Molecular nanosprings in spider capture-silk threads

Abstract

Spider capture silk is a natural material that outperforms almost any synthetic material in its combination of strength and elasticity. The structure of this remarkable material is still largely unknown, because spider-silk proteins have not been crystallized. Capture silk is the sticky spiral in the webs of orb-weaving spiders. Here we are investigating specifically the capture spiral threads from Araneus, an ecribellate orb-weaving spider. The major protein of these threads is flagelliform protein, a variety of silk fibroin. We present models for molecular and supramolecular structures of flagelliform protein, derived from amino acid sequences, force spectroscopy (molecular pulling) and stretching of bulk capture web. Pulling on molecules in capture-silk fibres from Araneus has revealed rupture peaks due to sacrificial bonds, characteristic of other self-healing biomaterials. The overall force changes are exponential for both capture-silk molecules and intact strands of capture silk.

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W
e often take pride in our ability to create materials that are
superior to the ones created by nature.Yet some of the materials
that nature creates outperform everything designed by the
human mind. The spider’s capture silk has tensile strength of ∼1GPa,
comparable in strength to Kevlar
1
or steel
2
.Unlike those two man-made
materials, however, spider capture silk is also extremely elastic and
stretches as much as 500–1,000%
3–5
. With its repeating structural
motifs,spider silk is also a natural block copolymer
6
.
Stretchy proteins are particularly interesting from a materials point
of view, given the value of elasticity in the design of fabrics, coatings,
ropes and fibres,and structural materials
7
.Some stretchy proteins, such
as elastin, stretch and relax without any net energy dissipation
8
.
These proteins are highly resilient. Other stretchy proteins dissipate
energy as heat in the process of stretching and relaxing and are thus less
resilient. One advantage of this is that there is less rebound when the
elastic protein relaxes. This is desirable in spider capture silk, because
excessive rebound would propel the insect away from the web, as if it
were on a trampoline
9
.
To capture insects, the two-dimensional (2D) webs of Araneidae
(orb-weaving spiders) need to be stronger than 3D web-networks of
spiders such as the black widow. In 3D webs, the energy required to stop
an insect is dissipated primarily by breaking some of the many silk
strands. In orb webs,the energy is dissipated not by breaking silk strands
but by stretching the elastic capture spiral, which must therefore be
strong so that it does not break and release the insect
9
.
Furthermore, the capture-silk spiral must contract readily on
stretching so that it does not sag and stick to things as it is blown about by
breezes. One of the ways in which capture silk from ecribellate
orb-weaving spiders maintains its elasticity is by remaining wet,through
the action of its hydroscopic gluey coating. Dry capture silk is much less
elastic than wet capture silk. Water acts as a plasticizer or mobilizer for
the molecules of capture silk, as shown by solution-state
13
C NMR
spectra
10
. Capture silk can also contract from its original length in the
web. When contracted to 50% of its original length, the capture silk
‘spools’ into aqueous droplets on the capture web, producing loops
∼100 µm long within the droplet
4
.
Spider capture silk is a natural material that outperforms
almost any synthetic material in its combination of strength
and elasticity. The structure of this remarkable material is still
largely unknown, because spider-silk proteins have not been
crystallized. Capture silk is the sticky spiral in the webs of
orb-weaving spiders. Here we are investigating specifically
the capture spiral threads from Araneus, an ecribellate orb-
weaving spider. The major protein of these threads is
flagelliform protein, a variety of silk fibroin. We present
models for molecular and supramolecular structures of
flagelliform protein, derived from amino acid sequences,
force spectroscopy (molecular pulling) and stretching of bulk
capture web. Pulling on molecules in capture-silk fibres from
Araneus has revealed rupture peaks due to sacrificial bonds,
characteristic of other self-healing biomaterials. The overall
force changes are exponential for both capture-silk
molecules and intact strands of capture silk.
Molecular nanosprings in spider
capture-silk threads
NATHAN BECKER
1
, EMIN OROUDJEV
1
, STEPHANIE MUTZ
1
, JASON P. CLEVELAND
2
,
PAUL K. HANSMA
1
, CHERYL Y. HAYASHI
3
, DMITRII E. MAKAROV
4
AND HELEN G. HANSMA*
1
1
Department of Physics,University of California, Santa Barbara,California 93106, USA
2
Asylum Research,341 Bolay Drive, Santa Barbara,California 93117, USA
3
Department of Biology, University of California,Riverside, California 92521, USA
4
Department of Chemistry and Biochemistry and Institute for Theoretical Chemistry, University of Texas at Austin,Austin, Texas 78712,USA
*e-mail: hhansma@physics.ucsb.edu
Published online: 23 March 2003; doi:10.1038/nmat858
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Our new discoveries about the sequence and structure of natural
spider capture silk are especially timely with the recent discovery of a
way to produce a high-quality synthetic spider dragline silk
11
,
something that has had “scientists scheming [to accomplish] for more
than 100 years”
12
. We used a relative of the atomic force microscope
(AFM) to investigate the molecular and multi-molecular properties of
spider capture silk. This instrument, the molecular force probe
(MFP), is optimized for the pulling mode and uses AFM cantilevers.
Previous AFM-based force spectroscopy
13–16
has revealed the elastic
behaviour in molecules of modular proteins such as the muscle
protein titin
17,18
, polysaccharides such as cellulose, dextran and
amylose
16,19,20
,DNA
21,22
, spider dragline silk
23
and silkworm silk
24
.
In addition, spider dragline silk
23,25
and cobweb silk
26
have been
imaged by AFM.
We present here the force spectra, or pulling curves, of spider
capture-silk molecules (Fig. 1). These force spectra show some of the
longest structures seen by force spectroscopy: the longest pulls are over
3 µm long, as compared with typical pulls of several hundred
nanometres or less for titin
18
and bone
27
, pulls of ∼200 nm or less for a
dragline silk construct
23
, and pulls of 300–1,200 nm for regenerated
fibroin from silkworm silk
24
. The capture-silk force spectra also show
the patterns of elasticity of this spider silk on the smallest force scale yet
seen,with pulling forces in the subnanonewton range, as compared with
previous forces on capture silk
4
in the range of 1–50 µN. This sensitive
force detection was made possible by new instrumentation—MFP and
AFM. Pulling results on webs from Araneus sp. were combined with
capture-silk sequences from Nephila clavipes
28
and Araneus gemmoides
(see Methods) to build molecular and multi-molecular models for
capture silk.
In our experiments, a region of unstretched orb web on glass was
covered with a drop of fluid (Fig. 1, top). Fluid was important in these
pulling experiments, because it eliminated the large meniscus forces
occurring in air
29
.It was also appropriate,because capture-silk is coated
with a hydroscopic glue that keeps it wet in its natural state
9
.A tip on the
end of a cantilever was pressed onto a strand of capture silk lying on the
glass slide (see Methods).When the tip picked up one or more molecules
from the strand of capture silk, these molecules were then stretched
between the tip and the surface of the slide.
After spider-silk molecules were attached to the tip, the tip was
raised above the sample surface (nearly 0.6 µm in Fig. 1) to prevent the
tip from picking up additional molecules in subsequent cycles of
stretching and relaxation. Successive plots of force against extension
were then recorded, as in Fig. 1. Thus,the pair of curves in Fig. 1a shows
the stretching and relaxation of the same silk molecules. The area
between the two curves is a measure of the energy dissipated during one
cycle of stretching and relaxing.
Pulls on capture-silk molecules showed a number of saw-teeth
(Fig. 1). These saw-teeth were reminiscent of the saw-teeth seen when
pulling on the modular proteins titin from muscle
18
, tenascin from the
extracellular matrix
17
, spectrin from red blood cells
13
, lustrin in an
abalone shell
30
, a synthetic construct of a spider dragline-silk protein
23
and also a non-modular protein,collagen
27
.The saw-teeth in the pulls of
these proteins correspond to rupture events, in which sacrificial bonds
are broken, exposing previously hidden polypeptide length, and thus
reducing the tension on the protein molecules
18,23,30–32
.
The saw-tooth rupture events in pulls on capture-silk persist as the
molecule or molecules are cycled through successive pulling and
relaxation (Fig. 1). This indicates that sacrificial bonds are being
broken and reformed, which suggests that the silk molecules are
refolding on relaxation. Thus, spider capture silk seems to be a self-
healing biomaterial. Like lustrin in abalone shells, it shows a more
irregular pattern of ruptures than titin
18
or synthetic dragline silk
23
.
The irregular saw-tooth patterns may be due to a variability in the exact
submolecular structure of refolded structures or to the greater
complexity of spider capture silk and abalone shell as compared with
purified titin and synthetic dragline silk. In each capture-silk pull, the
successive rupture peaks usually occurred at increasing forces.
The force change between successive rupture peaks showed a broad
distribution with a mean of 60 pN (data not shown), equivalent to the
rupture force for several hydrogen bonds
33
.
In previously published pulls on titin, spectrin, dragline silk and
other proteins, there were nonlinear increases in force preceding the
rupture peaks. These nonlinear increases were fitted to the worm-like
chain (WLC) model, which is based on the elastic theory of an ideal
(non-self-avoiding) polymer stretched in its entropic regime
34–36
.
In contrast, the increases in force preceding the rupture peaks in
capture silk were often linear for the intervals of 20–100 nm between
two rupture peaks. This linear behaviour was observed in over half of
the ∼200 rupture peaks analysed. Hookian springs have linear
force–distance curves, raising the question of whether some rupture
peaks in the spider-silk pulls may occur after the stretching of
molecular hookian springs.
Hayashi and Lewis predict that molecular springs are the basis for
the elasticity of spider capture silk
28,37,38
. Their prediction comes from
the spring-like β-spiral sequences of flagelliform protein in capture silk.
The flagelliform proteins of Araneus (see Methods) and Nephila
28
both
a
b
Force
200 pN
0.6 0.8 1.0 µm
Distance above surface
Figure 1 Force spectroscopy of spider capture-silk molecules. Top: diagram of
experimental setup,showing section of a spider web (Araneus sp.) deposited onto a glass
microscope slide,with a drop of CaCl
2
solution (10 mM) covering the cantilever and the
region of the web that is being pulled. Enlargement (circle) shows detail of cantilever pulling
molecules from a region of capture web. a, b, Two from a series of 36 consecutive pulls on
capture silk. Persistence lengths from worm-like chain (WLC) curve fits were typically
0.3–0.5 nm,as observed by other groups when pulling on single protein molecules
18,31
,but
this does not exclude the possibility that there was more than one molecule between the tip
and surface in these pulls.Another indication of single-molecule pulls is that the force
drops to zero at the end of the pull, suggesting that the last molecule stretched between the
tip and the surface has just broken away. By this criterion,the pulls here and in Fig. 3 are for
a few silk molecules.Upper (red) curve in each pair of curves shows stretching (pulling) of
the silk molecules; lower (blue) curve shows retraction (relaxation).
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contain high percentages of glycine and proline, often in the repetitive
GPGGX
n
motifs characteristic of β-spirals.
Model structures for flagelliform proteins in Fig. 2 were designed
with the assumptions that β-spirals were present and that the
unstretched proteins were compact, allowing for considerable
elongation,as observed from the extreme elasticity of both bulk capture
silk
3–5,39
and its molecules (Fig. 1). β-spiral models of flagelliform-
protein sequences from Araneus gemmoides (Fig. 2a) reflect the fact that
its sequence is less regular than the Nephila clavipes sequence, modelled
in Fig. 2b.Relaxed and extended conformations (Fig. 2c) show the large
change in length that can occur with extension. Single molecules of
capture silk are composed of tandem repeats of the entire ensemble,
consisting of repetitive β-spiral sequences plus non-repetitive spacer
sequences,as shown in the diagram in Fig. 2d.
Even if the elasticity of capture silk is due to β-spirals acting as
molecular springs, these springs are not necessarily hookian.They may,
instead, function primarily as entropic springs
37
. In work in progress,
we are carrying out simulated pulling, using steered molecular
dynamics, to investigate the molecular origins of the unique
mechanical properties of the β-spiral sequences in flagelliform protein.
Unlike a metal spring, a polypeptide ‘spring’ has a backbone that is, in
theory, free to rotate around its C–C and C–N single-bonds.
The prevalence of prolines in the flagelliform sequence, and the side
chains of the amino acids, will hinder the free rotation of the
polypeptide backbone; but this does not ensure that there is any
hookian component to the backbone elasticity.
The WLC was a bad fit for many of the force increases preceding
rupture peaks; it was also a bad fit for the overall force changes during
pulling. To a first approximation, the forces increased exponentially
with pulling, and decreased exponentially with relaxation, over a range
of forces from several piconewtons to as high as 800–900 pN (Fig. 3).
The rupture peaks appeared as force maxima, followed by force drops,
Figure 2 Molecular models for relaxed and extended flagelliform protein
sequences from spider capture silk. a, b,Side and end views for possible flagelliform
protein conformations of (a) Araneus gemmoides, 85 amino acids long (sequence is
VGPGGAYGPGGVYGPGAGGLSGPGGAGPYGPGGVGPGGAGPYGPGGVGPGGAGPYGPGGVGPG
GAGPYGPGGVGPGGAGPYGPGG),and (b) Nephila clavipes
28
,75 amino acids long,
(GPGGX)
15
,where X is Y or V, alternately. c, Scale models for extended and relaxed GPGGX
sequences,75 amino acids long.The extended model is at the maximum extension of the
protein,without deforming bond angles.The highly repetitive β-spiral sequence forms
almost 90% of the ∼440—amino-acid-long domains of Nephila flagelliform protein.
The remaining 10% is a short highly conserved spacer sequence rich in acidic amino
acids.There are at least 13 of these β-spiral domains in series in a single molecule of
spider capture silk, based on DNA sequences from Nephila clavipes and N.
madagascariensis
28
.The flagelliform protein in spider capture silk is coated with an
aqueous glue.The glue contains a high concentration of positively charged organic
amines and diamines,and also glycoproteins rich in N-acetylgalactosamine
43,44
.
Force (pN)
0.6 0.8
Distance above surface
1.0 µm
10
10
10
100
100
10
100
10
100
100
a
b
c
d
e
Figure 3 Force changes for stretching and relaxation of spider capture-silk
molecules.Force changes are exponential over a force range from a few pN to
∼500–800 pN.The force scales are on alternate sides of the y-axis such that scales for a,
c and e are on the inside and scales for b and d are on the outside of the y-axis.a and b
show the same data sets as Fig. 1a and b,and c–e are the next three data sets in this
series.As in Fig. 1,upper and lower curves of each force-distance pair show stretching and
relaxation, respectively. Exponential curve fits for relaxation curves had correlation
coefficients R > 0.990.The length constants were calculated for the relaxation curves from
the exponential equation, F= F
0
exp(x/x
0
) where F is the force at extension x.; F
0
is the initial
force and x
0
is the length constant.The length constant x
0
is longer for pulls further from
the surface.
Relaxed
Extended
a
b
c
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along the exponential pull curves. The length constants calculated for
the relaxation curves were 110 ± 30 nm (n = 31). WLC fits (not shown)
were significantly worse than exponential fits for all of the relaxation
curves of Fig. 3 and many segments of the pulling curves.WLC fits have
been used to characterize pulling and relaxation curves for the proteins
titin, tenascin and spectrin
13,17,18
; capture-silk molecules thus show a
different behaviour.
On the largest scale—intact capture silk—we also obtained
exponential force–distance curves (Fig. 4). The distance constant was
11 ± 3 mm (n = 12) for stretching intact spider capture silk in air.
This exponential force increase is consistent with Vollrath’s early work
on stretching intact spider capture silk
4
.Vollrath’s capture-silk pull also
shows an initial length increase at very low force before the roughly
exponential force increase. This length increase may be due to the
unwinding of extra capture-silk length from the ‘windlass’ or ‘spool’
that Vollrath observes in the glue droplets of capture silk
4
. In contrast,
intact spider dragline silk does not show an exponential force increase
on stretching
40
.
What picture emerges for capture silk that might explain the
exponential force–distance curves seen both when pulling on one or a
few molecules and when stretching intact capture silk? These two sets of
exponential data are easier to reconcile if we propose that the force-
spectroscopy pulls are of more than one molecule, because the intact
capture silk is clearly composed of multiple molecules.
One possible model is that we are stretching several of these sticky
proteins that successively rupture or detach,sometimes even in multiple
steps because of looping. At the high extensions where we analyse the
data, the proteins might be unfolded, in which case the slope would be
determined by the elasticity of the protein backbones.Standard models
for the elasticity of unfolded proteins are based on entropic arguments
in combination with enthalpic contributions. Freely jointed chain
(FJC), WLC and exponential curves all converge at high forces and
high extensions
41
. The exponential fit of the data is therefore necessary
but not sufficient to claim a non-entropic nature of the elastic response.
Additional information, such as the temperature dependence, would
help to strengthen this claim.
An alternate model is a molecular network composed of
interconnected springs. To see how such a network can lead to an
exponential force–extension curve, consider the system of identical
springs shown in Fig. 5a. Each spring has a spring constant kand unfolds
at force f; its length increases by a length Lwhen it unfolds.As the force F
applied to the system is increased, unfolding events will take place at
F = f,2f,4f,…,2
n
f,with the resulting extension of the system being L,2L,
3L,…, (n +1)L. The overall force constant after the rupture events will
be k,2k,4k,…, 2
n
k. Thus the overall dependence of the force on the
extension is exponential.In spider webs, this exponential force increase
may occur if one pulls not on a single chain but on a collection of chains
joined by crosslinks,as in Fig. 5b.If such a crosslinked network is pulled,
its properties may be similar to the model in Fig. 5a. This model assumes
the existence of hookian springs, whose presence in capture silk is highly
hypothetical,as discussed above.The problem of capture silk’s structure
is not a simple one, but these two different models can serve as
springboards for designing better models and new experiments.
Crosslinks between molecules may come from spacer sequences in
the capture silk, as shown in Fig. 2b. The spacer sequences in Nephila
clavipes are highly conserved and are relatively rich in the acidic amino
acids Asp and Glu
28,38
; only one spacer sequence is known for Araneus
gemmoides, and it also has Asp (D) and Glu (E) (see Methods section
on molecular modelling; spacer sequence is in bold type). Salt bridges
between these acidic amino acids could provide attachments between
adjacent flagelliform molecules. In mussel byssus threads, crosslinks
form between histidine residues chelated to divalent inorganic
cations
42
. In flagelliform protein, no divalent inorganic cations were
detected by either SIMS (secondary ion mass spectrometry) or XPS
(X-ray photoelectron spectroscopy); Na
+
and K
+
were the only
10
–3
10
–4
10
–5
10 15 20 25
Length (mm)
Force (N)
30 35 40
Figure 5 Schematic diagram for a network of identical springs that gives
exponential force– distance curves on stretching. Each spring,of length L
0
and spring
constant k,increases by length L under a stretching force f.F is total force applied to a
network,N is number of springs in network (see text). a, Idealized network; b,example of a
hypothetical network in spider capture silk.
L
0
L
0
+ Lf
a
b
k
k
k
F
F
Figure 4 Force increases are exponential for stretching intact spider capture silk
in air. Circles and triangles are data from two regions of capture webs.
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inorganic cations observed (S.M. and E.O., unpublished results).
In capture silk, the crosslinks may be components of the capture-silk
glue, such as the organic diamines or the amino-sugars of the
glue glycoproteins
43,44
.
Spider capture silk is not the only biopolymer that shows
exponential force increases with pulling. Single-stranded DNA also
stretches exponentially, especially at large extensions or when it is in a
fluid of low ionic strength
41
. This is, as Bensimon says, a ‘real’ polymer,
more complex than the ideal (non-self-avoiding) polymers that are
modelled nicely by WLC or FJC models
34
. These deviations from WLC
and FJC polymer models have been attributed to excluded volume
effects caused by polymer self-avoidance
34,41,45
.
In conclusion,spider silk is an extraordinary natural material that has
been around for almost 400 million years
46
, since long before the time of
dinosaurs. Many silk protein sequences have remained highly conserved
for an extremely long time
47
. There must be a strong reason for the
sequences to be so highly conserved,and it probably relates to the essential
materials properties of spider silks and spider webs.With a relatively new
technique—force spectroscopy—and extensive analysis of the pulls and
the amino-acid sequence, we have proposed models for both the
molecular and the multimolecular structures of capture silk. Capture silk
is harder to investigate than the less-elastic dragline silk, because capture
silk is found only in orb webs and is thus difficult to isolate from dragline
silk. Therefore force spectroscopy is especially valuable, because it can be
used to investigate capture silk in situ in orb webs.
METHODS
PULLING CAPTURE-SILK MOLECULES UNDER FLUID
A section of spider web from Araneus sp. was deposited onto a clean glass microscope slide by holding the
slide against the web and carefully breaking off silk strands at the edges of the slide. Webs on slides were
stored in a dust-free container under ambient conditions until used for pulling. For pulling, the slide was
mounted in the MFP (MFP-SA, Asylum Research, Santa Barbara), and a few drops of aqueous CaCl
2
(10 mM) were pipetted onto a V-shaped silicon nitride cantilever (spring constant k ≈ 30 pN nm
–1
) and
onto a strand of capture web attached to the glass slide. The MFP head with the cantilever was lowered
onto the MFP base with the glass slide. The optics of the MFP are readily able to image the capture silk
and its glue-droplets, which were spaced at intervals of ∼100 µm.The glue surrounding the capture web
was typically washed away by the aqueous fluid covering the web. The cantilever was manually lowered to
the capture web,and the MFP was operated such that the cantilever moved away from and then towards
the sample surface during the course of each pull. Simply pressing the cantilever’s tip onto a capture-silk
strand was sufficient for stretching molecules between the tip and the surface, as has been observed
previously with other biomaterials
23,32,48
.
MOLECULAR MODELLING OF FLAGELLIFORM PROTEIN SEQUENCES
Modelling was done with Insight II (Molecular Simulations) essentially as described previously
49
.The
sequence from Nephila clavipes was modelled as β-strands with Type II turns with modified psi and phi
angles for G3–G4 in GPGG repeats. This model forms folds for the Nephila sequence with many hydrogen
bonds stabilizing the β-spiral, and with all side chains facing towards the inside of the spiral, thus providing
additional stabilization for this fold in an aqueous environment. Other folds also gave compact model
structures for the Nephila sequence, but some had problems such as a low number of hydrogen bonds and
side chains exposed to the outside rather than the inside of spirals. The sequence from Araneus gemmoides
was modelled with Type II′ turns. The entire cDNA sequence for Araneus gemmoides, whose middle 85
amino acids were modelled in Fig. 2b,is GPGGV[GPGGAGV]
4
GPGGAYGPGGVYGPGAGGLS[GPG-
GAGPYGPGGV]
5
GPGGAGFGPGGAPGAPG[GPG]
3
GPGGVGGPLGPGAGGVGPGGAGPYGPG-
GAGPGGVGPGGAGPYGPGGPGGAGPGGEGPVTVDVEVNVGGAPGG; the spacer sequence is in bold
type. This is the sequence for one of the tandemly arrayed ensemble repeats from the cDNA library made
from the flagelliform gland mRNA: The amino acid composition of this sequence is similar to the com-
position reported
50
for Araneus diadematus, which has 44% G, 21% P, 8% A, 7% V, 3% each of D, G, S, T;
and 1% each of R, L.
PULLING INTACT CAPTURE-SILK IN AIR
A region of web was captured onto an open plastic grid with 1-cm squares.Regions of capture web that
spanned a square were fixed with epoxy to the square at each end, to prevent slippage. The plastic grid was
suspended above the stage of a dissecting microscope. Force on the web was increased by hooking small
wire weights to the web. Length increases were measured from the changes in height of the microscope
head when the lowest point of the web was in focus. Data for force against extension were calculated from
these measurements.
Received 22 October 2002; accepted 18 February 2003; published 23 March 2003.
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Acknowledgements
We thank D. Bensimon for helping us discover that the force–distance curves are exponential, and H.
Gaub, J. Fernandez, S. Fossey, M.Viani, R. Proksch, H. Li, and B. Smith for discussions. This work was
supported by NSF MCB grants to H.G.H., NSF DMR grants to P.K.H. and to UCSB’s Materials Research
Laboratory; ARO DAAG55-98-1-0262 (C.Y.H.), the Robert A.Welch Foundation (D.E.M.), and Asylum
Research, Santa Barbara.
Correspondence and requests for materials should be addressed to H.G.H.
Competing financial interests
The authors declare that they have no competing financial interests.
© 2003 Nature Publishing Group