Molecular Dynamics of Synthetic Flagelliform Silk Fiber Assembly

Abstract

In order to better understand the relationship between Flagelliform (Flag) spider silk molecular structural organization and the mechanisms of fiber assembly, it was designed and produced the Nephilengys cruentata Flag spidroin analogue rNcFlag2222. The recombinant proteins are composed by the elastic repetitive glycine-rich motifs (GPGGX/GGX) and the spacer region, rich in hydrophilic charged amino acids, present at the native silk spidroin. Using different approaches for nanomolecular protein analysis, the structural data of rNcFlag2222 recombinant proteins were compared in its fibrillar and in its fully solvated states. Based on the results was possible to identify the molecular structural dynamics of NcFlag2222 prior to and after fiber formation. Overal rNcFlag2222 shows a mixture of semiflexible and rigid conformations, characterized mostly by the presence of PPII, β-turn and β-sheet. These results agree with previous studies and bring insights about the molecular mechanisms that might driven Flag silk fibers assembly and elastomeric behavior.

Graphical Abstract

Using different approaches for nanomolecular protein analysis, the molecular structural dynamics of Flagelliform spidroin’s analogue rNcFlag2222 was analyzed prior to and after fiber formation. The results indicate that the structural features adopted by the Flagelliform repetitive glycine-rich and spacer regions play important roles in fiber assemble and elastomeric behavior.

1. Introduction

Aiming for the possible use of spider silks in the design and development of novel biomaterials, several laboratories have attempted to produce recombinant spider silks proteins [1-5]. Their molecular structure has direct implications on material strength and stability and allows the modular engineering of spider silks for a wide range of industrial and medical purposes [6-8]. Consequently, the determination of the structural organization of fiber silk and silk analogs have been a subject of great interest.

Among the silks produced by orb weaver spiders, the Flagelliform (Flag) silk is extremely elastic and is characterized by a very high hysteresis [7-9]. In nature, Flag silk is used in the core of the web for prey-capturing. It is coated by an aqueous glycoprotein stick glue which keeps the fibers hydrated. The presence of water in native Flag silks has been shown to supplement extensibility to the fiber. Apart from water’s contribution to the mechanical properties of Flag silk fibers, the primary sequence of spider silk proteins has also been correlated with its remarkable strength and extensibility [10-14].

Prior to fiber formation, the Flag silk proteins are stored as a highly concentrated protein solution in the Flag silk specific gland. Like other elastomeric proteins, such as collagen, elastin and abductin [15-17], the Flag protein has a high content of glycine residues. Its amino acid content is organized into large ensemble repeats composed of individual repetitive motifs (GPGGX and GGX) and a “spacer” sequence containing charged and hydrophilic amino acids [9, 18]. While it is accepted that the elastic nature of Flag silk fiber originates from the unusually elevated number of GPGGX motifs, the structural function of the spacer region is unclear [19]. However, its remarkably high sequence conservation among species and exclusive presence of negatively charged residues (D and E) among its primary amino acid population suggest that the spacer region is a critical component of the Flag silk protein [9].

According to previous research, the GPGGX pentapeptides in spider silk proteins confer fiber elasticity through the adoption of β-turn structures acting as spring-like spirals, with each turn of the spiral containing two pentapeptide motifs [19-22]. Others have described the GPGGX and GGX repeated sequences as an amorphous region, without any crystalline structure in the fibers, and conformations with no preferential orientation [23, 24]. In contrast, studies by Perea and coleagues [25] have demonstrated the presence of polyglycine II nanocrystals in Argiope trifasciata native Flag silk, which was supposed to be conferred by the dominant presence of the GPGGX and GGX motifs in its sequence. On the other hand, the spacer region secondary structure is still not clear. Nevertheless, it was proposed that the spacer may function as a strength-motif in Flag-like proteins due in part to β-sheet formation, through proteins intermolecular interactions [26, 27].

In our previous study we have identified the primary sequence of Flag spidroin from the Brazilian spider Nephilengys cruentata [28]. To investigate the relationship between Flag spidroin structural organization and the mechanisms of fiber assembly, we produced the recombinant spidoin rNcFlag2222. The Flag silk-like protein was based on key structural regions from N. cruentata spider’s Flag spidroin, the glycine-rich structural motifs and the spacer region. Using several approaches for nanomolecular analysis, including circular dichroism (CD) spectroscopy, atomic force (AFM) and scanning electron (SEM) microscopies, Raman spectromicroscopy and X-ray diffraction (WAXS), we have compared structural data of rNcFlag2222, in its fibrillar and in its fully solvated states. Based on the results, we were able to identify the structural dynamics of NcFlag2222 and bring new insights about the molecular mechanisms that might driven Flag silk fibers assembly and elastomeric behavior.

2. Results

2.1. Recombinant flagelliform proteins production and analysis

Following the protocol described by Teulé and others [29] we were able to construct an expression vector containing two of each Flag-like synthetic double stranded oligonucleotides modules spanning the consensus repeat of N. cruentata flagelliform silk spidroin [28] (Figure 1). The resulting 765 bp silk sequence was cloned into the pET19b vector and introduced into BL21(DE3) pLysS E. coli cells for expression (Figure 2a). The His-tagged rNcFlag2222 recombinant silk protein was purified from the total protein extract through metal affinity chromatography. The predicted molecular weight of rNcFlag2222 was 23 kDa, however according to the SDS PAGE analysis the recombinant protein ran at ~30 kDa (Figure 2b). Western blot analysis was performed using a polyHistidine antibody and confirmed the rNcFlag2222 molecular weight (Figure 2c). The reduced electrophoresis’ mobility shown by rNcFlag2222 is explained by the negatively charged amino acids composing the spacer region of the flagelliform protein (unit 3) [30-32].

Figure 1.

(a) Amino acid sequence of the flagelliform protein of the orbicular spider N. cruentata and its different structural motifs (GPGGX in red, GGX in green and the spacer region in blue). (b) The four synthesized units used in the construction of the synthetic silk sequence. (c) Primary protein sequence rNcFlag2222.

Figure 2.

(a) Electrophoretic analysis on agarose gel of the vector prNcFlag 2222 (insert of 765 bp). The plasmid was digested with the enzymes NdeI and BamHI. The smallest band corresponds to the Flag insert and the largest band is the linearized pET19b-kan vector (5.8 kpb). 1 Kb DNA Ladder Invitrogen marker. (b) SDS-PAGE analysis showing the purified Flag protein detected by Coomassie blue staining. Mass standard Molecular Precision Plus Protein ™ Dual Color Standards Bio Rad (c) Analysis of western blot using anti-polyHistidine antibody. The arrows point to the protein rNcFlag2222.

In order to analyze the structural dynamics of Flag domains in solution, the secondary structures present in the rNcFlag2222 solubilized in 100% methanol, 100% acetonitrile and distilled water solutions were investigated using CD spectroscopy (Figure 3). Interestingly, the rNcFlag2222 CD spectras recorded clearly indicated that besides β-turns, other conformations such as helices and random coil were present simultaneously. However, the content of each secondary structure present in cNcFlag2222 shifted significantly depending on the solvent used

Figure 3.

The FAR-UV CD spectra obtained for rNcFlag2222 diluted in the different solvents used, water (FlagWt), acetonitrile (FlagAcet) and methanol (FlagMet).

In distilled water, rNcFlag2222 showed a high amount of helix content (71.1%), and lower amount of random coil (16%), β-turn (10.7%) and β-sheet (3.9%) structures. In addition, the CD spectra of the protein in water indicated a typical polyproline type II helix (PPII) conformation with a negative peak around 215 nm (Figure 3). In acetonitrile the same negative peak was observed thus is indicative of a major presence of helical structures (56.1%), though random coil (22.5%), β-turn (13%) and β-sheet (6.4%) conformations were also observed.

In methanol the secondary structure of rNcFlag2222 exhibited a considerable decrease in helical structures (16.4%) and an increase in random coil structures (32.4%), as evidenced by the two weaker negative peaks observed around 208 and 222 nm (Figure 3). Additionally, in methanol the β-turn content in this protein slightly increased to 14.2% compared to those observed in water or acetonitrile. Curiously, the β-sheet content is the main conformation observed for this protein in methanol (30.3% antiparallel and 6.7% parallel), but rNcFlag2222 practically lacked any significant amount of β-sheets structures when diluted in the other solvents.

2.2. The synthetic spun Flag-like fibers

To evaluate the spinning potential of the rNcFlag2222 recombinant protein, a silk dope was prepared by dissolving this pure lyophilized protein in an organic solvent (HFIP). The extrusion of this silk dope into a 100% isopropyl alcohol coagulation bath resulted in the production of synthetic Flag-like silk fibers. The rNcFlag2222 silk fiber has a uniform cylindrical appearance under the light microscope, with approximately a 40 μm diameter (Figure 4a). SEM and AFM techniques were used in an attempt to better understand the ultra-structural and nano-structural organization of the synthetic Flag-like fibers. Figure 4b and Figure 4c shows the SEM analysis of the synthetic Flag-like rNcFlag2222 fiber. The images indicate that the fibers were compact and possessed a rough surface. AFM data complement the SEM data (Figure 4d) and show that the topographic surface of these synthetic fibers is granular, characterized by the presence of nanoglobular structures comparable to those found in other synthetic silk fibers [33]. Perea and others [25] also observed a nanoglobular microstructure in native Flag silk. However, Flag-like rNcFlag2222 fiber have areas with imperfections and an unstructured surface, with the nanoglobules loosely associated; which contrasts with AFM data for native spiders and silkworm silk [34].

Figure 4.

Structural analysis of the recombinant rNcFlag2222 silk fiber. (a) The Flag-like fiber acquired by light microscopy (40x; scale bar = 30 μm) (b) and (c) Scanning electron micrographs of synthetic fiber at different magnifications. The image in (b) shows the general appearance of the fiber (1000x; scale bar = 20 μm), and in (c) it is possible to observe the granules present on the fiber surface (5000x; scale bar = 5 μm); and (d) 3D atomic force microscopy (AFM) images shows topographic details of the Flag-like fiber surface.

Furthermore, no longitudinal organization could be observed in the synthetic fiber. Previous studies have provided evidence of the presence of nanofibrils in silkworm and spider fibers [35-37]. SEM imaging of B. mori, A. pernyii, N. edulis fibers suggests the presence of nanofibrils assembled to form tightly packed microfibrils, with some longitudinally oriented voids or cracks between them [38]. Although the rNcFlag2222 fiber has shown apparently packed areas with numerous irregularities, nanofibrils formation could not be observed in any of the scanned areas.

2.3. Molecular Structural analyses of the synthetic Flag-like silk

Raman spectromicroscopy and WAXS were employed to further investigate the synthetic Flag-like rNcFlag2222 fiber molecular structure. As seen in Figure 5, the conformation sensitive amide I (1600-1700 cm⁻¹) and amide III (1200-1300 cm⁻¹) bands from the polarized Raman spectra of the Flag-like fiber are particularly informative. Other bands can also be seen in agreement with the rNcFlag2222 amino acid sequence, the spectra show the presence of amino acids residues such as Phe (1003 cm⁻¹), Tyr (1615 cm⁻¹) and Gly (1415 cm⁻¹), which is very abundant in the recombinant spidroin (46%).

Figure 5.

Raman spectrum (above) and spectral decomposition of the amide I region (below) of the synthetic fiber rNcFlag2222.

Consistent with previous results from the native Flag silk, the spectra of the rNcFlag2222 fiber shows the presence of a broad amide I band with a peak at 1659 cm⁻¹, indicating the presence of a heterogeneous mixture of secondary structures [26, 39]. In this region, signals of random coil (1641 cm −1), PPII (1656 cm-1), β-sheets (1670 cm −1) and β-turns (1685 cm −1) structures were found. The most prominent and defined peak of all structures identified in the amide I region represents the helical structures (α-helices / PPII helices). These structures were also observed in the CD spectra of the rNcFlag2222 protein diluted in methanol, acetonitrile and distilled water, showing that the PPII conformations are retained before and after the spinning process, probably due to GGX and GPGGX motifs. In agreement with our data, is the observation that polyglycine II nanocrystals are present in the glycine rich regions of Argiope trifasciata native Flag silk [25]. Polyglycine II is also recognized as a PPII helix, because it generates a left-handed helix with CD conformational parameters similar to those for PPII [40].

The WAXS patterns of rNcFlag2222 fiber is characterized by the presence of a diffuse ring centered at 4.4Å and an additional reflection at 10.5Å (Figure 6). These patterns are consistent with the Raman spectra, and indicate the presence of an amorphous phase composed mainly by helices and stacked β-sheets type structures, respectively [41]. Previous study analyzing synthetic Flag-like fibers [27], also observed this double reflection WAXS pattern only for the synthetic fibers composed by recombinant proteins containing the spacer region in its primary structure, which clearly produced the strongest fibers with high extensibility. Our data agree with this finding, suggesting that the spacer motif might be involved in the formation of β-sheets type structures in Flag silk.

Figure 6.

Wide angle X-ray scattering (WAXS) analysis of fiber synthetic Flag-like rNcFlag2222 processed. The arrows point to two amorphous rings of different intensity with the centers at 4.4Å and 10.5Å.

3. Discussion

In this work, we designed and produced in E. coli the rNcFlag2222 Flag silk-like protein to investigate the structural behavior of Flag spidroin repetitive domain and their contribution to fiber assembly and elastomeric behavior. Composed of the repetitive core and the spacer region sequences found in the native N. cruentata Flag spidroins [28], rNcFlag2222 was analysed prior to and post fiber formation.

In its solvated state, rNcFlag2222 showed different secondary structural content, composed by helix, random coil, β-turn and β-sheet conformations. A recent study has shown that native spider silk proteins also presented specific secondary structure profiles in solution [42]. According with the authors, Flag proteins were partially folded and presented β-turn conformational structures. Their results suggest that spider silk proteins in general behave in solution like elongated semiflexible polymers with locally rigid sections, agreeing with the multipe conformational structure found for rNcFlag2222.

The CD spectra acquired for rNcFlag2222 also shifted significantly depending on the solvent used (methanol, acetonitrile and distilled water). The most significant difference observed was the presence of substantial β-sheets conformations when diluted in methanol (37%). The rNcFlag2222 also presented β-turns, helices and random coil conformations simultaneously, but in acetonitrile and distilled water solutions the amount of β-sheets structures was significantly lower (6.4% and 3.9%, respectively). However, the conformational features in aqueous solution indicated a major presence of PPII helix together with α-helix structures. Methanol seems to promote the transition between PPII/α-helix to β-sheet structures in rNcFlag2222 protein.

In agreement with this structure is the observation that the β-sheet regions are poorly hydrated [19]. Since methanol is a highly dehydrating solvent, this would explain the predominant β-sheet secondary structure present in rNcFlag2222 detected by CD analysis. The presence of hydroxyl groups in methanol also facilitates β-sheet formation in the rNcFlag2222 protein by decreasing solvent polarity and strengthening the local hydrogen bonds that result in the stabilization of the β-sheets. This also makes sense for flagelliform silks in general, where elasticity is supposed to be enhanced by two factors: the presence of GPGGX and GGX motifs and the exposure of molecular chains to moisture, which would increase hydrophobic forces and promote interchain hydrogen bond formation [8, 27, 43, 44].

In vivo, dehydration of the spinning solution leads to a structural transition from coil and helix to β-sheet structures resulting in fiber formation [45-47]. Recently, it was proposed that the PPII helix population in the glycine-rich region of Major Ampullate spidroins (Masp) might serve as the prefibrillar form of spider dragline silk. It may also contribute to the efficiency of the spinning process, because the PPII helix can easily undergo intramolecular interaction in response to shear forces and dehydration by forming reverse turns [47]. Considering our results, and that Masp and Flag proteins share common features [25, 48], we believe that the same mechanism might happen with rNcFlag2222.

To further investigate the conformational dynamics of Flag silk repetitive domains, rNcFlag2222 was spun into fibers. The recombinant spidroin was capable of producing synthetic Flag-like fibers. However, differing from native Flag silks [25, 35, 36, 49], the synthetic silk presented an amorphous surface with numerous irregularities and no nanofibril formation was observed. These characteristics might be explained by rNcFlag2222 size, amino acid composition and lack of the terminal domains. Another study, also using recombinant Flag silk, have shown that the length of the repetitive core domain as well as the presence of the carboxy-terminal (C-terminal) non-repetitive domain impacted on spidroin aggregation and fiber formation [50].

Indeed, the molecular size of rNcFlag2222protein is much smaller than the native ones thus conferring less opportunity for extensive molecular chain interactions and consequently a less stabilized fiber structure. Recently, Bowen and others [51] have produced recombinant spidroins of notable size (556 kDa), containing 192 repeat motifs of Nephila clavipes dragline spidroin. The fibers spun from these synthetic spidroins are the first to fully replicate the mechanical performance of their natural counterparts, showing that protein size does matter. On the other hand, Nileback and others [52] have demonstrated that each protein part is crucial for fiber silk assembly and, most importantly, organized nanofibrillar structures were formed only when the C-terminal of spider silk spidroin were present in the recombinant protein.

In our case, rNcFlag2222 was engineered without the C-terminal region, which might explain the absence of nanofibrils in the synthetic fiber produced. Other aspects such as pH, ionic conditions, fiber spinning speed, and the presence of His tag may also have influenced the spinning process and the success of fiber assembly [5, 42, 53, 54].

According to Raman spectromicroscopy and WAXS structural analyses, the synthetic rNcFlag2222 spun fiber is a mixture of secondary structures, mainly composed of helices (α-helices / PPII helices), β-turns and stacked β-sheets. These data concur with the CD spectra obtained for rNcFlag2222 diluted in different solvents. According to the conformational dynamics observed for the recombinant spidroin, the PPII helix is maintained in the synthetic silk, probably conferring a loose alignment of the protein chains. This structure together with the presence of β-turns, typically formed by glycine rich protein regions [55], are consistent with the elastomeric property of the natural Flag silk fibers [14, 25].

Bochicchio and others [56] proposed a multiconformational equilibrium common to all studied elastomeric proteins such as elastin, lamprin and abductin. Agreeing with our results, all of those elastomeric proteins would show a dynamic equilibrium between extended and folded conformations. The PPII helix is a flexible structure because of its particular dihedral angles that allows the protein chain to progress directly into a reverse turn [57]. In its turn, the β-turns structures also formed by the GPGGX motif would work as an elastic spring, with the Pro residue presenting a strong interaction with water [58].

Interestingly, recent proteomic data from Nephila clavipes native Flag silk have shown the presence of 600 intramolecular hydrogen bonds in presence of water, indicating that native Flag silk is structured and stable in aqueous solution [59]. The authors also identified hydroxyproline as the major post translational modification present in the glycine rich repetitive domain of Flag silk, just as it occurs in collagen found in the mussel Mytilus edulis, also warranting protein stability [60-62].

Additionally, it has been shown that native Flag silks works better when wet, either tested with the viscous coating or cleaned and immersed in water. When tested in dry environments, their mechanical behavior was no longer elastomeric [63]. These features would favor the equilibrium between open and folded structures, thus pointing to a common molecular mechanism for the origin of elasticity.

Together with the repeated elastic sequences containing highly flexible monomeric chains and a high glycine content, this elasticity would also depend on the extent of cross-linking of these elastomeric proteins [64]. For Flag silks, these cross-linking domains might be represented by the spacer regions between the elastic GPGGX and GGX repeated sequences. Although the spacer motif remains not clearly defined structurally, it is supposed that it provides strength to the fiber in order to support a flying prey [27]. This feature would favor the protein to return safely to its original state after elongation, causing no damage to the silk’s mechanical and physical properties [64].

It has been reported that small quantities of oriented β-sheets structures (8% to 16%, according to the spider species) are presented in native Flag spider silk fibers [24]. The authors suggest that the spacer region might be involved in the formation of the β-sheets in the silk fibers. Adrianos and others [27] were also able to identify through X-ray diffraction the presence of β-sheet structures in synthetic silk produced only by recombinant Flag-like proteins that contained the spacer module. Since the rNcFlag2222 protein contains a total of two spacer regions among the typical silk-like sequences, our results also bring evidence that this region might explain the presence of β-sheet in rNcFlag2222 structural conformational analysis.

4. Conclusions

Here we report the molecular structural dynamics of the rNcFlag2222 recombinant proteins in its fibrillar and in its fully solvated states. rNcFlag2222 is composed by the elastic repetitive motifs (GPGGX/GGX) and the spacer region present in native N. cuentata Flag silk. Similar to natural spider silks, rNcFlag2222 behaved in solution like elongated semiflexible polymers with rigid sections [42], characterized mostly by the presence of helical structures, β-turn and β-sheet structural conformations.

Interesting, the structural data obtained for rNcFlag2222 showed that PPII helix were present in all analyzed solutions. The results indicate that rNcFlag2222 proteins mostly adopt PPII conformations by interaction with water. The loose conformation adopted by this structure must be critical for rNcFlag2222 fiber assemble. Furthermore, PPII structures was maintained after fiber formation, suggesting that the structural conformation adopted by the glycine rich motifs might also favor Flag silks elastomeric behavior (25). Accordingly, the most extensible spider silks types are the ones who have GPGXX motifs in their primary amino acid sequence, Flag and MaSp2 [21].

The structural conformational analysis of the rNcFlag2222, prior and after fiber formation, also indicated the presence of β-sheets conformations. These results agree with previous studies and adds to the idea that the C-terminal region might be involved in β-sheets formation [26, 27], possibly serving as a linker for the flexible spirals adopted by the repeated glycine-rich sequence, conferring strength to the fiber.

The structural data obtained for the synthetic spun Flag-like fibers indicates that overall, spidroin composition and spinning conditions have a major influence in fiber assembly and supramolecular structure organization. However, previous works have reported that individual domains of spider silks function independently and there are no stable interactions between them [47, 65]. Therefore, our study of the structural conformational dynamics of Flag protein repetitive domains individually, before and after fiber formation, might be highly relevant for the development of novel biomaterials.

In order to design a successful spinning strategy, it is crucial the comprehension of the biochemical mechanisms responsible for fiber formation and mechanical properties. More importantly, take these in consideration for further investigation on spider silks design to obtain a biomaterial with desirable structural properties.

5. Experimental procedures

Gene sequence engineering:

The engineered sequence is based on Nephilengys cruentata Flag spidroin cDNA sequence.^[28] Four double stranded DNA units were synthesized by Addgene DNA 2.0 (Watertown, MA):

Unit 1 - CTC GAG CAT ATG CCC GGG CCG GGT GGT GCT TAC GGT CCG GGT GGT CCG GGT GGT CCG GGT GGT CCG GGT TCC GGA TAA GGA TCC

Unit 2 - CTC GAG CAT ATG CCC GGG CCG GGT GGT GCT GGT CCG GGT GGT TAC GGT CCG GGT GGT TCC GGA TAA GGA TCC

Unit 3 (Spacer) - CTC GAG CAT ATG CCC GGG GGT TCC GGT GGT ACC ACC GTT ATC GAA GAC CTG GAC ATC ACC GTT AAC GGT CCG GGT GGT CCG ATC ACC ATC TCC GAA GAA CTG ACC GTT GGT TCC GGA TAA GGA TCC

Unit 4 - CTC GAG CAT ATG CCC GGG CCG GGT GCT GGT GGT TCC GGT CCG GGT GGT GCT GGT CCG GGT GGT GCT GGT CCG GGT GGT GCT GGT CCG GGT GGT GTT GGT CCG GGT GGT GCT GGT GGT CCG GGT GGT GCT GGT GGT CCG TTC GGT CCG GGT GGT TCC GGT CCG GGT GGT GCT GGT GGT GCT GGT TCC GGA TAA GGA TCC

The synthetic Flag sequence was designed to generate monomer units to build large synthetic spider silk-like tandem repeat sequences, from small double-stranded monomer DNAs, flanked by compatible but non-regenerable restriction sites.^[29] This strategy can be repeated to derive any number of units desired. The rNcFlag2222 encoding sequence contained each unit repeated twice.

Protein expression:

The final rNcFlag2222 double stranded DNA construct was ligated in the pET19b vector (MilliporeSigma, Burlington, MA) and cloned into E. coli BL21(DE3) pLysS bacteria for expression purposes. The bacterial clone was cultivated in Luria-Bertani (LB) medium containing ampicillin (100μg/μL) at 37 °C in a shaking incubator. When the culture reached an OD₆₀₀ of 0.6 - 0.8, addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma Aldrich, St. Louis, MO) to a final concentration of 1 mM, triggered the recombinant gene expression and a protein accumulation after 1 h. The cells were harvested by centrifugation at 2,000 g for 15 min. The cells pellet was resuspended in a 3:1 weight-to-volume ratio with lysis buffer (50 mM Tris – HCl, pH 8.0, 10 mM MgCl₂, 100 mM NaCl) and frozen at −80 °C until needed.

Protein Purification:

The cell suspension lysis protocol used here was described previously.^[27] The his-tagged Flag silk-like protein was recovered using Immobilized Metal Affinit Chromatography (IMAC) (HisTrap HP Ni+ 1mL, GE Healthcare). The cell suspension was thawed at room temperature, which allowed endogenus lysozyme from BL21(DE3) pLysS cells to act on unlysed cells. 2% deoxycholic acid was added while stirring and the suspension was placed at 37 °C for 30 min. 0.01% DNase was added to the suspension and incubation continued at room temperature for 30 min on a shaking platform. The soluble and insoluble fractions were separated by centrifugation (12000 rpm for 15 min), the supernatant was removed and diluted 1:1 with 1x binding buffer (20 mM imidazole, 50 mM NaCl, 20 mM Tris – HCl, pH 7.9). The solution was passed over the column of his-bind resin, followed by washes with 50 mM imidazole, 60 mM imidazole, 80 mM imidazole and then the protein was eluted striping the resin following manufacture procedures. The eluted fractions were dialyzed against Milli-Q (Millipore) water using a stirred cell with a MWCO 6-8kDa membrane and lyophilized.

SDS-PAGE analyses:

The heat-treated (100°C/5min) protein extracts and the purified protein fractions were quantified using a Bradford assay (Bio-Rad Protein Assay) and analyzed by SDS-PAGE. For all SDS-PAGE analyses, polyacrylamide gels (5% stacking gel and 12% resolving gel) containing 0.1% SDS were made in Tris–HCl buffers (Mini PROTEAN3 Cell protocol for SDS-PAGE buffer system, Bio-Rad, Hercules, CA). A dual color protein standard (Bio-Rad, Hercules, CA) was included on all gels. After SDS-PAGE analysis, the gels were stained with Coomassie Brilliant Blue (G-250) dye according to the method published.^[66]

Western blot analyses:

The proteins samples separated by SDS-PAGE were transferred to a PVDF/Immobilon™-P Membrane (Millipore) by electroblotting using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) and the Trans-Blot SD-eletrotransfer® (Bio-Rad, Hercules, CA). Blots were set up as specified by the manufacturer. All transfers were performed under constant 100 mA for 50 min. After fixing the proteins, the membranes were subjected to immunoblotting analyses using the Monoclonal Anti-polyHistidine–Alkaline Phosphatase antibody produced in mouse (Sigma, St. Louis, MO) directed against the (histidine)₁₀ tag ((His)₁₀) present in the amino terminus of the silk fusion proteins. All immunoblots and immunodetection analyses were performed according to the protocols described by the manufacturers. Immunodetections were performed using the CDP-Star® and CSPD® chemiluminescent substrates for alkaline phosphatase (Applied Biosystems, Foster City, CA) following the instructions specified by the manufacturer. The blots were exposed using Kodak Standard^® films (Kodak, Rochester, NY).

Circular dichroism (CD):

CD data were obtained using the Jasco J-810 spectropolarimeter (Jasco Inc., Tokyo, Japan) coupled with a Peltier-type temperature controller, a thermostated cell holder, and calibrated with ammonium d-10-camphorsulfonate. The protein stock was resolubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; TCI America, Portland OR) to make a 25–30% (w/v). For analysis, the samples were prepared by dissolving the silk-HFIP dope to a concentration of 0.05 mg/mL in 100% methanol, or 100% acetonitrile, or distilled water. The concentration of the protein was measured using the Qubit™ Protein Quantitation System (Invitrogen, Carlsbad, CA). The CD spectra were recorded using a quartz optical cell (path length of 0.1 cm) at 25 °C with a 0.5-nm bandwidth and a scan speed of 50 nm/min. For each solvent used, the final spectrum was the average of three scans recorded between 190 to 260 nm. The CD data were converted to molar ellipticity [θ] (degree.cm2/dmol) based on molecular mass of 115-Da per residue.^[67] The secondary structure of the enzymes were estimated using the CDNN deconvolution (CDNN) version 2.1 software.^[68]

Artificial fiber spinning:

The pure lyophilized protein was resolubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; TCI America, Portland OR) to prepare a 25–30% (w/v) spinning dope. The fibers were extruded using a 1 mL Hamilton Gastight® syringe (Hamilton Company, reno, NV) mounted with a 10 cm long “blue” PEEK tubing (Upchurch Scientific) with an internal diameter of 0,0635 mm. The silk dope was extruded at a 0.5 mm/min constant speed into a 100% isopropyl alcohol coagulation bath. The extruded fibers were collected, dried, cut into 3 cm pieces and stored for further analyses.

Light microscopy:

The synthetic fibers were placed over a microscopy slide and observed using a Nikon Eclipse E200 microscope using 40x objective lenses.

Atomic force microscopy (AFM):

AFM was performed on the dry silk protein fibers which were deposited onto a glass slide surface. AFM measurements were recorded using a SPM-9600 instrument (Shimadzu, Japan). The images were acquired in dynamic mode using 125 μm-length cantilever (spring constant of ~42 N/m, resonant frequency of ~330 kHz) with conical tips (curvature radius < 10 nm). The images were acquired as 512 x 512 pixels at scan rate of 0.5 Hz. The process consisted in an automatic global leveling and the images were displayed 2D and 3D solid perspective.

Scanning electron microscopy (SEM):

Synthetic Flag silk fiber was also examined using SEM. The sample was coated 1x with gold (Emitech K550) and viewed on a DSM 962 Zeiss. The fibers were imaged at magnifications of 1000x and 5000x.

Raman spectromicroscopy:

The synthetic fibers were cut into 3 cm long pieces, glued on quartz slides and analyzed with a New Dimension Raman microscope (Snowy Range Instruments) equipped with a 1064 nm laser. A polystyrene control was used to calibrate the instrument before each data collection and the peaks used in the calibration were: 620, 795.8, 1001.4, 1450.5 and 1602.3cm⁻¹. The Raman data were acquired 2 times for 10 s or 20 s using a long working distance (objective of 20x). The data were analyzed in GRAMS software (Thermo Scientific, Waltham, MA) and smoothed using the Savitzky-Golay method (polynomial 3 points =17), corrected by the baseline, and then compared.

X-ray Diffraction:

Wide angle X-ray scattering (WAXS) experiments were performed in sector 14 BM-C/BioCARS of the Advanced Photon Source (APS) at Argonne National Laboratory (Argonne, IL, USA). The WAXS data were recorded with a large area 9-chip CCD detector (ADSC Quatum-315) placed 33 mm behind the sample. The monochromatic X-ray beam was focused to 150 × 200 microns at incident energy of 12.67 keV and with an X-ray wavelength of 0.978 Å. The bundled fiber samples were placed vertically, perpendicular to the X-ray beam, in the same geometry as the beam stop. For each sample, 5 frames were collected with a 50 mm beamstop to detector distance and an exposure time of 60 s. The background (air scattering) was subtracted from all X-ray intensities shown.