perspective published online: 17 April 2015 | doi: 10.1038/nchembio.1789
Toward spinning artificial spider silk Anna Rising1,2* & Jan Johansson1,2*
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Spider silk is strong and extensible but still biodegradable and well tolerated when implanted, making it the ultimate biomaterial. Shortcomings that arise in replicating spider silk are due to the use of recombinant spider silk proteins (spidroins) that lack native domains, the use of denaturing conditions under purification and spinning and the fact that the understanding of how spiders control silk formation is incomplete. Recent progress has unraveled the molecular mechanisms of the spidroin N- and C-terminal nonrepetitive domains (NTs and CTs) and revealed the pH and ion gradients in spiders’ silk glands, clarifying how spidroin solubility is maintained and how silk is formed in a fraction of a second. Protons and CO2, generated by carbonic anhydrase, affect the stability and structures of the NT and CT in different ways. These insights should allow the design of conditions and devices for the spinning of recombinant spidroins into native-like silk.
T
he ability to make silk has made spiders successful; over 45,000 species have been characterized (http://www.wsc. nmbe.ch/), and they can be found in most habitats on Earth1. Spider silk fibers are formed in a fraction of a second from highly concentrated protein solutions (known as ‘dope’) in sophisticated spinning apparatuses. A female spider can spin up to seven types of silk that are used for a variety of purposes, ranging from prey capture to reproduction (Fig. 1a). All silks are composed of proteins that typically consist of an extensive repetitive central part flanked by smaller nonrepetitive domains (Fig. 1b). Spider silk is an astonishing material that shows a combination of high tensile strength and extensibility, which allows some of the fibers to absorb more energy per weight than the strongest of man-made materials, for example, Kevlar (Table 1)2. More surprising, considering the biological functions of spider silks (Fig. 1a), is the fact that the fibers are well tolerated when implanted; for example, they have successfully been used for regeneration of peripheral nerves in vivo3,4. These features make spider silk one of the most fascinating materials known, and if we can learn how to produce it on a large scale there will be numerous possibilities for its application, from the construction industry to medicine. Making artificial spider silk fibers with mechanical properties similar to the natural material, however, has been a major unmet goal in material science for decades. What are the reasons for this shortcoming? Large partial spidroins can be produced in heterologous hosts5, but they, like many short recombinant spidroins6–8, require the use of denaturing conditions during purification and/or fiber formation, which probably explains why most fibers show disappointing mechanical properties (Supplementary Table 1). How to keep the spidroins correctly folded during production and soluble at high concentrations without the use of organic solvents or other chaotropic agents and how to make them form silk fibers without the use of nonphysiological coagulation baths are major issues that remain to be solved. Moreover, problems in making fibers with mechanical properties similar to those of the natural material may also arise due to the fact that the exact chemicophysical conditions of the glands are unknown and that the rheological properties of the dope and physical stresses are difficult to reproduce in vitro. Recent advances in the understanding of the natural silk spinning process9,10, in combination with detailed molecular studies of
partial spider silk proteins9,11–15, have revealed that spider silk formation is governed by mechanisms that have not yet been observed in any other physiological system. Scientific evidence points to the importance of the terminal domains in the regulation of spidroin assembly into fibers9,11–13,15,16 and suggests that current spinning procedures are inadequate because they rely on nonphysiological conditions and/or recombinant spidroins that lack one or both of the terminal domains. Herein, we review recent advances about the chemical biology of the natural spinning process, from which we outline new ways to design optimal protein constructs and biomimetic spinning devices.
Spidroins are conserved but also highly diverse
All spiders spin silk, and some can spin up to seven different types of silks or glues (for example, female orb-weaving spiders)17 (Fig. 1a and Table 1). The dragline silk is most studied and is well known for its high tensile strength, extreme toughness, light weight and favorable properties when implanted in living tissue3,4. This fiber is used to make the framework of the web and is used as a safety line during falls. The flagelliform silk fiber is coated with glue (aggregate silk) and forms the extremely extensible capture spiral of the orb web. The minor ampullate silk is used to reinforce the web. For making the egg sac, female spiders use two types of silks: cylindriform (also referred to as tubuliform) silk forms the durable outer shell of the sack, and aciniform silk is used as a soft inner lining. Aciniform silk is also used for wrapping prey. To attach silk fibers to a surface, the spider uses pyriform silk17. The different mechanical properties of the silks make them attractive for a variety of applications, from superglues to extremely strong and extendible ropes and fibers. The inherent differences of the silks also open up the possibility of designing composites with combined features for advanced technical and medical applications, for example, exact matching of the mechanical properties of a specific tissue for tissue engineering applications. Each silk type is made in a specific gland (Fig. 1a) and mainly consists of spidroins whose repetitive motif has a characteristic primary structure. However, this general view is complicated by the following factors: the presence of variable repetitive motifs within the same spidroin from different species18, non-gland-specific expression of some spidroins19,20 and the facts that spidroins can even be expressed in venom glands21 and that some spider silks contain
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. 2Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Division of Neurogeriatrics, Karolinska Institutet, Huddinge, Sweden. *e-mail: [email protected] or [email protected]
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Nature chemical biology doi: 10.1038/nchembio.1789 Though the repetitive parts and mechanical properties vary a lot between silk types2, the regulatory domains (NT and CT)11,12 and the conditions of the silk glands9 are highly conserved (details below), which means that they have important roles in the regulation of fiber formation. Conditions that allow artificial spinning procedures are therefore likely to be found in the natural counterpart and should be independent of what specific type of silk is to be made.
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How do the silk glands work?
Because most data available are from experiments on major ampullate glands and their product, the dragline silk, we will focus on this particular silk. The major ampullate gland has a long, winding and narrow tail and a wider and shorter ampulla or sac (Fig. 2a). The tail and sac contain three different single layered epithelial cell types that all contain numerous granules. The epithelial cell types have distinct distributions and thereby divide the gland into three zones, A–C10 (Fig. 2a). The cells in the A and B zones produce spidroins that form two separate layers in the dope10. Surprisingly, Cylindrical glands; outer silk of egg sac the C-zone epithelium does not produce spiAggregate gland; aqueous coating of capture spiral droins but was recently found to be important Flagelliform gland; core fibers of capture spiral for the physiological regulation of silk producMajor ampullate gland; dragline and structural silk tion (described below)9. The S-shaped narMinor ampullate gland; auxiliary spiral rowing duct is lined by a cuticular intima layer Aciniform gland; soft inner silk of egg sac and silk for swathing prey that probably contributes structural support to the duct and protects the underlying epithelial Pyriform gland; cement for joints and attachments cells from the fiber, but it has also been sugNT Repetitive region CT b gested to act similarly to a hollow fiber dialysis membrane, thereby allowing dehydration of the dope25. The sac is connected to the duct via the funnel, which has an unknown function Figure 1 | Spider silks and spidroins. (a) Illustration of a spider’s spinning glands, different silks and but appears to be associated to the cuticular intima of the duct10. their applications. Detailed descriptions of the different silks are in the main text. (b) A canonical The making of spider silk poses chalspider silk protein is composed of a nonrepetitive NT, a repetitive region and a nonrepetitive CT. lenges at molecular biological, biochemical and physiological levels. The spidroin genes nonspidroin proteins22. Spidroin genes and proteins are most are large, repetitive and very GC-rich, as glycine and alanine can commonly abbreviated by two letters indicating the gland where constitute more than 60% of spidroins26 and are coded for by the they are mainly expressed, followed by Sp for spidroin and a number referring to the different paralogs (for example, MaSp1 for Major ampullate spidroin 1). Table 1 | Mechanical properties of five types of spider silk, Spidroins vary in size21 but generally share a tripartite composiKevlar and high-tensile steel. tion of a nonrepetitive globular NT (~130 residues23), an extensive Strength Extensibility Toughness region made up of silk-specific alanine- and/or glycine-rich repeat Material (GPa) (%) (MJ m−3) 12 units and a nonrepetitive globular CT of ~110 residues (Fig. 1b) . 21–27 136–194 Dragline silk (Araneus diadematus, 0.88–1.5 The NT and CT are evolutionarily conserved and have important Araneus sericatus, Araneus roles in the regulation of spider silk formation (described in detail gemmoides, Argiope trifasciata, below). The spidroin repetitive parts, in contrast, show great variArgiope argentata)2,66–68 ability between silk types. The repetitive part is usually large and Flagelliform silk (A. diadematus, 0.50–1.3 119–270 75–283 can encompass up to hundreds of repeat units. The nature of the A. sericatus, A, argentata)2,66–68 repeat units correlates with the mechanical properties of the fiber; Cylindriform silk (A. argentata, 0.48–2.3 19–29 95 for example, long polyalanine segments give strong fibers, and long A. gemmoides)66,69 glycine-rich segments give more extendible fibers24. However, there is no linear correlation between protein length and mechanical Minor ampullate silk 0.92–1.4 22–33 137 properties5. In fact, the extensive arrays of almost-identical repeats (A. argentata, A. gemmoides)66,69 may be inadvertent consequences of unequal crossing over and Aciniform silk (A. argentata)66 1.1 40 230 homologous recombination events during replication18, which Kevlar 49 (ref. 2) 3.6 2.7 50 would implicate that shorter and less repetitive spidroins may be 2 High-tensile steel 1.5 0.8 6 useful for artificial production of spider silk. 310
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d
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Figure 2 | Overview of the natural spinning process and role of the terminal domains. (a) Silk gland with its winding tail, central sac and threelimbed duct. The three histologically different regions are depicted; zones A and B contain epithelium that produces and secretes spidroins into the glandular lumen, and zone C contains active carbonic anhydrase. (b) Blue represents the tail and the part of the sac where spidroin synthesis takes place10, and green represents the sac and duct regions where carbonic anhydrase is found9. (c) The continuous lowering of pH from 7.5–8 in the proximal tail to presumably close to 5.0 at the end of the duct9,13. (d) Thermodynamic stabilities of NT and CT as a function of pH, measured as urea concentrations for half-denaturation. The gray area represents the pH region in the gland where carbonic anhydrase is found. (e) Summary of the main structural features of NT and CT in three different pH intervals, according to the pH scale in c. (f) Schematic representation of the lock-and-trigger mechanism of spider silk formation, where NT (red) acts as a lock and forms dimers that initially are dynamic but become increasingly stable as pH continues to drop, and CT (blue) gets destabilized, unfolds and forms amyloid-like fibrils that may trigger fiber formation9,13. The black lines represent the repetitive regions of spidroins.
codons GGX and GCX. This puts high demands on spidroin gene replication and transcription26–28. To meet the high need of glycine and alanine during translation, the epithelial cells of the glands have unusually large alanyl- and glycyl-tRNA pools29. Despite the spidroins being able to assemble into a solid fiber within a fraction of a second, the spider manages to keep them soluble at very high concentrations (30–50%, w/v) for long-term storage30,31. This implies that the tertiary and quaternary structures of the soluble spidroins are optimized to prevent aggregation. There are currently two different models for how spidroins are organized when stored in the gland: in micelles, where the terminal domains form a hydrophilic outer shell and the repetitive region is shielded in the center32, or as a liquid crystalline feedstock33. These two models are not mutually exclusive and may both occur. The structures of soluble native spidroins are difficult to determine as the proteins are prone to change their conformation upon being manipulated as required to experimentally study the gland content. In spite of this, by using 13 C NMR on whole major ampullate glands, the repetitive part of the proteins was found to adopt a random34 and/or helical conformation31. Polyproline type II segments have also been observed by vibrational CD spectroscopy35. In the final silk fiber, the repetitive part has converted to polyalanine β-sheet crystals flanked by more amorphous glycine-rich repeats36. Although often referred to as amorphous, the glycine-rich regions in the fiber contain 31-helical Gly-Gly-X motifs37–40 and Gly-Pro-Gly-X-X motifs that form type II β-turns41. Finally, the fiber-forming process enables fiber formation in a defined segment of the duct, thus avoiding the fatal spread of the assembly process to the dope in the gland. Understanding how silk formation is regulated requires determination of how the composition of silk dope varies throughout the gland, but the small size of the gland makes this technically challenging. The duct is particularly difficult to work with experimentally owing to the narrow lumen and tough cuticular intima, which is hard to penetrate. Despite these difficulties, the pH from the proximal part of the tail to halfway through the spinning duct was recently determined to span from 7.6 to at least 5.7 (ref. 9). To uphold this broad and steep pH gradient, the spiders must have developed efficient methods to generate protons, and the almostnonexistent variation in pH between individual silk glands9 suggests
that the underlying mechanisms are tightly controlled. By using enzyme activity staining of histological sections, we recently discovered that carbonic anhydrase is responsible for generating and maintaining the pH gradient9. Carbonic anhydrases are ubiquitous enzymes found in all animals and photosynthesizing organisms42 and catalyze the following chemical reaction with an exceptionally high turnover rate of up to 106 s−1 (ref. 42): CO2 + H2O ↔ H + + HCO3−
In the silk gland, active carbonic anhydrase is present from zone C to the end of the duct (Fig. 2a,b). The pH decreases along the gland (Fig. 2c), and, surprisingly, the concentrations of both HCO3− and CO2 rise9. The hydrophobic nature of CO2 implies it should diffuse –
–
+ +
– –
–
2H+
– + +
– + + H+
Figure 3 | Schematic representation of charge interactions, protonation events and structural rearrangements that accompany NT monomer-todimer conversion and stabilization. In each subunit, helix 1 (H1) is brown, H2 is yellow, H3 is green, H4 is light blue and H5 is dark blue. The left side depicts two antiparallel NT subunits held together by charge attractions between the positively charged N-terminal end and the negatively charged C-terminal end. A tryptophan residue (red) is wedged in between H1 and H3. The orientations of H3 and H5, together with charge repulsions between acidic residues in the H3 and H5 of different subunits prevent close interactions. As pH is lowered, the two carboxylates of H3 and H5 become protonated, and the tryptophan side chain is relocated to a superficial position whereupon H3 and H5 change orientation, allowing close interactions between the subunits, but the subunits are mobile with respect to each other. Finally, a third carboxylate residue is protonated, which is linked to full stabilization of the dimer. Only the charged residues at one pole of the antiparallel dimer are shown.
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Figure 4 | A photograph of an artificial spider silk fiber. The fiber was formed in tilting tubes by self-assembly. Scale bar, 1 cm.
freely across cell membranes, but the spider glands have apparently developed a mechanism to uphold a high intraluminal pCO2 (ref. 9). A similar phenomenon has been described for parietal cells in the ventricle43, but the underlying mechanisms are poorly investigated. The high pCO2 in the silk gland is probably of physiological importance, as in vitro studies show that acidic pH in combination with a high CO2 concentration leads to marked structural changes of the terminal domains that can trigger fiber formation (described further below). In the sac, the concentrations of Na+, K+ and Cl− are 199 mM, 6 mM and 164 mM, respectively9. The intraluminal concentrations of Na+ and Cl− are slightly higher than they are in the hemolymph (about 140 mM and 125 mM, respectively, though these values were measured in two different orb-weaving spider species44), whereas the silk gland K+ concentration is lower (about 20 mM in hemolymph44). The concentrations of these ions have been suggested to change along the silk production pathway45, but the concentrations in the duct and hence at the site of fiber formation remain unknown.
Coordinated molecular events govern silk formation
by the addition of preformed fibrils, i.e., seeds (nuclei)51. The spiders apparently have adopted the seeding phenomenon to trigger ultrafast fiber formation. The structural transition of CT into β-sheet fibrils in the duct of the silk gland results in nuclei that may trigger the conversion of the repetitive region into β-sheet polymers, a new and fascinating functional use of amyloid fibrils9 (Fig. 2e,f). The NT is the most conserved part of spidroins and confers solubility to recombinant spidroins at pH 7 and rapid fiber formation when the pH is lowered to 6 (refs. 11,23). It is mainly monomeric at pH levels greater than 7 (refs. 16,52–54) but dimerizes when the pH is lowered below ~6.4 (the exact pKa of dimerization depends on the salt concentration)13,16,52. A relocation of helix 3 closer to helix 1 is a prerequisite for the formation of the NT dimer interface53. The residues responsible for sensing pH were recently identified for MaSp1 NT from the Euprosthenops australis spider13. During storage in the proximal sac (pH ~7.5; Fig. 2), the NT is mainly monomeric, but the dipolar subunits can interact by long-range electrostatic interactions (Fig. 3). Dimerization is not possible at this stage because the tilting of helices 3 and 5 sterically hinders close subunit interactions. When the pH is lowered to around 6.5, as seen in the most distal part of the sac near the funnel (Fig. 2), two conserved glutamic acid residues will each pick up one proton. The loss of these two negative charges allows the rearrangement of mainly helices 3 and 5 and a transition into a subunit conformation that is compatible with dimerization53. To accomplish the structural transition, a tryp tophan residue (or phenylalanine in some NTs) will leave its wedged interhelical position in the monomer and swing out to a position where its side chain is more solvent exposed. This allows helices 1 and 3 to be more tightly packed and leads to the formation of a rather flat dimer interface (Fig. 3). Although NT is dimeric at this stage, it is not until the pH reaches 5.7 and below, corresponding to the pH halfway through the duct and beyond, that a structurally defined and fully stable NT dimer is formed by protonation of a third glutamic acid residue (Figs. 2d,e and 3)13. Some of the residues that control NT dimerization and stabilization in E. australis MaSp1 are replaced by nontitratable residues in other spidroins, but all NTs appear to harbor acidic residues at the dimer interface11,46,55. It seems that the NT has conserved the ability to control dimerization and stabilization in response to a pH gradient, but that the exact residues that titrate during this process are variable is a supposition that must be verified by analyses of wild-type and site-directed mutants of NTs from additional spidroins. The dimerization and stabilization process of NT may seem overly complicated, but the multistep mechanism is probably vital for the control of silk polymerization: the silk fiber is pulled from the spider, and pulling forces can be propagated via the protein chains when they are firmly interconnected via the stable NT dimers and constitutive CT dimers (Fig. 2f). The pulling, as such, may promote refolding of helical or random repetitive segments into extended β-sheet conformations. As the spidroins flow through the duct, the pulling also causes shearing, which has been shown to contribute to the structural transition of the CT into β-sheet nuclei9,12, and this transition can be further accelerated by lowered pH and elevated
The terminal domains of spider silk proteins act as regulatory elements that control spidroin solubility and assembly9,11,12. They are structurally conserved, unique to spiders and present in almost all spidroins, even though spidroins diverged in evolution several hundred million years ago23,46. Both domains are bundles of five α-helices, but they do not share any primary or tertiary structure similarities, which indicates that they fulfill different functions. CT is a constitutive, often disulfide-linked homodimer9,12,47 and thereby probably links the spidroins in pairs already in the oxidizing environment of the endoplasmic reticulum of the glands’ epithelial cells. At pH levels greater than 6.5, as in the lumen of the synthesis and storage parts of the gland (zones A and B; Fig. 2a–c), the CT is stable and highly soluble9,47. When the dope travels down the duct, the decreased pH and simultaneously increased pCO2 markedly affect the CT’s structure and stability9. The low pH destabilizes the CT (Fig. 2d), owing at least in part to the loss of a conserved salt bridge9. Simultaneously, CO2 interacts with partly buried residues in CT, which can facilitate unfolding9. The physiological use of CO2 to destabilize a protein has not been described before and may even be a unique feature of the spiders. At pH levels less than 5.5, the CT loses its native helical structure and forms β-sheet fibrils. This behavior resembles that seen in the formation Figure 5 | A schematic biomimetic spinning device. A highly concentrated aqueous spidroin of amyloid fibrils, where proteins that have lost solution (blue) is pumped into a series of pulled glass capillaries. Aqueous buffers (green and (or never gained) their native conformation yellow) are serially introduced into the device and form laminar flows that cause increased flow instead adopt a very regular structure of cross- rate and shearing of the spidroin solution (i.e., the jet gets thinner). The laminar flow will allow for β-sheets (amyloid fibrils)48–50. The kinetics of diffusion across the solution interfaces, which will lead to a gradual lowering of pH and elevation amyloid fibril formation are greatly accelerated of pCO2 in the spidroin solution when it travels through the device. 312
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Nature chemical biology doi: 10.1038/nchembio.1789 pCO2 (Fig. 2e,f), leading to rapid polymerization of the spidroins. Fast polymerization kinetics are a prerequisite for the spiders’ ability to spin silk at speeds >1 m s−1, but for the spider it is also vital to ensure that the polymerization process is confined to the duct and prevented from spreading up to the sac, as this would prematurely coagulate the contents of the gland. The dynamically associated NT dimers present in the beginning of the duct apparently provide the solution to both these problems: they ensure prealignment of the NTs so that the interlocking of the silk proteins in the distal parts of the duct is independent of diffusion (i.e., their association is ultrafast)15, and, at the same time, they act as a safety mechanism that keeps the pulling forces from propagating up to the gland (i.e., the loosely associated dimers dissociate; Fig. 2f).
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Shortcomings of current methods to spin artificial silk
The insights into the ion and pH gradients of the silk gland and importance of the terminal domains as regulators of spider silk assembly have not yet been translated into a biomimetic spinning process. As outlined in Supplementary Table 1, several recombinant spidroin variants have been produced and spun or self-assembled into fibers. The nature of the produced proteins varies greatly, and none correspond to a full-length natural spidroin. Typically they are much smaller than their natural counterparts and are composed of a repetitive part with or without a CT. There is only one reported case of fibers made from a recombinant spider silk protein that includes all three parts of a canonical spidroin—the NT, a repetitive part and the CT—but their mechanical properties were not analyzed11. Most techniques used for fiber formation require a highly concentrated spinning dope, typically 10–20% (w/v), and to achieve this, the proteins are commonly dissolved in organic solvents such as hexafluoroisopropanol. Notably, even when harsh solvents are used, the solubility of the recombinant spidroins does not match that seen in the native dope56. The techniques used for fiber spinning include wet spinning (ejection of the dope into a coagulation bath, most often containing methanol or isopropanol)5, electrospinning57,58, self-assembly at airwater interfaces59,60 and spinning in microfluidic devices8. Organic solvents or coagulation baths are used to obtain both electrospun (nonwoven) silk fibers and wet spun fibers. Fiber formation through denaturation and aggregation will probably not lead to truly silk-like replicas as the spidroins’ functions are lost. The natural spinning process involves intricate molecular mechanisms and results in fibers with specific secondary structures, i.e., the spidroins are assembled into fibers, not aggregated. Self-assembly of spider silk proteins into meter-long fibers under nondenaturing conditions can be achieved in tubes that are tilted from side to side, which induces shear forces in the dope59 (Fig. 4). However, the method is difficult to scale up, ion and pH gradients are not easily achieved, and the fibers are variable in structure and mechanical properties. Interestingly, though, by including the NT in the recombinant spidroins, the rate of the fiber-forming process can be controlled by pH11. Microfluidic spinning is perhaps the most attractive fiber spinning method as it has successfully been used to obtain fibers without the use of denaturing steps and can be used to create ion and pH gradients as well as shear forces. Fiber formation of a MaSp2 analog has been achieved by applying elongational flow, dropping the pH to 6 and increasing the K+ concentration to 500 mM8. Apart from the high K+ concentration, this approach represents the most biomimetic spinning procedure presented so far. The resulting fibers were not tested for mechanical properties, and their structural properties were not described, making it difficult to evaluate whether this particular biomimetic spinning technique gives rise to functional fibers. Observed effects of K+ on spidroin aggregation are not unequivocal; native spidroin dope has been shown to aggregate into nanofibrils in response to increased potassium concentration30, whereas a recombinant
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spidroin composed of repetitive segments and CT did not respond to potassium concentrations up to 300 mM (ref. 61). Taken together, much effort has been put into spinning artificial spider silk fibers, but few or none of the produced fibers are usable for practical purposes owing to poor mechanical performance, low reproducibility or both. We believe that these shortcomings are most likely due to insufficient mimicking of the natural spinning processes.
How to spin biomimetic spider silk?
To establish a bimimetic spinning technique, the first problem to solve is how to obtain a stable aqueous solution of recombinant spider silk proteins that matches the native solution (30–50% w/w), and the second is how to mimic the conditions of the spider’s spinning apparatus. Microfluidics or similar methods appear well suited for mimicking the gradients of the silk gland and are therefore highly interesting for future development of the spinning technique (Fig. 5). In such devices, laminar flow may be created, allowing for diffusion across the liquid interfaces and thus a gradual change of the conditions in the spinning dope as it travels through the spinning apparatus. Furthermore, increased flow rates generate shear forces, another important factor needed for biomimetic spinning. A lowering of pH from around 7.5 to 5.0–5.5 will be required, and increasing pCO2 will most likely facilitate the nucleation of the polymerization process. An increasing concentration of K+ could lead to faster polymerization of the spidroins. For this biomimetic spinning device to have the intended impact on the spidroins to be spun, the spidroins must encompass both terminal domains. Including the terminal domains in the recombinant spidroins could actually also solve the first problem, as both the NT and the CT, at least in certain species, are very stable and can be concentrated to around 300 mg ml−1 in aqueous buffers11,47. However, when these domains are coupled to repeats from the minor ampullate spidroin, the solubility markedly decreases47. This is surprising as the repetitive segments of spidroins are generally not hydrophobic or very aggregation prone as such. For example, alanine has the highest α-helix propensity of all of the residues62, and this in combination with its biological hydrophilicity63 suggest that polyalanine segments are unsuited to spontaneously aggregate into crystalline β-sheets. Along the same line, the repetitive segments of the aciniform spidroins are α-helical and highly soluble and form globular, folded domains that in the fiber are at least partially transformed into β-sheet conformations64,65. We propose that a main feature of the repetitive segments is to be soluble to avoid premature aggregation of the spidroins and that the unique NT and CT have evolved to allow rapid conversion of the repetitive segments into β-sheet aggregates at a confined place in the spinning duct. If this hypothesis is correct, it implies that the exact sequence of the repetitive segments may be less important than their solubility, suggesting that recombinant spidroin analogs designed from simple repeat units capped by NT and CT could be used for production of silk-based biomaterials.
Future perspectives
During the last few years, important progress has been made toward the accomplishment of generating artificial spider silk mimics: fulllength spidroins have been characterized, two spider genomes have been sequenced, and the milieu in different parts of silk glands and how it affects spidroin domains at a molecular level have been revealed. Hopefully, these advances in basic knowledge will allow the design of proteins, methods and devices that can be used to produce biomimetic spider silk for various purposes in the near future. A challenge that remains is how to keep recombinant spidroins soluble at high concentrations without the use of harsh solvents. We also need to understand, in detail, the unprecedented effects of CO2 on spidroins to be able to harness them for biomimetic spinning. Finally, we envision customized microfluidic spinning devices; for
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example, carbonic anhydrase, an efficient and robust enzyme, could be introduced in a spinning apparatus to achieve a mimic of the glands’ physiology. Received 16 January 2015; accepted 2 March 2015; published online 17 April 2015
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References 1. Kovoor, J. in Ecophysiology of Spiders (ed. Nentwig, W.) 160–186 (SpringerVerlag, Berlin, 1987). 2. Gosline, J.M., Guerette, P.A., Ortlepp, C.S. & Savage, K.N. The mechanical design of spider silks: from fibroin sequence to mechanical function. J. Exp. Biol. 202, 3295–3303 (1999). 3. Allmeling, C. et al. Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration. Cell Prolif. 41, 408–420 (2008). 4. Radtke, C. et al. Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep. PLoS ONE 6, e16990 (2011). 5. Xia, X.X. et al. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Natl. Acad. Sci. USA 107, 14059–14063 (2010). This is the first report on recombinant production of native-sized spidroin. 6. Wong Po Foo, C. et al. Novel nanocomposites from spider silk-silica fusion (chimeric) proteins. Proc. Natl. Acad. Sci. USA 103, 9428–9433 (2006). 7. Teulé, F. et al. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat. Protoc. 4, 341–355 (2009). 8. Rammensee, S., Slotta, U., Scheibel, T. & Bausch, A.R. Assembly mechanism of recombinant spider silk proteins. Proc. Natl. Acad. Sci. USA 105, 6590–6595 (2008). 9. Andersson, M. et al. Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains. PLoS Biol. 12, e1001921 (2014). This paper characterizes pH regulation of spider silk formation at the molecular level. 10. Andersson, M., Holm, L., Ridderstrale, Y., Johansson, J. & Rising, A. Morphology and composition of the spider major ampullate gland and dragline silk. Biomacromolecules 14, 2945–2952 (2013). 11. Askarieh, G. et al. Self-assembly of spider silk proteins is controlled by a pH-sensitive relay. Nature 465, 236–238 (2010). This paper presents the first high-resolution structure of the NT. 12. Hagn, F. et al. A conserved spider silk domain acts as a molecular switch that controls fibre assembly. Nature 465, 239–242 (2010). This paper present the first high-resolution structure of the CT. 13. Kronqvist, N. et al. Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation. Nat. Commun. 5, 3254 (2014). This paper presents the molecular mechanism of NT pH-sensitive dimerization. 14. Ries, J., Schwarze, S., Johnson, C.M. & Neuweiler, H. Microsecond folding and domain motions of a spider silk protein structural switch. J. Am. Chem. Soc. 136, 17136–17144 (2014). 15. Schwarze, S., Zwettler, F.U., Johnson, C.M. & Neuweiler, H. The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening. Nat. Commun. 4, 2815 (2013). This paper presents the molecular mechanism of ultra-fast NT association. 16. Gaines, W.A., Sehorn, M.G. & Marcotte, W.R. Jr. Spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly. J. Biol. Chem. 285, 40745–40753 (2010). 17. Candelas, G.C. & Cintron, J. A spider fibron and its synthesis. J. Exp. Zool. 216, 1–6 (1981). 18. Rising, A. et al. Major ampullate spidroins from Euprosthenops australis: multiplicity at protein, mRNA and gene levels. Insect Mol. Biol. 16, 551–561 (2007). 19. Garb, J.E. & Hayashi, C.Y. Modular evolution of egg case silk genes across orb-weaving spider superfamilies. Proc. Natl. Acad. Sci. USA 102, 11379–11384 (2005). 20. Lane, A.K., Hayashi, C.Y., Whitworth, G.B. & Ayoub, N.A. Complex gene expression in the dragline silk producing glands of the Western black widow (Latrodectus hesperus). BMC Genomics 14, 846 (2013). 21. Sanggaard, K.W. et al. Spider genomes provide insight into composition and evolution of venom and silk. Nat. Commun. 5, 3765 (2014). This paper presents the first characterized spider genome. 22. Clarke, T.H. et al. Multi-tissue transcriptomics of the black widow spider reveals expansions, co-options, and functional processes of the silk gland gene toolkit. BMC Genomics 15, 365 (2014). 23. Rising, A., Hjalm, G., Engstrom, W. & Johansson, J. N-terminal nonrepetitive domain common to dragline, flagelliform, and cylindriform spider silk proteins. Biomacromolecules 7, 3120–3124 (2006).
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24. Hayashi, C.Y., Shipley, N.H. & Lewis, R.V. Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins. Int. J. Biol. Macromol. 24, 271–275 (1999). 25. Vollrath, F. & Knight, D.P. Structure and function of the silk production pathway in the spider Nephila edulis. Int. J. Biol. Macromol. 24, 243–249 (1999). 26. Ayoub, N.A., Garb, J.E., Tinghitella, R.M., Collin, M.A. & Hayashi, C.Y. Blueprint for a high-performance biomaterial: full-length spider dragline silk genes. PLoS ONE 2, e514 (2007). This paper presents the first sequence of full-length spidroin. 27. Hinman, M.B. & Lewis, R.V. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. J. Biol. Chem. 267, 19320–19324 (1992). 28. Mita, K., Ichimura, S., Zama, M. & James, T.C. Specific codon usage pattern and its implications on the secondary structure of silk fibroin mRNA. J. Mol. Biol. 203, 917–925 (1988). 29. Candelas, G.C., Arroyo, G., Carrasco, C. & Dompenciel, R. Spider silkglands contain a tissue-specific alanine tRNA that accumulates in vitro in response to the stimulus for silk protein synthesis. Dev. Biol. 140, 215–220 (1990). 30. Chen, X., Knight, D.P. & Vollrath, F. Rheological characterization of Nephila spidroin solution. Biomacromolecules 3, 644–648 (2002). 31. Hijirida, D.H. et al. 13C NMR of Nephila clavipes major ampullate silk gland. Biophys. J. 71, 3442–3447 (1996). 32. Jin, H.J. & Kaplan, D.L. Mechanism of silk processing in insects and spiders. Nature 424, 1057–1061 (2003). 33. Vollrath, F. & Knight, D.P. Liquid crystalline spinning of spider silk. Nature 410, 541–548 (2001). 34. Hronska, M., van Beek, J.D., Williamson, P.T., Vollrath, F. & Meier, B.H. NMR characterization of native liquid spider dragline silk from Nephila edulis. Biomacromolecules 5, 834–839 (2004). 35. Lefèvre, T. et al. In situ conformation of spider silk proteins in the intact major ampullate gland and in solution. Biomacromolecules 8, 2342–2344 (2007). 36. Simmons, A.H., Michal, C.A. & Jelinski, L.W. Molecular orientation and two-component nature of the crystalline fraction of spider dragline silk. Science 271, 84–87 (1996). 37. van Beek, J.D., Hess, S., Vollrath, F. & Meier, B.H. The molecular structure of spider dragline silk: folding and orientation of the protein backbone. Proc. Natl. Acad. Sci. USA 99, 10266–10271 (2002). 38. Holland, G.P., Creager, M.S., Jenkins, J.E., Lewis, R.V. & Yarger, J.L. Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy. J. Am. Chem. Soc. 130, 9871–9877 (2008). 39. Kümmerlen, J., vanBeek, J.D., Vollrath, F. & Meier, B.H. Local structure in spider dragline silk investigated by two-dimensional spin-diffusion nuclear magnetic resonance. Macromolecules 29, 2920–2928 (1996). 40. Holland, G.P., Jenkins, J.E., Creager, M.S., Lewis, R.V. & Yarger, J.L. Quantifying the fraction of glycine and alanine in β-sheet and helical conformations in spider dragline silk using solid-state NMR. Chem. Commun. (Camb.) 5568–5570 (2008). 41. Jenkins, J.E. et al. Solid-state NMR evidence for elastin-like β-turn structure in spider dragline silk. Chem. Commun. (Camb.) 46, 6714–6716 (2010). 42. Lindskog, S. Structure and mechanism of carbonic anhydrase. Pharmacol. Ther. 74, 1–20 (1997). 43. Waisbren, S.J., Geibel, J.P., Modlin, I.M. & Boron, W.F. Unusual permeability properties of gastric gland cells. Nature 368, 332–335 (1994). 44. Cohen, A.C. Hemolymph chemistry of two species of araneid spiders. Comp. Biochem. Physiol. 66A, 715–717 (1980). 45. Knight, D.P. & Vollrath, F. Changes in element composition along the spinning duct in a Nephila spider. Naturwissenschaften 88, 179–182 (2001). 46. Garb, J.E., Ayoub, N.A. & Hayashi, C.Y. Untangling spider silk evolution with spidroin terminal domains. BMC Evol. Biol. 10, 243 (2010). 47. Gao, Z. et al. Structural characterization of minor ampullate spidroin domains and their distinct roles in fibroin solubility and fiber formation. PLoS ONE 8, e56142 (2013). 48. Makin, O.S., Atkins, E., Sikorski, P., Johansson, J. & Serpell, L.C. Molecular basis for amyloid fibril formation and stability. Proc. Natl. Acad. Sci. USA 102, 315–320 (2005). 49. Nelson, R. et al. Structure of the cross-β spine of amyloid-like fibrils. Nature 435, 773–778 (2005). 50. Westermark, P. Aspects on human amyloid forms and their fibril polypeptides. FEBS J. 272, 5942–5949 (2005). 51. Jarrett, J.T. & Lansbury, P.T. Jr. Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB. Biochemistry 31, 12345–12352 (1992). 52. Landreh, M. et al. A pH-dependent dimer lock in spider silk protein. J. Mol. Biol. 404, 328–336 (2010). 53. Jaudzems, K. et al. pH-Dependent dimerization of spider silk N-terminal domain requires relocation of a wedged tryptophan side chain. J. Mol. Biol. 422, 477–487 (2012).
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54. Hagn, F., Thamm, C., Scheibel, T. & Kessler, H. pH-dependent dimerization and salt-dependent stabilization of the N-terminal domain of spider dragline silk—implications for fiber formation. Angew. Chem. Int. Edn Engl. 50, 310–313 (2011). 55. Chen, G. et al. Full-length minor ampullate spidroin gene sequence. PLoS ONE 7, e52293 (2012). 56. Lin, Z., Deng, Q., Liu, X.Y. & Yang, D. Engineered large spider eggcase silk protein for strong artificial fibers. Adv. Mater. 25, 1216–1220 (2013). 57. Stephens, J.S. et al. Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via Fourier transform Raman spectroscopy. Biomacromolecules 6, 1405–1413 (2005). 58. Lang, G., Jokisch, S. & Scheibel, T. Air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins. J. Vis. Exp. doi:10.3791/50492 (8 May 2013). 59. Stark, M. et al. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 8, 1695–1701 (2007). 60. Xu, L., Rainey, J.K., Meng, Q. & Liu, X.Q. Recombinant minimalist spider wrapping silk proteins capable of native-like fiber formation. PLoS ONE 7, e50227 (2012). 61. Huemmerich, D. et al. Primary structure elements of spider dragline silks and their contribution to protein solubility. Biochemistry 43, 13604–13612 (2004). 62. Kallberg, Y., Gustafsson, M., Persson, B., Thyberg, J. & Johansson, J. Prediction of amyloid fibril-forming proteins. J. Biol. Chem. 276, 12945–12950 (2001). 63. Hessa, T. et al. Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377–381 (2005).
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64. Lefèvre, T., Paquet-Mercier, F., Rioux-Dube, J.F. & Pezolet, M. Review structure of silk by Raman spectromicroscopy: from the spinning glands to the fibers. Biopolymers 97, 322–336 (2012). 65. Wang, S., Huang, W. & Yang, D. NMR structure note: repetitive domain of aciniform spidroin 1 from Nephila antipodiana. J. Biomol. NMR 54, 415–420 (2012). 66. Blackledge, T.A. & Hayashi, C.Y. Silken toolkits: biomechanics of silk fibers spun by the orb web spider Argiope argentata (Fabricius 1775). J. Exp. Biol. 209, 2452–2461 (2006). 67. Denny, M. The physical properties of spider’s silk and their role in the design of orb-webs. J. Exp. Biol. 65, 483–506 (1976). 68. Köhler, T. & Vollrath, F. Thread biomechanics in the two orb weaving spiders Araneus diadematus (Araneae, Araneidae) and Uloborus walckenaerius (Araneae, Uloboridae). J. Exp. Zool. 271, 1–17 (1995). 69. Stauffer, S.L., Coguill, S.L. & Lewis, R.V. Comparison of physical properties of 3 silks from Nephila clavipes and Araneus gemmoides. J. Arachnol. 22, 5–11 (1994).
Acknowledgments
We are grateful to S. Knight for valuable discussions and helpful comments on this manuscript. The Swedish Research Council supported work in the authors’ laboratory.
Competing financial interests
The authors declare no competing financial interests.
Additional information
Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Correspondence should be addressed to A.R. or J.J.
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Toward spinning artificial spider silk Anna Rising1,2* & Jan Johansson1,2*
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Spider silk is strong and extensible but still biodegradable and well tolerated when implanted, making it the ultimate biomaterial. Shortcomings that arise in replicating spider silk are due to the use of recombinant spider silk proteins (spidroins) that lack native domains, the use of denaturing conditions under purification and spinning and the fact that the understanding of how spiders control silk formation is incomplete. Recent progress has unraveled the molecular mechanisms of the spidroin N- and C-terminal nonrepetitive domains (NTs and CTs) and revealed the pH and ion gradients in spiders’ silk glands, clarifying how spidroin solubility is maintained and how silk is formed in a fraction of a second. Protons and CO2, generated by carbonic anhydrase, affect the stability and structures of the NT and CT in different ways. These insights should allow the design of conditions and devices for the spinning of recombinant spidroins into native-like silk.
T
he ability to make silk has made spiders successful; over 45,000 species have been characterized (http://www.wsc. nmbe.ch/), and they can be found in most habitats on Earth1. Spider silk fibers are formed in a fraction of a second from highly concentrated protein solutions (known as ‘dope’) in sophisticated spinning apparatuses. A female spider can spin up to seven types of silk that are used for a variety of purposes, ranging from prey capture to reproduction (Fig. 1a). All silks are composed of proteins that typically consist of an extensive repetitive central part flanked by smaller nonrepetitive domains (Fig. 1b). Spider silk is an astonishing material that shows a combination of high tensile strength and extensibility, which allows some of the fibers to absorb more energy per weight than the strongest of man-made materials, for example, Kevlar (Table 1)2. More surprising, considering the biological functions of spider silks (Fig. 1a), is the fact that the fibers are well tolerated when implanted; for example, they have successfully been used for regeneration of peripheral nerves in vivo3,4. These features make spider silk one of the most fascinating materials known, and if we can learn how to produce it on a large scale there will be numerous possibilities for its application, from the construction industry to medicine. Making artificial spider silk fibers with mechanical properties similar to the natural material, however, has been a major unmet goal in material science for decades. What are the reasons for this shortcoming? Large partial spidroins can be produced in heterologous hosts5, but they, like many short recombinant spidroins6–8, require the use of denaturing conditions during purification and/or fiber formation, which probably explains why most fibers show disappointing mechanical properties (Supplementary Table 1). How to keep the spidroins correctly folded during production and soluble at high concentrations without the use of organic solvents or other chaotropic agents and how to make them form silk fibers without the use of nonphysiological coagulation baths are major issues that remain to be solved. Moreover, problems in making fibers with mechanical properties similar to those of the natural material may also arise due to the fact that the exact chemicophysical conditions of the glands are unknown and that the rheological properties of the dope and physical stresses are difficult to reproduce in vitro. Recent advances in the understanding of the natural silk spinning process9,10, in combination with detailed molecular studies of
partial spider silk proteins9,11–15, have revealed that spider silk formation is governed by mechanisms that have not yet been observed in any other physiological system. Scientific evidence points to the importance of the terminal domains in the regulation of spidroin assembly into fibers9,11–13,15,16 and suggests that current spinning procedures are inadequate because they rely on nonphysiological conditions and/or recombinant spidroins that lack one or both of the terminal domains. Herein, we review recent advances about the chemical biology of the natural spinning process, from which we outline new ways to design optimal protein constructs and biomimetic spinning devices.
Spidroins are conserved but also highly diverse
All spiders spin silk, and some can spin up to seven different types of silks or glues (for example, female orb-weaving spiders)17 (Fig. 1a and Table 1). The dragline silk is most studied and is well known for its high tensile strength, extreme toughness, light weight and favorable properties when implanted in living tissue3,4. This fiber is used to make the framework of the web and is used as a safety line during falls. The flagelliform silk fiber is coated with glue (aggregate silk) and forms the extremely extensible capture spiral of the orb web. The minor ampullate silk is used to reinforce the web. For making the egg sac, female spiders use two types of silks: cylindriform (also referred to as tubuliform) silk forms the durable outer shell of the sack, and aciniform silk is used as a soft inner lining. Aciniform silk is also used for wrapping prey. To attach silk fibers to a surface, the spider uses pyriform silk17. The different mechanical properties of the silks make them attractive for a variety of applications, from superglues to extremely strong and extendible ropes and fibers. The inherent differences of the silks also open up the possibility of designing composites with combined features for advanced technical and medical applications, for example, exact matching of the mechanical properties of a specific tissue for tissue engineering applications. Each silk type is made in a specific gland (Fig. 1a) and mainly consists of spidroins whose repetitive motif has a characteristic primary structure. However, this general view is complicated by the following factors: the presence of variable repetitive motifs within the same spidroin from different species18, non-gland-specific expression of some spidroins19,20 and the facts that spidroins can even be expressed in venom glands21 and that some spider silks contain
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. 2Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Division of Neurogeriatrics, Karolinska Institutet, Huddinge, Sweden. *e-mail: [email protected] or [email protected]
1
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Nature chemical biology doi: 10.1038/nchembio.1789 Though the repetitive parts and mechanical properties vary a lot between silk types2, the regulatory domains (NT and CT)11,12 and the conditions of the silk glands9 are highly conserved (details below), which means that they have important roles in the regulation of fiber formation. Conditions that allow artificial spinning procedures are therefore likely to be found in the natural counterpart and should be independent of what specific type of silk is to be made.
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How do the silk glands work?
Because most data available are from experiments on major ampullate glands and their product, the dragline silk, we will focus on this particular silk. The major ampullate gland has a long, winding and narrow tail and a wider and shorter ampulla or sac (Fig. 2a). The tail and sac contain three different single layered epithelial cell types that all contain numerous granules. The epithelial cell types have distinct distributions and thereby divide the gland into three zones, A–C10 (Fig. 2a). The cells in the A and B zones produce spidroins that form two separate layers in the dope10. Surprisingly, Cylindrical glands; outer silk of egg sac the C-zone epithelium does not produce spiAggregate gland; aqueous coating of capture spiral droins but was recently found to be important Flagelliform gland; core fibers of capture spiral for the physiological regulation of silk producMajor ampullate gland; dragline and structural silk tion (described below)9. The S-shaped narMinor ampullate gland; auxiliary spiral rowing duct is lined by a cuticular intima layer Aciniform gland; soft inner silk of egg sac and silk for swathing prey that probably contributes structural support to the duct and protects the underlying epithelial Pyriform gland; cement for joints and attachments cells from the fiber, but it has also been sugNT Repetitive region CT b gested to act similarly to a hollow fiber dialysis membrane, thereby allowing dehydration of the dope25. The sac is connected to the duct via the funnel, which has an unknown function Figure 1 | Spider silks and spidroins. (a) Illustration of a spider’s spinning glands, different silks and but appears to be associated to the cuticular intima of the duct10. their applications. Detailed descriptions of the different silks are in the main text. (b) A canonical The making of spider silk poses chalspider silk protein is composed of a nonrepetitive NT, a repetitive region and a nonrepetitive CT. lenges at molecular biological, biochemical and physiological levels. The spidroin genes nonspidroin proteins22. Spidroin genes and proteins are most are large, repetitive and very GC-rich, as glycine and alanine can commonly abbreviated by two letters indicating the gland where constitute more than 60% of spidroins26 and are coded for by the they are mainly expressed, followed by Sp for spidroin and a number referring to the different paralogs (for example, MaSp1 for Major ampullate spidroin 1). Table 1 | Mechanical properties of five types of spider silk, Spidroins vary in size21 but generally share a tripartite composiKevlar and high-tensile steel. tion of a nonrepetitive globular NT (~130 residues23), an extensive Strength Extensibility Toughness region made up of silk-specific alanine- and/or glycine-rich repeat Material (GPa) (%) (MJ m−3) 12 units and a nonrepetitive globular CT of ~110 residues (Fig. 1b) . 21–27 136–194 Dragline silk (Araneus diadematus, 0.88–1.5 The NT and CT are evolutionarily conserved and have important Araneus sericatus, Araneus roles in the regulation of spider silk formation (described in detail gemmoides, Argiope trifasciata, below). The spidroin repetitive parts, in contrast, show great variArgiope argentata)2,66–68 ability between silk types. The repetitive part is usually large and Flagelliform silk (A. diadematus, 0.50–1.3 119–270 75–283 can encompass up to hundreds of repeat units. The nature of the A. sericatus, A, argentata)2,66–68 repeat units correlates with the mechanical properties of the fiber; Cylindriform silk (A. argentata, 0.48–2.3 19–29 95 for example, long polyalanine segments give strong fibers, and long A. gemmoides)66,69 glycine-rich segments give more extendible fibers24. However, there is no linear correlation between protein length and mechanical Minor ampullate silk 0.92–1.4 22–33 137 properties5. In fact, the extensive arrays of almost-identical repeats (A. argentata, A. gemmoides)66,69 may be inadvertent consequences of unequal crossing over and Aciniform silk (A. argentata)66 1.1 40 230 homologous recombination events during replication18, which Kevlar 49 (ref. 2) 3.6 2.7 50 would implicate that shorter and less repetitive spidroins may be 2 High-tensile steel 1.5 0.8 6 useful for artificial production of spider silk. 310
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d
Zone A
B
e NT CT
C 5.0
b
Monomer α-Helical
Loose dimers Destabilized
Stable dimers β-sheet fibrils
f
NT
Stability
4.0 3.0 2.0
c
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pH 8
pH 7
pH 6
7.0
6.5
5.5
6.0 pH
5.0
pH 5
Figure 2 | Overview of the natural spinning process and role of the terminal domains. (a) Silk gland with its winding tail, central sac and threelimbed duct. The three histologically different regions are depicted; zones A and B contain epithelium that produces and secretes spidroins into the glandular lumen, and zone C contains active carbonic anhydrase. (b) Blue represents the tail and the part of the sac where spidroin synthesis takes place10, and green represents the sac and duct regions where carbonic anhydrase is found9. (c) The continuous lowering of pH from 7.5–8 in the proximal tail to presumably close to 5.0 at the end of the duct9,13. (d) Thermodynamic stabilities of NT and CT as a function of pH, measured as urea concentrations for half-denaturation. The gray area represents the pH region in the gland where carbonic anhydrase is found. (e) Summary of the main structural features of NT and CT in three different pH intervals, according to the pH scale in c. (f) Schematic representation of the lock-and-trigger mechanism of spider silk formation, where NT (red) acts as a lock and forms dimers that initially are dynamic but become increasingly stable as pH continues to drop, and CT (blue) gets destabilized, unfolds and forms amyloid-like fibrils that may trigger fiber formation9,13. The black lines represent the repetitive regions of spidroins.
codons GGX and GCX. This puts high demands on spidroin gene replication and transcription26–28. To meet the high need of glycine and alanine during translation, the epithelial cells of the glands have unusually large alanyl- and glycyl-tRNA pools29. Despite the spidroins being able to assemble into a solid fiber within a fraction of a second, the spider manages to keep them soluble at very high concentrations (30–50%, w/v) for long-term storage30,31. This implies that the tertiary and quaternary structures of the soluble spidroins are optimized to prevent aggregation. There are currently two different models for how spidroins are organized when stored in the gland: in micelles, where the terminal domains form a hydrophilic outer shell and the repetitive region is shielded in the center32, or as a liquid crystalline feedstock33. These two models are not mutually exclusive and may both occur. The structures of soluble native spidroins are difficult to determine as the proteins are prone to change their conformation upon being manipulated as required to experimentally study the gland content. In spite of this, by using 13 C NMR on whole major ampullate glands, the repetitive part of the proteins was found to adopt a random34 and/or helical conformation31. Polyproline type II segments have also been observed by vibrational CD spectroscopy35. In the final silk fiber, the repetitive part has converted to polyalanine β-sheet crystals flanked by more amorphous glycine-rich repeats36. Although often referred to as amorphous, the glycine-rich regions in the fiber contain 31-helical Gly-Gly-X motifs37–40 and Gly-Pro-Gly-X-X motifs that form type II β-turns41. Finally, the fiber-forming process enables fiber formation in a defined segment of the duct, thus avoiding the fatal spread of the assembly process to the dope in the gland. Understanding how silk formation is regulated requires determination of how the composition of silk dope varies throughout the gland, but the small size of the gland makes this technically challenging. The duct is particularly difficult to work with experimentally owing to the narrow lumen and tough cuticular intima, which is hard to penetrate. Despite these difficulties, the pH from the proximal part of the tail to halfway through the spinning duct was recently determined to span from 7.6 to at least 5.7 (ref. 9). To uphold this broad and steep pH gradient, the spiders must have developed efficient methods to generate protons, and the almostnonexistent variation in pH between individual silk glands9 suggests
that the underlying mechanisms are tightly controlled. By using enzyme activity staining of histological sections, we recently discovered that carbonic anhydrase is responsible for generating and maintaining the pH gradient9. Carbonic anhydrases are ubiquitous enzymes found in all animals and photosynthesizing organisms42 and catalyze the following chemical reaction with an exceptionally high turnover rate of up to 106 s−1 (ref. 42): CO2 + H2O ↔ H + + HCO3−
In the silk gland, active carbonic anhydrase is present from zone C to the end of the duct (Fig. 2a,b). The pH decreases along the gland (Fig. 2c), and, surprisingly, the concentrations of both HCO3− and CO2 rise9. The hydrophobic nature of CO2 implies it should diffuse –
–
+ +
– –
–
2H+
– + +
– + + H+
Figure 3 | Schematic representation of charge interactions, protonation events and structural rearrangements that accompany NT monomer-todimer conversion and stabilization. In each subunit, helix 1 (H1) is brown, H2 is yellow, H3 is green, H4 is light blue and H5 is dark blue. The left side depicts two antiparallel NT subunits held together by charge attractions between the positively charged N-terminal end and the negatively charged C-terminal end. A tryptophan residue (red) is wedged in between H1 and H3. The orientations of H3 and H5, together with charge repulsions between acidic residues in the H3 and H5 of different subunits prevent close interactions. As pH is lowered, the two carboxylates of H3 and H5 become protonated, and the tryptophan side chain is relocated to a superficial position whereupon H3 and H5 change orientation, allowing close interactions between the subunits, but the subunits are mobile with respect to each other. Finally, a third carboxylate residue is protonated, which is linked to full stabilization of the dimer. Only the charged residues at one pole of the antiparallel dimer are shown.
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Figure 4 | A photograph of an artificial spider silk fiber. The fiber was formed in tilting tubes by self-assembly. Scale bar, 1 cm.
freely across cell membranes, but the spider glands have apparently developed a mechanism to uphold a high intraluminal pCO2 (ref. 9). A similar phenomenon has been described for parietal cells in the ventricle43, but the underlying mechanisms are poorly investigated. The high pCO2 in the silk gland is probably of physiological importance, as in vitro studies show that acidic pH in combination with a high CO2 concentration leads to marked structural changes of the terminal domains that can trigger fiber formation (described further below). In the sac, the concentrations of Na+, K+ and Cl− are 199 mM, 6 mM and 164 mM, respectively9. The intraluminal concentrations of Na+ and Cl− are slightly higher than they are in the hemolymph (about 140 mM and 125 mM, respectively, though these values were measured in two different orb-weaving spider species44), whereas the silk gland K+ concentration is lower (about 20 mM in hemolymph44). The concentrations of these ions have been suggested to change along the silk production pathway45, but the concentrations in the duct and hence at the site of fiber formation remain unknown.
Coordinated molecular events govern silk formation
by the addition of preformed fibrils, i.e., seeds (nuclei)51. The spiders apparently have adopted the seeding phenomenon to trigger ultrafast fiber formation. The structural transition of CT into β-sheet fibrils in the duct of the silk gland results in nuclei that may trigger the conversion of the repetitive region into β-sheet polymers, a new and fascinating functional use of amyloid fibrils9 (Fig. 2e,f). The NT is the most conserved part of spidroins and confers solubility to recombinant spidroins at pH 7 and rapid fiber formation when the pH is lowered to 6 (refs. 11,23). It is mainly monomeric at pH levels greater than 7 (refs. 16,52–54) but dimerizes when the pH is lowered below ~6.4 (the exact pKa of dimerization depends on the salt concentration)13,16,52. A relocation of helix 3 closer to helix 1 is a prerequisite for the formation of the NT dimer interface53. The residues responsible for sensing pH were recently identified for MaSp1 NT from the Euprosthenops australis spider13. During storage in the proximal sac (pH ~7.5; Fig. 2), the NT is mainly monomeric, but the dipolar subunits can interact by long-range electrostatic interactions (Fig. 3). Dimerization is not possible at this stage because the tilting of helices 3 and 5 sterically hinders close subunit interactions. When the pH is lowered to around 6.5, as seen in the most distal part of the sac near the funnel (Fig. 2), two conserved glutamic acid residues will each pick up one proton. The loss of these two negative charges allows the rearrangement of mainly helices 3 and 5 and a transition into a subunit conformation that is compatible with dimerization53. To accomplish the structural transition, a tryp tophan residue (or phenylalanine in some NTs) will leave its wedged interhelical position in the monomer and swing out to a position where its side chain is more solvent exposed. This allows helices 1 and 3 to be more tightly packed and leads to the formation of a rather flat dimer interface (Fig. 3). Although NT is dimeric at this stage, it is not until the pH reaches 5.7 and below, corresponding to the pH halfway through the duct and beyond, that a structurally defined and fully stable NT dimer is formed by protonation of a third glutamic acid residue (Figs. 2d,e and 3)13. Some of the residues that control NT dimerization and stabilization in E. australis MaSp1 are replaced by nontitratable residues in other spidroins, but all NTs appear to harbor acidic residues at the dimer interface11,46,55. It seems that the NT has conserved the ability to control dimerization and stabilization in response to a pH gradient, but that the exact residues that titrate during this process are variable is a supposition that must be verified by analyses of wild-type and site-directed mutants of NTs from additional spidroins. The dimerization and stabilization process of NT may seem overly complicated, but the multistep mechanism is probably vital for the control of silk polymerization: the silk fiber is pulled from the spider, and pulling forces can be propagated via the protein chains when they are firmly interconnected via the stable NT dimers and constitutive CT dimers (Fig. 2f). The pulling, as such, may promote refolding of helical or random repetitive segments into extended β-sheet conformations. As the spidroins flow through the duct, the pulling also causes shearing, which has been shown to contribute to the structural transition of the CT into β-sheet nuclei9,12, and this transition can be further accelerated by lowered pH and elevated
The terminal domains of spider silk proteins act as regulatory elements that control spidroin solubility and assembly9,11,12. They are structurally conserved, unique to spiders and present in almost all spidroins, even though spidroins diverged in evolution several hundred million years ago23,46. Both domains are bundles of five α-helices, but they do not share any primary or tertiary structure similarities, which indicates that they fulfill different functions. CT is a constitutive, often disulfide-linked homodimer9,12,47 and thereby probably links the spidroins in pairs already in the oxidizing environment of the endoplasmic reticulum of the glands’ epithelial cells. At pH levels greater than 6.5, as in the lumen of the synthesis and storage parts of the gland (zones A and B; Fig. 2a–c), the CT is stable and highly soluble9,47. When the dope travels down the duct, the decreased pH and simultaneously increased pCO2 markedly affect the CT’s structure and stability9. The low pH destabilizes the CT (Fig. 2d), owing at least in part to the loss of a conserved salt bridge9. Simultaneously, CO2 interacts with partly buried residues in CT, which can facilitate unfolding9. The physiological use of CO2 to destabilize a protein has not been described before and may even be a unique feature of the spiders. At pH levels less than 5.5, the CT loses its native helical structure and forms β-sheet fibrils. This behavior resembles that seen in the formation Figure 5 | A schematic biomimetic spinning device. A highly concentrated aqueous spidroin of amyloid fibrils, where proteins that have lost solution (blue) is pumped into a series of pulled glass capillaries. Aqueous buffers (green and (or never gained) their native conformation yellow) are serially introduced into the device and form laminar flows that cause increased flow instead adopt a very regular structure of cross- rate and shearing of the spidroin solution (i.e., the jet gets thinner). The laminar flow will allow for β-sheets (amyloid fibrils)48–50. The kinetics of diffusion across the solution interfaces, which will lead to a gradual lowering of pH and elevation amyloid fibril formation are greatly accelerated of pCO2 in the spidroin solution when it travels through the device. 312
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Nature chemical biology doi: 10.1038/nchembio.1789 pCO2 (Fig. 2e,f), leading to rapid polymerization of the spidroins. Fast polymerization kinetics are a prerequisite for the spiders’ ability to spin silk at speeds >1 m s−1, but for the spider it is also vital to ensure that the polymerization process is confined to the duct and prevented from spreading up to the sac, as this would prematurely coagulate the contents of the gland. The dynamically associated NT dimers present in the beginning of the duct apparently provide the solution to both these problems: they ensure prealignment of the NTs so that the interlocking of the silk proteins in the distal parts of the duct is independent of diffusion (i.e., their association is ultrafast)15, and, at the same time, they act as a safety mechanism that keeps the pulling forces from propagating up to the gland (i.e., the loosely associated dimers dissociate; Fig. 2f).
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Shortcomings of current methods to spin artificial silk
The insights into the ion and pH gradients of the silk gland and importance of the terminal domains as regulators of spider silk assembly have not yet been translated into a biomimetic spinning process. As outlined in Supplementary Table 1, several recombinant spidroin variants have been produced and spun or self-assembled into fibers. The nature of the produced proteins varies greatly, and none correspond to a full-length natural spidroin. Typically they are much smaller than their natural counterparts and are composed of a repetitive part with or without a CT. There is only one reported case of fibers made from a recombinant spider silk protein that includes all three parts of a canonical spidroin—the NT, a repetitive part and the CT—but their mechanical properties were not analyzed11. Most techniques used for fiber formation require a highly concentrated spinning dope, typically 10–20% (w/v), and to achieve this, the proteins are commonly dissolved in organic solvents such as hexafluoroisopropanol. Notably, even when harsh solvents are used, the solubility of the recombinant spidroins does not match that seen in the native dope56. The techniques used for fiber spinning include wet spinning (ejection of the dope into a coagulation bath, most often containing methanol or isopropanol)5, electrospinning57,58, self-assembly at airwater interfaces59,60 and spinning in microfluidic devices8. Organic solvents or coagulation baths are used to obtain both electrospun (nonwoven) silk fibers and wet spun fibers. Fiber formation through denaturation and aggregation will probably not lead to truly silk-like replicas as the spidroins’ functions are lost. The natural spinning process involves intricate molecular mechanisms and results in fibers with specific secondary structures, i.e., the spidroins are assembled into fibers, not aggregated. Self-assembly of spider silk proteins into meter-long fibers under nondenaturing conditions can be achieved in tubes that are tilted from side to side, which induces shear forces in the dope59 (Fig. 4). However, the method is difficult to scale up, ion and pH gradients are not easily achieved, and the fibers are variable in structure and mechanical properties. Interestingly, though, by including the NT in the recombinant spidroins, the rate of the fiber-forming process can be controlled by pH11. Microfluidic spinning is perhaps the most attractive fiber spinning method as it has successfully been used to obtain fibers without the use of denaturing steps and can be used to create ion and pH gradients as well as shear forces. Fiber formation of a MaSp2 analog has been achieved by applying elongational flow, dropping the pH to 6 and increasing the K+ concentration to 500 mM8. Apart from the high K+ concentration, this approach represents the most biomimetic spinning procedure presented so far. The resulting fibers were not tested for mechanical properties, and their structural properties were not described, making it difficult to evaluate whether this particular biomimetic spinning technique gives rise to functional fibers. Observed effects of K+ on spidroin aggregation are not unequivocal; native spidroin dope has been shown to aggregate into nanofibrils in response to increased potassium concentration30, whereas a recombinant
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spidroin composed of repetitive segments and CT did not respond to potassium concentrations up to 300 mM (ref. 61). Taken together, much effort has been put into spinning artificial spider silk fibers, but few or none of the produced fibers are usable for practical purposes owing to poor mechanical performance, low reproducibility or both. We believe that these shortcomings are most likely due to insufficient mimicking of the natural spinning processes.
How to spin biomimetic spider silk?
To establish a bimimetic spinning technique, the first problem to solve is how to obtain a stable aqueous solution of recombinant spider silk proteins that matches the native solution (30–50% w/w), and the second is how to mimic the conditions of the spider’s spinning apparatus. Microfluidics or similar methods appear well suited for mimicking the gradients of the silk gland and are therefore highly interesting for future development of the spinning technique (Fig. 5). In such devices, laminar flow may be created, allowing for diffusion across the liquid interfaces and thus a gradual change of the conditions in the spinning dope as it travels through the spinning apparatus. Furthermore, increased flow rates generate shear forces, another important factor needed for biomimetic spinning. A lowering of pH from around 7.5 to 5.0–5.5 will be required, and increasing pCO2 will most likely facilitate the nucleation of the polymerization process. An increasing concentration of K+ could lead to faster polymerization of the spidroins. For this biomimetic spinning device to have the intended impact on the spidroins to be spun, the spidroins must encompass both terminal domains. Including the terminal domains in the recombinant spidroins could actually also solve the first problem, as both the NT and the CT, at least in certain species, are very stable and can be concentrated to around 300 mg ml−1 in aqueous buffers11,47. However, when these domains are coupled to repeats from the minor ampullate spidroin, the solubility markedly decreases47. This is surprising as the repetitive segments of spidroins are generally not hydrophobic or very aggregation prone as such. For example, alanine has the highest α-helix propensity of all of the residues62, and this in combination with its biological hydrophilicity63 suggest that polyalanine segments are unsuited to spontaneously aggregate into crystalline β-sheets. Along the same line, the repetitive segments of the aciniform spidroins are α-helical and highly soluble and form globular, folded domains that in the fiber are at least partially transformed into β-sheet conformations64,65. We propose that a main feature of the repetitive segments is to be soluble to avoid premature aggregation of the spidroins and that the unique NT and CT have evolved to allow rapid conversion of the repetitive segments into β-sheet aggregates at a confined place in the spinning duct. If this hypothesis is correct, it implies that the exact sequence of the repetitive segments may be less important than their solubility, suggesting that recombinant spidroin analogs designed from simple repeat units capped by NT and CT could be used for production of silk-based biomaterials.
Future perspectives
During the last few years, important progress has been made toward the accomplishment of generating artificial spider silk mimics: fulllength spidroins have been characterized, two spider genomes have been sequenced, and the milieu in different parts of silk glands and how it affects spidroin domains at a molecular level have been revealed. Hopefully, these advances in basic knowledge will allow the design of proteins, methods and devices that can be used to produce biomimetic spider silk for various purposes in the near future. A challenge that remains is how to keep recombinant spidroins soluble at high concentrations without the use of harsh solvents. We also need to understand, in detail, the unprecedented effects of CO2 on spidroins to be able to harness them for biomimetic spinning. Finally, we envision customized microfluidic spinning devices; for
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example, carbonic anhydrase, an efficient and robust enzyme, could be introduced in a spinning apparatus to achieve a mimic of the glands’ physiology. Received 16 January 2015; accepted 2 March 2015; published online 17 April 2015
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Acknowledgments
We are grateful to S. Knight for valuable discussions and helpful comments on this manuscript. The Swedish Research Council supported work in the authors’ laboratory.
Competing financial interests
The authors declare no competing financial interests.
Additional information
Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Correspondence should be addressed to A.R. or J.J.
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