Please read the article below, (Spinning of Spider Silk) and write a critical summary report of your reading.
The 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 âdopeâ) 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 spidroins6â8,
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 proteins9,11â15, 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 fibers9,11â13,15,16 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 mainlyconsists 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 containThough 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.
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 numerousgranules. The epithelial cell types have distinct
distributions and thereby divide the gland into
three zones, AâC10 (Fig. 2a). The cells in the
A and B zones produce spidroins that form
two separate layers in the dope10. Surprisingly,
the C-zone epithelium does not produce spidroins
but was recently found to be important
for the physiological regulation of silk production
(described below)9. The S-shaped narrowing
duct is lined by a cuticular intima layer
that probably contributes structural support to
the duct and protects the underlying epithelial
cells from the fiber, but it has also been suggested
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
but appears to be associated to the cuticular
intima of the duct10.
The making of spider silk poses challenges
at molecular biological, biochemical
and physiological levels. The spidroin genes
are large, repetitive and very GC-rich, as glycine and alanine can
constitute more than 60% of spidroins26 and are coded for by thenonspidroin proteins22. Spidroin genes and proteins are most
commonly abbreviated by two letters indicating the gland where
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).
Spidroins vary in size21 but generally share a tripartite composition
of a nonrepetitive globular NT (~130 residues23), an extensive
region made up of silk-specific alanine- and/or glycine-rich repeat
units and a nonrepetitive globular CT of ~110 residues (Fig. 1b)12.
The NT and CT are evolutionarily conserved and have important
roles in the regulation of spider silk formation (described in detail
below). The spidroin repetitive parts, in contrast, show great variability
between silk types. The repetitive part is usually large and
can encompass up to hundreds of repeat units. The nature of the
repeat units correlates with the mechanical properties of the fiber;
for example, long polyalanine segments give strong fibers, and long
glycine-rich segments give more extendible fibers24. However, there
is no linear correlation between protein length and mechanical
properties5. In fact, the extensive arrays of almost-identical repeats
may be inadvertent consequences of unequal crossing over and
homologous recombination events during replication18, which
would implicate that shorter and less repetitive spidroins may be
useful for artificial production of spider silk.codons GGX and GCX. This puts high demands on spidroin gene
replication and transcription26â28. 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
(30â50%, 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
13C 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 motifs37â40 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 throughoutthe 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 suggeststhat 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 sâ1 (ref. 42):
CO2 + H2OâH+ +HCO3â
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
â and
CO2 rise9. The hydrophobic nature of CO2 implies it should diffusefreely 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 Clâ
are 199 mM, 6 mM and 164 mM, respectively9. The intraluminal
concentrations of Na+ and Clâ 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
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 glandsâ 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. 2aâc), 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 CTâs structure and stability9. The low pH destabilizes the CTSimultaneously, 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
of amyloid fibrils, where proteins that have lost
(or never gained) their native conformation
instead adopt a very regular structure of cross-
β-sheets (amyloid fibrils)48â50. The kinetics of
amyloid fibril formation are greatly acceleratedby 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. 16,52â54) 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 tryptophan
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, correspondingto 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 elevatedleading to rapid polymerization of the spidroins.
Fast polymerization kinetics are a prerequisite for the spidersâ ability
to spin silk at speeds >1 m sâ1, 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 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 spidroinâthe NT, a repetitive part and
the CTâbut their mechanical properties were not analyzed11.
Most techniques used for fiber formation require a highly concentrated
spinning dope, typically 10â20% (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 spidroinsâ 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 intometer-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 recombinantspidroin 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 (30â50% w/w), and
the second is how to mimic the conditions of the spiderâs 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 5.0â5.5 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 mlâ1 inaqueous 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 uniqueNT 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 example, carbonic anhydrase, an efficient and robust enzyme, could
be introduced in a spinning apparatus to achieve a mimic of the
glandsâ physiology.
