicated that silk protein nanoparticles had the potential to become a powerful delivery platform for lysosomal drugs. Pancreatic cancer is malignant and is closely related to the nervous microenvironment. Lei et al.
Both of them could target pancreatic tumors via transferrin receptors. Because of the nature of ferritin, drugs would be released in the slightly acidic tumor microenvironment rather than in the neutral blood or alkalescent pancreatic juice.
Biabanikhankahdani et al. Gou et al. Thong et al. Both photothermal and photodynamic therapy are light-mediated therapy [ ]. Photothermal therapy generally uses nanomaterials themselves or photothermal agents to transform the absorbed light energy into heat energy to raise the temperature of a tumor to kill cancer cells.
Apoferritin AFN is a pH-sensitive spherical cage protein. He et al. Nanoparticles can target tumors and become internalized by cancer cells due to the overexpressed folate receptors on their membrane. After intravenous injection of the composite nanoparticles into breast cancer tumor-bearing mice, the temperature of tumor sites increased significantly with nm laser irradiation and thus inhibited the tumor growth through photothermal-chemotherapy. The thermal stability of ferritin makes ferritin nanoparticles competitive nanocarriers for photothermal cancer therapy.
Wang et al. The composite particles inhibited the growth of xenograft glioblastoma by PTT, realizing the integration of diagnosis and treatment. Photodynamic therapy is a kind of oxidative therapy. Photosensitizers are employed to transfer light energy to chemical energy and turn the surrounding oxygen molecules to highly active singlet oxygen 1 O 2 to kill cancer cells [ , ].
Photosensitizer Ce6 was covalently connected to BSA, and together with ferritin and ferroptosis-inducing drug sorafenib SRF they formed a nanoreactor. In addition to 1 O 2 produced by photosensitizers through photodynamic therapy, ferritin-induced Fenton reaction also produced hydroxyl radicals to kill cancer cells.
Also, SRF inhibited the synthesis of GSH, decreased the expression of glutathione peroxidase 4 GPX4 , accelerated the lipid peroxidation, thus finally inducing ferroptosis. Rhee et al. Yang et al. MnO 2 can act as a catalyst and promote the decomposition of endogenous hydrogen peroxide, producing oxygen to reduce tumor hypoxia and enhancing the effect of photodynamic therapy.
Schematic diagram of the synthesis, dual-model imaging, and combination therapy of multifunctional platform SMID. Upper: The silk fibroin SF is extracted from the silkworm cocoon and self-assembles into SF nanoparticles. There are many types of tumor immunotherapy, but most of them exert their anti-tumor effect through T cells in essence [ ].
The immune checkpoint is a class of immunosuppressive molecules. Blocking immune checkpoints is one of the effective strategies to enhance T cell activation. It is also a popular target for anti-tumor drug development in recent years. This effect realized the codelivery of photosensitizer and an immune checkpoint inhibitor.
The in vivo results showed that after in-situ injection of composite nanoparticles, drugs are released at an accelerated rate under acidic conditions. The consequent ROS and tumor-associated antigens inhibited tumor growth and increased T cell activity. This research combined PDT and immunotherapy and improved the efficacy of cancer therapy. Cheng et al. The hybrid VLP could accelerate the maturation of dendritic cells, induce a strong immune response in vivo and effectively inhibit the growth of subcutaneous melanoma and the formation of metastatic melanoma in the lung.
Schematic illustration of the therapy and the therapeutic effect of the tumor [ ]. Upper: Schematic illustration of the construction of dual antigens loaded HBc VLP and the subcutaneous inoculation; Bottom left: Results of treatment of lung metastases. Compared to saline and single antigen-loaded VLPs, hybrid VLPs significantly inhibited the lung metastasis of melanoma; Bottom right: Results of treatment of subcutaneous melanoma. Compared to saline and single antigen-loaded VLPs, hybrid VLPs inhibited tumor growth and reduced the tumor volume to the maximum extent.
Schneider et al. VLP displayed with the tumor-associated antigens triggered a powerful immune response due to the immunogenicity of VLP itself and effectively treated the tumor, showing the potential of VLP as a tumor vaccine. The ClinicalTrials. Among them, Abraxane is the most common clinical drug to treat many kinds of cancer. We have summarized some typical clinical trials of the albumin-based nanoparticles applied for cancer therapy in Table 3.
Optical imaging includes fluorescence imaging, bioluminescence imaging, and photoacoustic imaging. Protein nanoparticles can carry fluorescent dyes for in vivo fluorescence imaging. An et al. This composite nanoparticle can be injected into tumor mice intravenously to target the tumor via the EPR effect while imaging the tumor in vivo simultaneously. In addition, this fluorescent probe solved the problem of poor tissue penetration of green fluorescence. Hu et al. H-ferritin HFn has a specific affinity to transferrin receptor 1 TfR1.
It is specifically overexpressed in the majority of cancers, so the protein nanoparticles can target tumors. Bellini et al. The linker between HFn and luciferin was sensitive to GSH, so the complex could release luciferin in the cytoplasm of cancer cells and carried out bioluminescence imaging BLI of the luciferin-luciferase binding system.
The bioluminescence imaging in vitro and in vivo could directly demonstrate that protein nanoparticles were internalized into cancer cells with high efficiency, diminishing the background signal.
The composite nanoparticles are powerful imaging agents in luciferase tumor models. Schematic representation of the mechanism by which Luc-linker HFn is recognized by the transferrin receptor and carries out bioluminescence.
Luc-linker HFn is internalized by cancer cells through TfR1. Intracellular GSH can break the disulfide bond and release free luciferin. Luciferin is able to react with luciferase and perform bioluminescence imaging [ ]. Due to the good near-infrared absorbance of CuS—Fn, the nanoparticles could be applied in photoacoustic imaging PAI in vivo. When 64 Cu was introduced, the nanoparticles could also act as a positron emission tomography PET imaging contrast agent.
Therefore, PTT directed by dual-modal imaging was achieved, and the tumor was completely eliminated. Tao et al. Mandal et al. The in vitro MRI images showed that after antibody treatment, more nanoparticles were endocytosed by gastric cancer cells through receptor-mediated endocytosis.
Cai et al. The T1 weight signal at the tumor site was significantly enhanced only 10 min after intravenous injection. The in vivo MR imaging proved the ability of the composite particles to target tumors and achieve prolonged tumor retention Fig.
In vivo MRI results. A T1 weighted MR images of prostate cancer tumor-bearing mice at specific time points. B Quantitative analysis of T1 shortening in tumor. C Biodistribution of Gd complex in main organs and tumor h post injection [ ]. Ferritin can act as a MRI reporter gene as well. Lu et al. Also, Cohen et al. When the pH was returned to 7. The in vivo PET and near-infrared fluorescence images showed that after tail vein injection of the ferritin probes, the probes could enrich at the tumor as a result of both RGD targeting peptide and EPR effect.
Similarly, Aanei et al. Xie et al. Furthermore, iron oxide was an MRI imaging contrast agent. Therefore, the HSA nanoparticles are a new type of multifunctional theranostic platforms that could perform tri-modal imaging.
The encapsulation of iron oxide particles into HSA matrices to form the nanosystems was a process similar to drug cargo loading which took full advantages of the HSA. The surface of the dopamine modified iron oxide nanoparticles was rich in amine groups. Compact HSA coating could be realized just by adsorbing due to the natural multivalency and good solubility of protein-based nanoparticles. The hydrodynamic size of the nanoparticles changed from Although CT imaging is often used in combination with PET, it can act as an independent imaging modality [ ].
Chu et al. They manipulated the ratio of gold to silver in the synthesis of nanoparticles. Interestingly, they found that when the ratio of gold to silver is 3: 2, the protein particles displayed great CT imaging performance. Besides, the particles showed good biocompatibility both in vitro and in vivo. Zhang and her coworkers [ ] employed apoferritin to construct a novel nano-diagnosis and treatment platform; DOX was encapsulated inside the ferritin cavity, and radiation sensitizer bismuth sulfide Bi 2 S 3 crystals were embedded in the protein shell Dox AFBS.
These studies demonstrated that the combination of chemotherapy and radiotherapy could effectively diminish the volume of subcutaneous Hela tumor. In vitro and in vivo CT imaging results. As drug carriers for cancer diagnosis and treatment, protein nanoparticles have the advantages of biocompatibility and biodegradability compared with other carriers such as inorganic nanoparticles.
Facile functionalization of protein surfaces makes it easy to load drugs, targeting ligands or other functional small molecules onto them, thus constructing multi-functional theranostic platforms. Hence, we summarize the preparation methods of several most common protein nanoparticles and the latest advances of their use in cancer treatment and imaging. However, there are several problems with protein nanoparticles: 1 some proteins are not that stable, and their structures may change during synthesis of the nanoparticles; 2 some protein nanoparticles are not uniform in size and size adjustment during preparation is not as easy as inorganic nanoparticles [ ]; 3 the mechanism of protein nanoparticles mediated transport is unclear [ ].
Besides, most nanoparticles for tumor therapy in the clinical stage are liposome or lipid nanoparticles, and there are few protein nanoparticle based clinical drugs [ ]. The functional relevance of nitrotyrosinations observed on FSP in the present study remains unclear, but previous work revealed the effects of tyrosine nitration on mechanoelastic properties and on protein-protein interactions 21 , Hydroxyproline was the major PTM reported in the present study, with a total of 45 sites assigned to the FSP sequence.
This modification consists of the addition of hydroxyl groups to the proline residues catalyzed by prolyl hydroxylase PHD , warranting protein stability The N. Hydroxyproline is an essential amino acid residue present in collagen and studies indicate the presence of structural domains in the collagen of M.
Sutherland et al. Previous studies demonstrating the proline and the glycine content of elastomeric and amyloids proteins, indicate that elastin-like behaviour occurs above a threshold level of combined proline and glycine content; with proline as the primary determinant of elastin-like behaviour 49 , In Guinea et al.
Modelling of N. So far only parts of 3D structures of the N- and C-terminal regions of spidroin-1 have been reported 33 , 39 , 52 , In our own previous studies with N. The proposed 3D model of FSP indicates the predominance of stretched structures in the solid fibres of the silk protein like the type I collagen, molecule used as template , presenting a heterogeneous, disordered and randomly oriented structural conformation, even after the simulations by molecular dynamics.
In this protein model, the N-terminal region exhibits three complete helices despite the absence of the segment of the sequence at position — in the N-terminal domain of N. The C-terminal domain contains one small helical section, maintained by a disulphide bridge formed by two highly conserved cysteine residues.
According to Heim et al. MD simulations were performed in a virtual box in presence of water molecules and all model validation parameters revealed that the molecular model was reliable, despite of the predominance of random structures in the solid fibres of the silk.
Apparently, the FSP seems to be an intrinsically disordered protein IDP or natively unfolded proteins; and this property is a computational challenge for MD simulations, because by definition there are no available 3D structures of the whole molecule that is IDP 55 , 56 , The structural properties of IDPs are more sensitive to protein-water interactions than those of folded proteins Protein intrinsic disorder is a state related to protein function 58 , Studies have demonstrated that disorder is a metastable state susceptible to the changes in the environment; and that IDPs behaviour is often controlled by PTMs, such as phosphorylation It is important to mention that our findings pointed a series of PTMs such as phosphorylation and hydroxylation of proline on the FSP; these PTMs could potentially act on mechanoelastic properties and on protein-protein interactions in the web fibres.
The number of hydrogen bonds in a molecular structure may be considered as an indicator of its stability. FSP contains intramolecular hydrogen bonds in presence of water Figure S6d , indicating that FSP is structured and stable in presence of water.
The spider silk has a great potential as a biomaterial. To explore this potential, it is needing a better understanding about the structure-function relationship of the proteins present in N. In the present study a large amount of hydroxyproline was observed along the sequence of the FSP, which constitutes the silk of the capture spiral. This silk is known to be highly elastic and the observation of a large amount of hydroxylated proline residues could potentially characterize this elastic property presented by the fibres.
Sequence data reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based spider polymers, forms the basis for understanding previous work and for designing future studies of silks. Web silk radii and spiral from N. The web-silk samples were processed as mentionedpreviously by dos Santos-Pinto et al.
The protein concentration was estimated by the Bradford assay Peptide recovering was performed using 0. Thus, it was selected all , entries contained in the taxa Araneae for protein identification as reported previously by dos Santos-Pinto et al.
Afterwards the identified proteins were subjected to additional filtering by Scaffold 4. Using the advanced PTM-explorer search strategies it was possible to perform searches for amino acid substitution and unknown mass shifts. The Modiro software is complementary to the MASCOT software, using already identified sequences and has the advantage that also unknown mass shifts can be handled For the quantification of proteolytic peptides, it was used the spectral counting of the data generated during the mass spectrometry analysis 15 , 16 , 63 , 64 , 65 of the individual digests of N.
The extracted ion chromatograms of each peptide were manually inspected for spectral counting; and the results were normalized dividing the individual counting of each peptide by the lowest counting observed amongst all the peptides. The entire procedure was performed as described previously by dos Santos-Pinto et al.
Flagelliform silk protein was subjected to molecular modelling using a restrained-based modelling approach as implemented in the program MODELLER 9v Thus, we decided to search for templates using the THREADER program, which searches according to structural similarity, such that two proteins are considered homologous if they share similar sequences and structures. The output of these tools was formatted and used as input for the MODELLER program, which implements an automated approach for comparative modelling based on the fulfilment of spatial restraints.
Images of the three-dimensional structures of the models were generated using PyMOL Flagelliform silk protein was subjected to molecular dynamics simulation in a cubic box containing water, and a 1. In the initial MD simulations, all hydrogen atoms, ions, and water molecules were subjected to steps of energy minimization to remove close van der Waals contacts.
The water system was then subjected to a short MD simulation with position restraints for 1, pico-seconds ps. Energy minimization and MD were performed under periodic boundary conditions. Temperature and pressure were modulated using coupling techniques 74 with coupling and isothermal compressibility constants of 0. Electrostatic interactions among non-ligand atoms were evaluated using the particle-mesh Ewald method Cut-off distances for the calculation of the Coulomb and van der Waals interactions were 1.
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