310 10*1mm Stainless steel coiled tubing chemical component ,The N-terminal domains of spidroin form hydrogels based on amyloid fibrils and provide a platform for protein immobilization.

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Recombinant spider silk proteins (spider silk proteins) have many potential applications in the development of new biomaterials, but their multimodal and aggregation-prone nature makes them difficult to obtain and easy to use. Here we report that recombinant miniature spidroin proteins and, importantly, the N-terminal domain (NT) itself rapidly form self-supporting and transparent hydrogels at 37 °C. fusion proteins consisting of NT and green fluorescent protein or purine nucleoside phosphorylase form fully functional fusion proteins. Hydrogels. Our results show that recombinant NT and fusion proteins provide high expression yields and endow hydrogels with attractive properties such as transparency, gelation without crosslinking, and direct immobilization of active proteins at high density.
Spiders have as many as seven different sets of silk glands, each producing a specific type of silk. All seven silk species are composed of spider silk proteins (spidroins) approximately 6000 residues long and contain a large central repeat region surrounded by spherical N- and C-terminal domains (NT and CT)1,2. The most widely studied type of silk, the primary ampulla, is produced by the primary ampulla gland. In this gland, a monolayer of epithelial cells synthesizes spidroin proteins and secretes them into the lumen of the gland, where they are present in a soluble form (doping) at extremely high concentrations (30–50% w/v)3,4. The organization and conformation of the main ampullar spidroin proteins in the gland has been debated, but most experimental evidence indicates the presence of a generally helical and/or random helical conformation and micellar or lamellar structures5,6,7,8,9,10. While the repetitive domains regulate mechanical properties of silk fibers, forming β-sheet nanocrystals and amorphous structures11,12,13,14,15, end domains regulate silk fibers in response to changing conditions along the silk gland16,17,18. By controlling silk formation, 19. Terminal domains are evolutionarily conserved and their function may be common to all spidroin proteins 2,20,21. During passage through the gland, the pH of spidroin decreases from about 7.6 to < 5.716 and increases with shear and stretch mediated by movement through the gradually narrowing duct. In solution, CT is an α-helical constitutive parallel dimer17, but in response to low pH and shear forces, CT unfolds and switches β-layers16, 17, possibly triggering β-layers in the repetitive regions of Convert 16. NT are monomeric under conditions reflecting conditions in the lumen of the gland and mediate the solubility of spidroin, but at reduced pH, protonation of a number of carboxylic acid side chains leads to dimerization of NT with a pKa of approximately 6.5, thereby stabilizing NT and fixing spidroin in large quantities. networks16,18. Thus, NT plays a key role in filament formation, changing from a monomer in the coating to a dimer in the fiber23,24,25. NT remains highly soluble and helical under all conditions studied to date16, 18, 19, 20, 26, 27, 28, 29, which inspired its development as a solubility-enhancing label for the production of heterologous proteins.
Recombinant mini spider silk protein, consisting of one NT, one short repeat region, one CT, and a His6 tag (His-NT2RepCT) for purification, is as soluble in aqueous buffer as native spider silk protein and mimics native important characteristics of silk spider. coverage 25.31. His-NT2RepCT can be spun into continuous fibers using a biomimetic machine in which a pH 8 soluble coating is extruded into a pH 525,32,33,34,35 water bath. Bioreactor fermentation of E. coli expressing His-NT2RepCT and subsequent post-treatment resulted in >14 g/L yield after purification. The high yield, high solubility, and adequate response of His-NT2RepCT to acidic conditions are all attributed to NT23, 25, 34.
Here we report the rapid formation of transparent hydrogels from recombinant spidroin proteins, including NT alone, by incubating a protein solution at 37 °C. Using thioflavin T fluorescence (ThT), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) and transmission electron microscopy (TEM), we found that NT and microspider proteins undergo structural transformation into β-sheets and amyloid-like fibrils when gels are formed. In addition, fusion proteins of NT and green fluorescent protein (GFP) or purine nucleoside phosphorylase (PNP) form hydrogels with fully functional fusion fragments. High-throughput expression in heterologous hosts, coupled with rapid formation of hydrogels under physiological conditions, opens up the possibility of cost-effective production of hydrogels with engineered functions.
Unlike most reported recombinant spidroin proteins36, His-NT2RepCT is stable in Tris-HCl buffer at pH 8 and can be concentrated up to 500 mg/mL without precipitation25. Therefore, we were surprised to find that this protein rapidly forms optically clear, self-supporting hydrogels when incubated at 37°C (Fig. 1b-d). Further studies showed that His-NT2RepCT gelation occurred over a wide range of protein concentrations (10–300 mg/mL) and that this concentration was inversely correlated with gelation time (Fig. 1c and Supplementary Fig. 1). To find out which parts of His-NT2RepCT mediate hydrogel formation, we then examined each domain individually and in various combinations using a flask inversion assay (Figure 1a,b). All tested fractions of recombinant spidroin formed gels (at a protein concentration of 300 mg/mL) in less than 1 h, except for precipitated 2Rep (Fig. 1b). This suggests that NT and CT alone, in combination, or associated with repeats, can gel at 37°C and that the His6 tag does not affect this process to any significant extent. Given the common notion that NT is a highly soluble and stable protein, and that previous reports of recombinant spidroin hydrogels have attributed gelation effects to conformational changes in repeat regions and/or CTs, NT itself could. The discovery of gelation was unexpected. Supplementary Table 1) 37, 38, 39. Remarkably, NT already gelled within 10 minutes at a concentration of ≥ 300 mg/mL (Fig. 1c). Vial inversion experiments with various concentrations of NT showed that at >50 mg/mL the NT solution gelled faster than His-NT2RepCT at the corresponding concentration (w/v, Figure 1c).
Schematic representation of various spidroin constructs studied in this work. b Gel time at 37 °C for various recombinant spidroin proteins (300 mg/mL) verified by inverting the vial. CT gel immediately without incubation (<300 mg/mL), 2Rep precipitates (300 mg/mL, 5 mm scale). c Gel time of His-NT2RepCT and NT at indicated protein concentrations at 37°C. d Photographs of His-NT2RepCT and NT hydrogels with the spider and the letter “NT” printed underneath, respectively (both 200 mg/mL, scale bar 5 mm).
Hydrogels formed by various recombinant spidroin proteins have slightly different colors, and naked eye observation shows varying degrees of transparency (Fig. 1b). NT gels are exceptionally clear while other gels become opaque. His-NT2RepCT and NT gels cast into cylindrical tubes could be removed from the mold intact (Fig. 1d).
To test whether natural spider silk coatings gel under conditions now found to cause gelation of the recombinant spidroin proteins, coatings were collected from the large ampulla gland of the Swedish bridge spider (Larinioides sclopetarius). Coatings were stored in 20 mM Tris-HCl buffer at 50 mg/mL (based on measured dry weight), but no gelation was observed during the 21 day incubation at 37 °C (Supplementary Figure 2a).
To quantify these gels, rheological measurements can be used to study the gelation process and determine the overall mechanical properties. In particular, monitoring the storage modulus (elasticity) at elevated temperatures can provide information on the gelling temperature as well as the viscoelastic properties of the coating. Temperature rise experiments (using 1°C/min at 25-45°C, based on previous studies using natural silk stock solutions)40,41 showed that the storage moduli of His-NT2RepCT and NT solutions increased with increasing temperature . was increased (Fig. 2 and Supplementary Fig. 3). Notably, the NT module started growing at a lower temperature compared to His-NT2RepCT, consistent with the faster gel time observed when NT was directly incubated with His-NT2RepCT at 37°C (Figure 1). After a subsequent drop in temperature, the storage modulus did not return to lower values ​​and remained above the loss modulus (see Supplementary Fig. 3), indicating thermally irreversible stable gelation. After gelation, the final elastic modulus ranged from 15 to 330 kPa for His-NT2RepCT hydrogels at a concentration of 100–500 mg/mL, and the final elastic modulus for NT hydrogels (100–500 mg/mL) ranged from 2 to 1400 kPa (Fig. , 2 and complete ramp data) see Supplementary Fig. 3).
a Change in temperature during measurements of His-NT2RepCT (300 mg/mL) and b NT (300 mg/mL) with shaking. The arrows indicate the temperature trend, and the lighter shading of the storage module data depicts testing at lower torque values ​​for the instrument than specified by the manufacturer, which is the cause of the increased noise. c End-module accumulation of His-NT2RepCT and NT after elevated temperature (100, 300, and 500 mg/mL). All module readings are taken at a frequency of 0.1 Hz.
As a potential method for investigating conformational changes associated with gelation, we recorded FTIR spectra of His-NT2RepCT and NT before and after gelation at 37°C (Figure 3a,b). As expected, the spectra of His-NT2RepCT and NT solutions corresponded to proteins showing α-helix/random coil secondary structure, with a pronounced band at 1645 cm-1. For both hydrogels, gelation resulted in the formation of two arms in the middle I band at about 1617 cm-1 and 1695 cm-1 (Fig. 3a, b), indicating the formation of antiparallel β-sheet structures. These changes can also be clearly seen in the respective second derivative and difference gelation spectra (Supplementary Fig. 4b). The two bands of the NT β-layer were more pronounced than those of His-NT2RepCT, indicating that the total content of β-layer bands in the NT hydrogel was higher than that of the NT2RepCT hydrogel.
a FTIR absorption spectra of His-NT2RepCT and b NT (both 500 mg/mL) before (solution) and after (gel) incubation at 37°C. c TEM images of resuspended 50 mg/ml NT2RepCT gels and d NT. Scale bar 200 nm. e Fiber diameters of His-NT2RepCT and NT hydrogels. n = 100 measured fibrils, p < 0.0001. The error bars show the standard deviation. The center of the error bars is the mean. An unpaired t-test (two-tailed) was used for statistical analysis. f ThT fluorescence of various recombinant spidroin proteins (100 mg/mL) at 37 °C without shaking. g NT (100 mg/mL) inoculation experiments from 100 mg/mL NT gel with 0%, 5%, 10%, and 20% seeds.
Analysis of the gel using transmission electron microscopy (TEM) showed that the hydrogel consists of amyloid-like fibrils (Figs. 3c, 3d). NT-formed fibrils were elongated (5–12 nm in diameter) and unbranched, while His-NT2RepCT fibrils were shorter in length and significantly wider in diameter (7–16 nm) (Fig. 3e). These results allowed us to follow the kinetics of fibrosis using the thioflavin T (ThT) assay. For all recombinant spidroin proteins, the fluorescent signal increased when samples were incubated at 37 °C (Fig. 3f, Supplementary Fig. 5a). Consistent with this finding, microscopic examination of NT and His-NT2RepCT under gelling conditions revealed a uniform increase in ThT fluorescence without noticeable local accumulation of ThT-positive aggregates (Supplementary Fig. 5b,c). The formation of ThT-positive fibrils was not accompanied by an increase in NT and His-NTCT turbidity (Supplementary Fig. 5d), which means that a network of fibrils in the gel could form without compromising gel clarity. Seeding by adding small amounts of pre-formed fibrils can significantly accelerate fibril formation of some amyloids42,43,44 but adding 5%, 10% or 20% (w/w) NT to a solution of NT hydrocoagulants. seeding effect (Fig. 3g). Perhaps this is due to the fact that the fibrils in the hydrogel are relatively fixed and cannot be used as seeds.
The unexpected behavior of recombinant spidroin proteins at high temperatures prompted further nuclear magnetic resonance (NMR) spectroscopy studies to identify conformational changes associated with gel formation. NMR spectra of His-NT2RepCT solutions recorded over time at 37°C showed that CT was still partially folded, whereas NT and 2Rep signals had disappeared (Fig. 4a), suggesting that it was mainly NT and 2Rep that partially controlled formation of His-NT2RepCT hydrogel. The CT signal was also attenuated to 20% of its original intensity, suggesting that CT is also mostly fixed and incorporated into the hydrogel structure. For a smaller portion of the CT, which is as mobile as in the preincubated sample and thus observed by solution NMR, the spectra lack signals for the first 10 structured residues, possibly due to difficult immobilization of the attached portion of His-NT2Rep . The NMR spectra of the -state of hydrogels -NT2RepCT revealed the predominant presence of α-helices and β-layers and, to a lesser extent, the random coil conformation (Fig. 4b). Chemical shift analysis of methionine residues present only in NT showed that this domain had been converted to a β-sheet structure. The time-dependent spectra of NT in solution showed a uniform decrease in signal intensity (Fig. 4c), and solid-state NMR of NT hydrogels showed that most of the NT residues were converted to β-sheet structures (Fig. 4d). The conformation of 2Rep could not be determined separately due to its tendency to aggregate. However, the solid state NMR spectra of the NTCT and His-NT2RepCT hydrogels looked very similar (Fig. 4b; Supplementary Fig. 6b), suggesting that 2Rep contributed little to the structural part of the His-NT2RepCT hydrogel. For CT hydrogels, α-helices, β-sheets, and random helical secondary structures were found to exist (Supplementary Fig. 6d). This suggests that some parts of the CT remain α-helices while others become β-sheets. Thus, the results of NMR spectroscopy suggest that NT is important for hydrogel formation and also transforms into a β-sheet conformation upon fusion with 2Rep and CT. Consistent with this, we recently found that amyloid spatial zippers likely form in all five helices of the NT domain, and the Waltz algorithm predicted an amyloidogenic region in helix 1 (Fig. 4e).
2D spectra of 15N-HSQC 10 mg/mL His-NT2RepCT solution before (blue) and 19 hours after incubation (red) at 37°C. Individual cross peaks in the red spectrum and F24, G136, polyA in the blue spectrum are denoted by single letter amino acid symbols and residue numbers. The insets show the dependence of the signal intensity on time for selected residues from the NT, 2Rep, and CT domains. b Solid-state radiofrequency (RFDR) spectra of His-NT2RepCT hydrogels. Correlations of Cα/Cβ residues observed in RFDR spectra were determined by comparison with model peptide chemical shifts and values ​​derived from statistics82,83 and their secondary structures. SSB – rotating sideband. c One-dimensional spectra of 15N-HSQC 10 mg/mL NT solution during incubation at 37 °C for 36 hours. The inset shows volumetric intensity versus time. d Solid state RFDR spectra of NT hydrogels. The correlations of Cα/Cβ residues and their secondary structures observed in the RFDR spectra are indicated. e Based on the NT45.79 fibrillation propensity profile from the Zipper database (https://services.mbi.ucla.edu/zipperdb/). The Rosetta energy of the spatial lightning shift window of the hexapeptide is shown in kcal/mol. Red bars denote hexapeptides with a high fibrosis propensity (Rosetta energy below -23 kcal/mol; below the dotted line). Green bars indicate fragments with Rosetta energies above the threshold and therefore less likely to form steric zippers. Fragments containing proline were excluded from the analysis (without columns). Squares indicate areas of amyloidosis predicted by the Waltz algorithm81 (https://waltz.switchlab.org). The sequence of amino acid residues of NT is at the top, and the types of residues found in the β secondary structure (determined by solid-state NMR spectroscopy) are shown in red. The positions of the five NT α-helices are designated as (H1-H5)28.
At pH <6.5, HT dimerizes, being resistant to heat- or urea-induced denaturation18. To elucidate how NT dimerization and stability affect gelation, solutions containing 100 mg/ml NT were controlled at pH 8, 7, and 6 using the vial inversion test. NT samples incubated at pH 8 and 7 gelled after 30 min at 37 °C, but the pH 8 gel remained clear, while the pH 7 gel showed a visible precipitate (Fig. 5a). In contrast, a solution containing HT at pH 6 did not form a gel, and a large precipitate could be seen after 20 min at 37°C. This suggests that dimers themselves and/or their higher stability compared to monomers prevent gelation. The formation of a precipitate for NT at pH 7 and 6 was not expected, since it has been reported that NT is soluble at 200 mg/ml27, easily refolds after heat denaturation, and also retains an α-helix at lower values ​​of pH 18. A probable explanation for these discrepancies is that that the previously reported experiments were carried out at room temperature or below, or at relatively low protein concentrations16,18,19.
NT vial inversion test (100 mg/mL) at pH 8, 7, 6 and 154 mM NaCl (pH 8) after incubation at 37°C. b NT CD spectra with and without 154 mM NaF and 154 mM NaCl, respectively. Molar ellipticity at 222 nm is converted to a proportion of natural folds. c NT inversion assay (100 mg/mL) NT* (37 °C and 60 °C), NTA72R (37 °C), and His-NT-L6 (37 °C and 60 °C). d CD spectra of NT mutants NT*, NTA72R, and His-NT-L6. Molar ellipticity at 222 nm is converted to a proportion of natural folds. e Inversion test of NTFlSp, NTMiSp and reduced NTMiSp (100 mg/mL). Scale bar 5 mm. f CD spectra of NT, NTFlSp, NTMiSp and reduced NTMiSp. Molar ellipticity at 222 nm is converted to a proportion of natural folds. Full NT spectra at 25 °C and 95 °C are shown in Supplementary Figure 8.
Physiological salt concentration determines electrostatic interactions between NT subunits and dimerization of NT transfer to lower pH18. We found that the presence of 154 mM NaCl and NaF did indeed inhibit gelation, respectively (Fig. 5a, b; Supplementary Fig. 2b) and that these salts increased the thermal stability of NT monomers (Fig. 5b, Supplementary Fig. 8). It also suggests that stability enhancement, rather than dimerization, prevents gel formation.
To further explore the role of protein dimerization and stability in gelation, we used two mutants, NT* and NTA72R, which also remain monomeric at low pH28.30. NT* is a double charge reversal mutant in which the apparent dipolar charge distribution of the monomer is flattened, which prevents dimerization and drastically increases monomer stability. NTA72R is a charged dipole, but Arg-substituted Ala is located at the dimer boundary, so mutations interfere with subunit interactions required for dimerization. Upon incubation at 37°C, NT* did not form a hydrogel, while NTA72R formed an opaque gel for 15 min (Fig. 5c). Since both NT* and NTA72R cannot dimerize but differ in monomer stability (Fig. 5d), these results strongly suggest that high thermodynamic stability prevents NT from gelling. This is also supported by the fact that HT* forms a gel when it is unstable at high temperature (after 8 min at 60°C; Fig. 5c). It has previously been shown that the high content of methionine in NT liquefies its natural folding and that six Met to Leu substitutes (referred to here as His-NT-L6) strongly stabilize the NT46 monomer. Based on the assumption that structural flexibility is required for NT gel formation, we found that the His-NT-L6 stable mutant did not gel at 37 °C (Figure 5c, d). However, His-NT-L6 also formed a gel upon incubation at 60°С for 60 min (Fig. 5c).
The ability of NT to transform into β-sheet structures and form hydrogels appears to apply to some but not all of the NT domains of spidroin. NTs from different silk types and spider species, Trichonephila clavipes (NTFlSp), formed gels despite their relatively low methionine content and high thermal stability (Fig. 5e, f and Supplementary Table 2). In contrast, NT from the small ampullar protein spidroin from Araneus ventricosus (NTMiSp) with low thermal stability and high methionine content did not form hydrogels (Supplementary Table 2 and Fig. 5e, f). The latter may be associated with the presence of intramolecular disulfide bonds29,47. Consistently, when the disulfide bonds of NTMiSp were reduced, it formed a hydrogel after incubation at 37°C for 10 min (Fig. 5e). In conclusion, it should be noted that structural flexibility is an important, but not the only, criterion for the formation of a gel from NT. Another factor that may be relevant is the propensity to form amyloid fibrils, and analysis with the zipper database and the Waltz algorithm did show a correlation between the ability to form gels and the presence of amyloidogenic regions, as well as the extent of the regions predicted to form steric zippers. There was a correlation (Supplementary Table 2 and Supplementary Fig. 9).
The ability of NT to form fibrils and form gels under favorable conditions led us to hypothesize that NT fusions with other protein fragments can still form gels with full function of fusion partners. To test this, we introduced green fluorescent protein (GFP) and purine nucleoside phosphorylase (PNP) at the C-terminus of the NT, respectively. The resulting fusion proteins were expressed in E. coli with very high final yields (150 mg/L and 256 mg/L shake flask cultures for His-NT-GFP and His-NT-PNP, respectively), consistent with what has been shown for Other proteins fused to NT Ref. 30. His-NT-GFP (300mg/mL) and His-NT-PNP (100mg/mL) fusion proteins formed gels after 2 hours and 6.5 hours at 37°C and, importantly, the GFP fraction remained unchanged. observed after gelation, with >70% of the initial fluorescence intensity remaining after gelation (Fig. 6a). To measure PNP activity in his-NT-PNP solutions and gels, we had to dilute the fusion protein with NT because the enzymatic activity of the pure preparation was outside the detection range of the assay at gelling concentrations. The gel formed with a mixture containing 0.01 mg/mL His-NT-PNP and 100 mg/mL NT retained 65% of the initial enzymatic activity of the preincubated samples (Fig. 6b). The gel remained intact during the measurement (Supplementary Fig. 10).
a Relative fluorescence intensity before and after gelation of His-NT-GFP (300 mg/mL) and inverted vial containing His-NT-GFP hydrogel (300 mg/mL) under visible and UV light. Points show individual measurements (n = 3), error bars show standard deviation. The average value is shown in the center of the error bars. b PNP activity was obtained by fluorometric analysis using solutions and gels consisting of NT (100 mg/ml) and a mixture containing 0.01 mg/ml his-NT-PNP and 100 mg/ml New Taiwan dollars. The inset shows an inverted vial containing a hydrogel containing His-NT-PNP (5 mm scale bar).
Here, we report the formation of hydrogels from NT and other recombinant spidroin proteins by incubating a protein solution at 37°C (Figure 1). We show that gelation is associated with the transformation of α-helices into β-layers and the formation of amyloid-like fibrils (Figs. 3 and 4). This finding is surprising as NTs are coiled globular five-helix bundles known for their extremely high solubility and high stability at concentrations >200 mg/mL at 4°C for several days27. In addition, NTs readily refold after heat denaturation at low protein concentrations in µM. According to our results, fibril formation requires a combination of >10 mg/mL protein concentration and slightly elevated temperature (Fig. 1). This is consistent with the idea that amyloid fibrils can form from globularly folded proteins that are in a partially unfolded state due to thermal fluctuations under physiological conditions 48 . Examples of proteins that undergo this conversion include insulin49,50, β2-microglobulin, transthyretin and lysozyme51,52,53. Although NT is an α-helix in its native state, approximately 65% ​​of the polypeptide chain is compatible with steric zipper formation (Fig. 4e) 45 . Since the monomer is dynamically mobile46, it can expose these potential amyloidogenic regions at moderately elevated temperatures and at high concentrations of total protein can reach a critical concentration for amyloid fibril formation54. Following this reasoning, we found a negative correlation between spidroin concentration and gelation time (Fig. 1c), and if the monomeric NT conformation is stabilized either by mutations (NT*, His-NT-L6) or by salt addition, can prevent the formation hydrogels (Fig. 5).
In most cases, amyloid fibrils disappear from solution as a precipitate, but under certain conditions they can form hydrogels55,56,57. Hydrogel-forming fibrils typically have a high aspect ratio and form stable three-dimensional networks through molecular entanglement,55,58 consistent with our results. For hydrogel formation in vitro, proteins are often fully or partially unfolded, for example, by exposure to organic solvents, high temperature (70–90°C) and/or low pH (1.5–3.0)59,60,61,62. The spidroin hydrogels described here do not require harsh processing, nor do they require cross-linking agents to stabilize the hydrogels.
It has previously been reported that spidroin repeats and QDs, which appear to undergo β-sheet switching during silk spinning, form hydrogels. Compared to our findings, incubation times and/or incubation temperatures were significantly longer or higher, respectively, and the resulting hydrogels were often opaque (Figure 7 and Supplementary Table 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. In addition to fast gel times, NT hydrogels >300 mg/mL (30%) outperformed all other described recombinant spider silk protein hydrogels, as well as natural hydrogels such as gelatin, alginate (2%), agar (0.5 %) and collagen. (0.6%) (Figure 7 and Supplementary Tables 1 and 3)37,39,66,67,68,69,70,71,72,73,74.
The gel time and elastic modulus of the hydrogels in this study were compared with other spidroin-based hydrogels and selected natural hydrogels. References are given along with a description of the gelation conditions. APS Ammonium persulfate, room temperature. Data 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Spiders appear to have developed ways to prevent spidroin from gelling during storage. Despite the high concentration of protein in the silk gland, the large repeat region associated with the terminal domain means that the apparent concentration of NT and CT in the gland corresponds to approximately 10-20 mg/ml, at the boundary of this study. required for in vitro observed hydrogel formation. In addition, similar concentrations of salts 16 stabilized NT, as in silk glands (Fig. 5b). NT conformation has been studied in the E. coli cytosol and found to be more tightly folded than when examined in vitro, further indicating that salt or other factors prevent its aggregation in vivo. However, the ability of NTs to transform into β-sheet fibrils may be important for filament formation and should be investigated in future studies.
In addition to the novel aspects of NT-amyloid-like fibril and hydrogel formation observed in this study, we also show that this phenomenon may have biotechnological and biomedical applications (Fig. 8). As a proof of concept, we combined NT with GFP or PNP and showed that the fusion protein also forms hydrogels when incubated at 37 °C and that the GFP and PNP fractions largely retain their activity after gelation (Figure 6). Nucleoside phosphorylases are important catalysts synthesis of nucleoside analogues75, which makes our discovery relevant for the biopharmaceutical industry. The concept of expressing fusion proteins that form transparent hydrogels under favorable conditions allows the creation of functionalized hydrogels with favorable properties for a wide range of applications such as enzyme immobilization, controlled drug release and tissue engineering. In addition, NT and NT* are efficient expression markers30, which means that NT and its variants can be used for high-throughput production of soluble fusion proteins and subsequent creation of immobilized target proteins in 3D hydrogels.
NT is soluble, α-helical and stable at low concentrations (µM) and 37°C. At the same temperature, but at increasing concentrations (>10 mg/ml), NT forms gels consisting of amyloid-like fibrils. NT fusion proteins also form fibrillar gels with fully functional fusion fragments, allowing various proteins to be immobilized in 3D hydrogels using NT. Bottom: NT (PDB: 4FBS) and illustrations of fiber networks and associated protein structures (assumed and not drawn to scale, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/pdb4RJ2/pdb).
The constructs (see Supplementary Table 4 for a complete list including amino acid sequences) were cloned into plasmid pT7 and transformed into E. coli BL21 (DE3). E. coli containing engineered plasmids were inoculated in Luria broth supplemented with kanamycin (70 mg/l) and grown overnight at 30°C and 250 rpm. The culture was then inoculated 1/100 into LB medium containing kanamycin and cultured at 30°C and 110 rpm until the OD600 reached 0.8. For NMR studies, bacteria were grown in M9 minimal medium containing 2 g of D-glucose 13C (Aldrich) and 1 g of ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) for protein labeling with isotopes. Lower the temperature to 20 degrees Celsius and induce protein expression with 0.15 mM isopropylthiogalactopyranoside (final concentration). After overnight protein expression, cells were harvested at 7278×g, 4°C for 20 min. Cell pellets were resuspended in 20 mM Tris-HCl, pH 8, and frozen until further use. Thawed cells were lysed using a cell disruptor (TS series machines, Constant Systems Limited, England) at 30 kPa. Then the lysates were centrifuged at 25,000 g for 30 minutes at 4°C. For NTMiSp, the pellet was then resuspended in 2 M urea, 20 mM Tris-HCl, pH 8, and sonicated for 2 min (2 s on/off, 65%), then centrifuged again at 25,000 xg, 4° C. within 30 min. The supernatant was loaded onto a Ni-NTA column, washed with 20 mM Tris-HCl, 2 mM imidazole, pH 8, and finally the protein was eluted with 20 mM Tris-HCl, 200 mM imidazole, pH 8. To generate NT2RepCT and NTCT, thrombin digestion introduces the site (ThrCleav) between His and NT. Thrombin cleavage sites are also present in His-NT-ThrCleav-2Rep (produces 2Rep), His-thioredoxin-ThrCleav-NT (produces NT), His-thioredoxin-ThrCleav-CT (produces CT), His-Thioredoxin-ThrCleav-NT. * (produces NT*), His-Thioredoxin-ThrCleav-NTA72R (produces NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (produces NTF1Sp), and His-Sulphur Redoxin-ThrCleav-NTMiSp (produces NTMiSp). The constructs were digested with thrombin (1:1000) and dialyzed overnight at 4° C. with 20 mM Tris-HCl, pH 8, using a Spectra/Por dialysis membrane with a molecular weight threshold of 6-8 kDa. After dialysis, the solution is loaded onto a Ni-NTA column and the effluent containing the protein of interest is collected. Protein concentrations were determined by measuring UV absorbance at 280 nm using the extinction coefficient of each protein, except for NTF1Sp, which used the Bradford assay according to the manufacturer’s protocol. Purity was determined by SDS polyacrylamide (4–20%) gel electrophoresis and Coomassie brilliant blue staining. Proteins were concentrated using centrifuge filters (VivaSpin 20, GE Healthcare) at 4000 xg with a 10 kDa molecular weight cutoff in 20 minute cycles.
Thaw the protein solution and carefully pipet 150 µl into a 1 ml clear septum vial (8 x 40 mm Thermo Scientific). The tubes were capped and sealed with parafilm to prevent evaporation. Samples (n = 3) were incubated at 37°C or 60°C and periodically inverted to observe gelation. Samples that did not gel were incubated for at least one week. Reduce NTMiSp disulfide bonds with 10 mM DTT per 10 µM protein. To analyze the gelation of natural spider silk coatings, the Swedish bridge spider was cut, the two main ampullated glands were placed in 200 μl of 20 mM Tris-HCl buffer pH 8 and cut to allow the coating to separate from the glands. . The contents of the glands are dissolved in buffer, 50 µl for determination of dry weight (by incubation of open vials at 60 °C to constant weight) and 150 µl for gelation at 37 °C.
The measuring geometry/tool ​​is made of stainless steel using a parallel plate with a top diameter of 20 mm and a gap of 0.5 mm. Heat the sample from 25 °C to 45 °C and back to 25 °C at a rate of 1 °C per minute using a stainless steel bottom Peltier plate. Vibrational measurements were carried out at a frequency of 0.1 Hz and in the linear viscoelastic region of the material at a strain of 5% and 0.5% for samples of 100 mg/mL and 300–500 mg/mL, respectively. Use a custom humidity chamber to prevent evaporation. Data was analyzed using Prism 9.
For collecting infrared (IR) spectra at room temperature from 800 to 3900 cm–1. The ATR device, as well as the light path through the spectrometer, is purged with dry filtered air before and during the experiment. Solutions (500 mg/mL to minimize water absorption peaks in the spectra) were pipetted onto the crystals, and gels (500 mg/mL) were formed prior to measurement and then transferred to the crystals (n = 3). 1000 scans were recorded with a resolution of 2 cm-1 and zero duty cycle of 2. The second derivative was calculated using OPUS (Bruker) using a smoothing range of nine points. The spectra were normalized to the same integration region between 1720 and 1580 cm-1 using F. Menges “Spectragryph – Optical Spectroscopy Software”. In ATR-IR spectroscopy, the penetration depth of an infrared beam into a sample is wavenumber dependent, resulting in stronger absorption at lower wavenumbers than at higher wavenumbers. These effects have not been corrected for the spectra shown in Figs. 3 because they are very small (Supplementary Fig. 4). Corrected spectra for this figure were calculated using the Bruker OPUS software.
In principle, a comprehensive quantification of protein conformations is possible after reliable deconvolution of the components within the amide I peak. However, some obstacles arise in practice. Noise in the spectrum can appear as (false) peaks during deconvolution. In addition, the peak due to water bending coincides with the position of the amide I peak and may have a similar magnitude for samples containing a large amount of water, such as the aqueous gel studied here. Therefore, we did not attempt to completely decompose the amide I peak, and our observations should only be considered in support of other methods such as NMR spectroscopy.
Solutions of 50 mg/ml NT and His-NT2RepCT were gelled overnight at 37°C. The hydrogel was then diluted with 20 mM Tris-HCl (pH 8) to a concentration of 12.5 mg/ml, shaken well and pipetted to break the gel. Next, the hydrogel was diluted 10 times with 20 mM Tris-HCl (pH 8), 5 μl of the sample was applied to a copper grid coated with formvar, and the excess sample was removed with blotting paper. Samples were washed twice with 5 µl of MilliQ water and stained with 1% uranyl formate for 5 minutes. Remove excess stain with absorbent paper, then air dry the mesh. Imaging was performed on these grids using an FEI Tecnai 12 Spirit BioTWIN operating at 100 kV. The images were recorded at x 26,500 and x 43,000 magnifications using a Veleta 2k × 2k CCD camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany). For each sample (n = 1), 10–15 images were recorded. ImageJ (https://imagej.nih.gov/) was used for image analysis and measurement of fiber diameters (n = 100, different fibers). Prism 9 was used to perform unpaired t-tests (two-tailed). The mean His-NT2RepCT and NT fibrils were 11.43 (SD 2.035) and 7.67 (SD 1.389) nm, respectively. The confidence interval (95%) is -4.246 to -3.275. degrees of freedom = 198, p < 0.0001.
80 µl of liquid samples containing 10 µM thioflavin T (ThT) were measured in triplicate (n = 3) under static conditions using Corning 96-well black bottom clear bottom plates (Corning Glass 3881, USA). Fluorescence differences were recorded using a 440 nm excitation filter and a 480 nm emission filter (FLUOStar Galaxy from BMG Labtech, Offenburg, Germany). The ThT signal was neither saturated nor quenched, as experiments with different concentrations of ThT were performed without changing the signal intensity. Record absorbance at 360 nm for haze measurement. For seeding experiments, 100 mg/mL gels were formed at 37° C., resuspended, and used for seeding at molar ratios of 5%, 10%, and 20%. Data was analyzed using Prism 9.
Thaw stocks of His-NT2RepCT and NT >100 mg/mL on ice and filter through a 0.22 µm filter. Concentrations were calculated by measuring absorbance at 280 nm using Nanodrop. In wells of a 96-well black non-binding plate (Corning) with a clear bottom, samples were diluted to 20 mg/ml in 20 mM Tris-HCl pH 8 and mixed with 5 μM ThT (final concentration), total sample concentration 50 μl volume. Samples were imaged every 10 minutes at 37 °C on a CellObserver (Zeiss) microscope with transmitted light channel and FITC excitation and emission filter sets for ThT imaging. A 20x/0.4 lens is used for imaging. Zen Blue (Zeiss) and ImageJ (https://imagej.nih.gov/) were used for image analysis. Gels were also prepared from NT and His-NT2RepCT solutions at a concentration of 50 mg/mL containing 20 mM Tris pH 8 and 5 µM ThT and incubated at 37°C for 90 min. The gel pieces were transferred to a new well containing 20 mM Tris, pH 8, and 5 μM ThT in a non-binding black 96 well clear bottom plate. Acquire green fluorescence and bright field images at 20x/0.4 magnification. ImageJ was used for image analysis.
Solution NMR spectra were obtained at 310 K on a 600 MHz Bruker Avance Neo spectrometer equipped with a QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN). NMR samples containing 10 mg/mL homogeneous protein labeled with 13C, 15N, dissolved in 20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1). Chemical shifts of NT2RepCT at pH 6.7 were used to assign peak 23 in the 2D spectrum of 15N-HSQC.
Magic angle spinning solid NMR (MAS) spectra of 13C, 15N-labeled hydrogels were recorded on a Bruker Avance III HD spectrometer at 800 MHz equipped with a 3.2 mm 13C/15N{1H} electronless probe. Sample temperature was controlled using a variable temperature gas stream at 277 K. Two-dimensional dipole rotational resonance (DARR)76 and radio frequency reconnection (RFDR)77 spectra were acquired at MAS frequencies of 12.5 kHz and 20 kHz, respectively. Cross polarization (CP) from 1H to 13C was performed using a linear ramp from 60.0 to 48.0 kHz at 1H, 61.3/71.6 kHz at 13C (at 12.5/20 kHz MAS) and contact time 0.5–1 ms. Spinal6478 decoupling at 73.5 kHz was used during data collection. The acquisition time was 10 milliseconds and the cycle delay was 2.5 seconds. The single-linked Cα/Cβ correlations observed in the RFDR spectra were assigned based on the characteristic residue-type chemical shifts and multiply-linked correlations in the DARR spectra.
The Zipper79 database (https://services.mbi.ucla.edu/zipperdb/) was used to evaluate flutter tendencies and Rosetta energy for NT, NTFlSp, and NTMiSp. The Zipper database computes Rosetta Energy80, which combines several free energy functions to model and analyze protein structure. An energy level of -23 kcal/mol or lower indicates a high tendency to fibrillate. The lower energy means more stability of the two β-strands in the zipper conformation. In addition, the Waltz algorithm was used to predict amyloidogenic regions in NT, NTFlSp and NTMiSp Ref. 81. (https://waltz.switchlab.org/).
The NT protein solution was mixed with 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 5.5 and 6.0 to lower the pH to pH 6 and 7, respectively. The final protein concentration was 100 mg/ml.
Measurements were performed on a J-1500 CD spectrometer (JASCO, USA) using a 300 μL cuvette with an optical path of 0.1 cm. Proteins were diluted to 10 μM (n = 1) in 20 mM phosphate buffer (pH 8). To analyze protein stability in the presence of salt, proteins were analyzed at the same concentration (n = 1) in 20 mM phosphate buffer (pH 8) containing 154 mM NaF or NaCl, respectively. Temperature scans were recorded at 222 nm from 25°C to 95°C with a heating rate of 1°C/min. The proportion of natively folded proteins was calculated using the formula (KDmeasure – KDfinal)/(KDstart – KDfinal). In addition, five spectra were recorded for each sample from 260 nm to 190 nm at 25°C and after heating to 95°C. Five spectra were averaged, smoothed and converted to molar ellipticity. Data was analyzed using Prism 9.
The fluorescence intensity of His-NT-GFP (300 mg/mL, 80 µL) was measured in triplicate (n = 3) in 96-well Corning plates with a black transparent bottom (Corning Glass 3881, USA) under static conditions. Measure samples with a fluorescence-based plate reader with an excitation wavelength of 395 nm and record the emission at 509 nm before gelation and 2 hours later at 37°C. Data was analyzed with Prism 9.
Purine nucleoside phosphorylase activity assay kit (fluorometric method, Sigma Aldrich) was used according to the manufacturer’s instructions. To measure activity in gels and solutions containing His-NT-PNP, mix 10 ng of His-NT-PNP with 100 mg/mL NT to a total volume of 2 µL because the gel gave a signal above the detection interval of the set. Controls for gels and solutions without His-NT-PNP were included. The measurements were carried out twice (n = 2). After the activity was measured, the reaction mixture was removed and the gel photographed to ensure that the gel remained intact during the measurement. Data was analyzed using Prism 9.
For more information on study design, see the Nature study abstract linked to this article.
Figures 1 and 2 present the initial data. 1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, and 6, Supplementary Figs. 3, supplementary fig. 5a, d, supplementary fig. 6 and supplementary fig. 8. Data Data from this study is hosted in the Zenodo database https://doi.org/10.5281/zenodo.6683653. The NMR data obtained in this study were posted to the BMRBig repository under the entry ID bmrbig36. The structures of GFP and PNP were taken from PDB (GFP 2B3Q, PNP 4RJ2).
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Babb, P.L. et al. The Nephila clavipes genome highlights the diversity of spider silk genes and their complex expression. National Genette. 49, 895–903 (2017).