The Journal of Experimental Biology 202, 3295–3303 (1999) 3295

Printed in Great Britain © The Company of Biologists Limited 1999

JEB2216

THE MECHANICAL DESIGN OF SPIDER SILKS: FROM FIBROIN SEQUENCE TO

MECHANICAL FUNCTION
J. M. GOSLINE*, P. A. GUERETTE, C. S. ORTLEPP AND K. N. SAVAGE
Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
*e-mail: [email protected]

Accepted 3 May; published on WWW 16 November 1999

Summary
Spiders produce a variety of silks, and the cloning of fibroins into a polymer network with great stiffness,
genes for silk fibroins reveals a clear link between strength and toughness. As illustrated by a comparison of
protein sequence and structure–property relationships. MA silks from Araneus diadematus and Nephila clavipes,
The fibroins produced in the spider’s major ampullate variation in fibroin sequence and properties between spider
(MA) gland, which forms the dragline and web frame, species provides the opportunity to investigate the design
contain multiple repeats of motifs that include an 8–10 of these remarkable biomaterials.
residue long poly-alanine block and a 24–35 residue long
glycine-rich block. When fibroins are spun into fibres, the Key words: spider, Araneus diadematus, Nephila clavipes, silk,
poly-alanine blocks form β-sheet crystals that crosslink the fibroin, structure/property relationship.

Introduction
Orb-web-spinning spiders produce a variety of high- unique blend of structural polymers and produces a fibre with
performance structural fibres with mechanical properties a unique set of functional properties (Gosline et al., 1986;
unmatched in the natural world and comparable with the very Vollrath, 1992). Unfortunately, our knowledge of functional
best synthetic fibres produced by modern technology. As a relationships for most of these materials is very limited, with
result, there is considerable interest in the design of these the exception of the major ampullate (MA) gland fibres and to
materials as a guide to the commercial production of protein- some extent the viscid silk fibres produced by the flagelliform
based structural polymers through genetic engineering. To (FL) gland. The organization of these fibres in the spider’s orb-
understand the design of silks, we must understand the web is illustrated in Fig. 1, where it can be seen that MA silk
relationships between structure and function, and this will fibres form the web frame and the spider’s dragline. The viscid
require information from molecular biology, polymer physics, silk forms the glue-covered catching spiral.
materials science and spider biology. We now have good
information on the amino acid sequence motifs present in Mechanical properties of MA and viscid silks
spider fibroins, and our understanding of the molecular Fig. 1 shows typical tensile test data for MA and viscid silk
architecture of spider silks is growing daily. Unfortunately, in from the spider Araneus diadematus plotted as stress–strain
many cases, our understanding of silk design is flawed because curves. The stress (σ) is the normalized force (F), defined as
we do not know which mechanical properties are crucial to its σ=F/A, where A is the initial cross-sectional area of the silk
function. To quote Wainwright (1988), ‘Identification of the fibre. The strain (ε) is the normalized deformation, defined as,
properties that are important to a particular function requires ε=∆L/L0, where L0 is the fibre’s initial length, and ∆L is the
all the intuitive insights and creative abilities of objective change in fibre length. The slope of the stress–strain curve
scientists.’ Only when we understand the true function of gives the stiffness of the material, and the maximum values of
spider silks will be able determine whether a spider’s dragline stress and strain at the point where the material fails give the
silk offers an appropriate model for man-made materials strength (σmax) and extensibility (εmax), respectively. The area
produced through genetic engineering. under the stress–strain curve gives the energy required to break
the material, and this variable can be used to quantify
toughness. Values for the stiffness, strength, extensibility and
The function of spider silks toughness of these two spider silks, along with values for other
The orb-web-weaving araneid spiders provide ideal model biomaterials and selected man-made materials, are presented
organisms for studying the functional design of protein-based in Table 1.
structural materials. These spiders have seven different Note that MA silk is stiffer than the other polymeric
gland–spinneret complexes, each of which synthesizes a biomaterials listed, although mineralized biomaterials may
3296 J. M. GOSLINE AND OTHERS
1.4
Araneus diadematus MA silk:
dragline and web frame
1.2

1.0

Stress (GPa)
0.8
Araneus diadematus viscid silk:
0.6
catching spiral of orb-web
0.4
Einit=10 GPa
0.2 Einit=0.003 GPa
Fig. 1. Stress–strain curves for major ampullate
(MA) gland (red line) silk and viscid silks (blue line) 0
from the spider Araneus diadematus. Einit, initial 0 0.5 1 1.5 2 2.5 3
stiffness. Strain

achieve greater stiffness than this silk. The strength of MA silk, Looking further along Table 1, we see that Araneus MA silk
however, is clearly superior to that of all other biomaterials in is quite extensible, failing at a maximal strain of approximately
this list, suggesting that strength, and to a lesser degree 0.27 (i.e. 27 % extension), whereas the engineering materials
stiffness, may be important indicators of its mechanical fail at strains of the order of 0.01–0.03. The large extensibility
performance. This list of biomaterials is incomplete, but it is of MA silk, in spite of its somewhat inferior strength, makes
fair to say that spider MA silk is amongst the stiffest and MA silk tougher than the engineering materials. Indeed, with
strongest polymeric biomaterials known. Note, however, that an energy to breakage (toughness) of 160 MJ m−3, MA silk is
the stiffness of MA silk is well below that of Kevlar, carbon 3–10 times tougher than its engineering counterparts. This
fibre and high-tensile steel, engineering materials that are suggests that energy absorption and toughness may be the
commonly employed to transmit and support tensile forces. crucial properties that will lead us to an understanding of the
Note also that the strength of MA silk is somewhat less than function of MA silk.
that of these engineering materials. At first glance, we might The viscid silk provides an interesting contrast in properties,
interpret this information as indicating that MA silk is superior and yet it also is a material with exceptional toughness. Its
to other biomaterials, such as collagen, but not as ‘good’ as initial stiffness, at 0.003 GPa, is three orders of magnitude
Kevlar and carbon fibres. However, this interpretation is based lower than that of MA silk and is comparable with that of a
on the assumption that ‘good’ means stiff and strong. lightly crosslinked rubber. Indeed, viscid silk is best thought

Table 1. Tensile mechanical properties of spider silks and other materials

Stiffness, Einit Strength, σmax Extensibility, Toughness Hysteresis
Material (GPa) (GPa) εmax (MJ m−3) (%)
Araneus MA silk 10 1.1 0.27 160 65
Araneus viscid silk 0.003 0.5 2.7 150 65
Bombyx mori cocoon silk 7 0.6 0.18 70
Tendon collagen 1.5 0.15 0.12 7.5 7
Bone 20 0.16 0.03 4
Wool, 100 % RH 0.5 0.2 0.5 60
Elastin 0.001 0.002 1.5 2 10
Resilin 0.002 0.003 1.9 4 6
Synthetic rubber 0.001 0.05 8.5 100
Nylon fibre 5 0.95 0.18 80
Kevlar 49 fibre 130 3.6 0.027 50
Carbon fibre 300 4 0.013 25
High-tensile steel 200 1.5 0.008 6

The data for Araneus silks are from Denny (1976) and our laboratory. Other data have been taken from Wainwright et al. (1982), Gordon
(1978, 1988) and Vincent (1982), without specific references because they are intended to indicate the relative magnitude rather than exact
values.
MA silk, silk from the major ampullate gland; RH, relative humidity.
 The mechanical design of spider silks 3297
of as a rubber-like material. With a maximum strain of comparison, a Kevlar fibre that had exactly the same breaking
approximately 2.7, viscid silk is not exceptionally stretchy tension, but with εmax=0.027, would support a load of less than
compared with other rubbery materials, but its strength, at 40 % of that supported by the MA silk. Fibre extensibility can
approximately 0.5 GPa, makes viscid silk roughly 10 times compensate for strength when fibres are loaded normal to their
stronger than any other natural or synthetic rubber. In this long axis.
context, viscid silk is a truly remarkable material, and the A similar situation holds in prey capture when a flying insect
combination of high strength and extensibility gives it a strikes a silk fibre in the web, again usually approximately at
toughness virtually identical to that of MA silk fibres. right angles to the fibre axis (Fig. 2B). The role of the web fibre
is to absorb the kinetic energy of the insect’s forward velocity,
Mechanical function of MA and viscid silks bringing the insect to a halt so that the spider can capture it.
To understand the roles that extensibility and toughness play Clearly, the extreme toughness of both MA and viscid silks
in the function of MA and viscid silks, we must consider the allows the spider to absorb a large amount of the prey’s kinetic
way that these fibres are used in the normal activities of energy with a minimal volume of silk, but another important
spiders. As documented by Denny (1976), the MA silk fibres consideration is the manner in which this energy is absorbed.
in an orb-web often experience loads at right angles to their The energy could either be stored through elastic deformation
long axis, and this imposes an interesting constraint on the or it could be dissipated as heat through friction. Load cycle
properties of the fibre. For example, the weight (mg, where m experiments by Denny (1976) (Fig. 2C) indicate that the
is mass and g is the acceleration due to gravity) of a spider hysteresis for both MA and viscid silks is approximately 65 %
hanging from a horizontal guy line (Fig. 2A) will cause a load (Table 1). That is, 65 % of the kinetic energy absorbed is
(l) that deflects the web fibre downwards, stretching the fibre transformed into heat and is not available to catapult the prey
and causing a force (Fsilk) to develop. Fsilk=l/2sinθ, where θ is out of the web through elastic recoil.
the deflection angle. For fibres with low extensibility, θ will be The key properties of web silks that emerge from our
small, and Fsilk can be much larger than the load l. Denny analysis of web function involve a balance between strength
(1976) showed that the load is maximized with fibres having and extensibility, giving enormous toughness, and a high level
an extensibility εmax of 0.42, and that the εmax of 0.27 for MA of internal molecular friction, giving high levels of hysteresis.
silk allows the guy line to support a near-maximal load. For Taken together, these properties are more consistent with a
viscoelastic material than with the engineering materials listed
in Table 1, and one of the key features of viscoelastic materials
A B
is that their properties vary strongly with the rate of
θ deformation. In contrast, engineering materials are selected to
Force have high stiffness and strength at the expense of extensibility,
Weight
and they achieve this by having an extremely rigid molecular
structure that virtually excludes the possibility of large-scale
molecular motion.
C Could the viscoelasticity of web silks be an important
functional property? The answer is very probably yes, if strain-
The energy lost in these
rate-dependent changes in properties enhance the functional
Stress capabilities of the web materials. Denny’s (1976) analysis of
load–unload cycles is
indicated by the shaded areas. the strain-rate-dependence of MA silk demonstrated that
Hysteresis is approximately
65% for both silks stiffness, strength, extensibility (extension to failure) and
MA silk
toughness (energy to failure) all increase as strain rate
Viscid silk increases from 0.0005 s−1 to 0.024 s−1 (Table 2), suggesting
that the performance of MA silk may indeed be enhanced by
Strain increasing strain rate. At the highest strain rate (0.024 s−1), a
test to failure would take approximately 11 s. The events
Fig. 2. Functional loading of Araneus diadematus major ampullate involved in prey capture or during the fall of a spider against
(MA) gland silk. (A) When a static load is applied at right angles to its dragline will certainly occur over a much shorter time scale
the fibre, fibre extension allows a large deflection angle θ, and the and therefore at a much higher strain rate. We recently
fibre is able to support a large load. (B) When a flying insect impacts developed an impact apparatus that loads MA silk fibres
a web element and is brought to a halt by the deflection of a silk
normal to the fibre axis with a falling object travelling at a
fibre, the silk absorbs the kinetic energy (EK=0.5mV2, where EK is
kinetic energy, m is mass and V is velocity) of the insect. (C) The
velocity of approximately 1 m s−1. Impact at this velocity
viscoelastic nature of this MA silk transforms much of this energy causes failure after approximately 0.02 s, with strain rates of
into heat through molecular friction. The energy dissipated is the order of 30 s−1. Our experiments are currently in progress,
indicated by the area of the shaded loops in the stress–strain curves but preliminary results indicate strong strain-rate-dependence,
for load cycle experiments. Hysteresis is the ratio of the energy as shown in Fig. 3 and Table 2. The initial stiffness rises
dissipated to the energy absorbed. dramatically, as does the strength, and in exceptional tests the
3298 J. M. GOSLINE AND OTHERS
Table 2. Strain-rate-dependent material properties of spider MA silks
Araneus Nephila clavipes
Strain rate
Properties 0.0005 s−1 0.002 s−1 0.024 s−1 20–50 s−1 0.1 s−1 ≈3000 s−1
Initial stiffness, Einit 9.8 8.9 20.5 25–40 22 20
(GPa)
Strength, σmax 0.65 0.72 1.12 2.0–4.0 1.3
(Gpa)
Extension to failure, εmax 24 24 27 20–50 12 10
(%)
Energy to failure (toughness) 91 106 158 500–1000 80
(MJ m−3)

The first three columns of Araneus data are for Araneus seriaticus, taken from Denny (1976), and the strength data have been converted to
engineering stress. The fourth column is for A. diadematus, from this study.
The Nephila clavipes data are from Cunnliff et al. (1994).
MA silk, silk from the major ampullate gland.

strength now virtually matches that of Kevlar and the it shrinks by approximately 40–50 %, and its mechanical
toughness reaches the astronomical level of approximately properties change markedly. The initial stiffness drops by three
1000 MJ m−3. Thus, to summarize, our analysis of the orders of magnitude, and the material becomes rubber-like in
mechanical properties and mechanical function of MA and its behaviour (Gosline et al., 1984). Viscid silk also contracts
viscid silk fibres indicates that these materials have probably when immersed in water, although the effect on its mechanical
been selected through evolution to achieve a harmonious properties is less dramatic (Gosline et al., 1994, 1995). Work
balance (Wainwright, 1988) of strength, extensibility and (1977) called this transition supercontraction, and to date
viscoelasticity, which together produce materials of incredible no-one has discovered or described a function for
toughness. supercontraction. Supercontraction will occur in air if the
One final set of properties remains to be considered. These relative humidity reaches a level of approximately 90 % or
are the responses of silk fibres to environmental variables such more and, therefore, it is likely that the silk fibres in the webs
as temperature, moisture, etc. To date, little is known about the of spiders may experience conditions for supercontraction in
temperature-dependence of the mechanical properties of silks, their normal use. The consequences of supercontraction in the
although the strong strain-rate-dependence suggests that orb-web are not known but, because the web is always tethered
temperature changes will alter the mechanical properties. to rigid external structures and is laid down in tension (Denny,
Water content, however, is known to have a major effect on 1976), neither the MA silk nor the viscid silk will be able to
MA silks (Work, 1977). When MA silk is immersed in water, shorten significantly. They will, however, develop a small

4.5

4.0 High strain rate tests

≈ 30 s-1
3.5

3.0
Stress (GPa)

Horizontal
Fig. 3. Selected high-strain-rate impact tests of
2.5 transducer
Araneus diadematus major ampullate (MA)
gland silk that spans the range of behaviour 2.0 Verti cal L0
observed. The low-strain-rate curve illustrates transducer
the magnitude of the increase in stiffness and 1.5
Fsilk Fv
strength. The inset diagram shows the
experimental arrangement with the silk fibre 1.0 Fh
supported between two force transducers, one Low strain rate test
0.5
that senses vertical force (Fv) and the other that ≈ 0.002 s-1 Load, l
senses horizontal force (Fh). Fibre force (Fsilk) 0.0
and fibre strain are calculated trigonometrically. 0 0.1 0.2 0.3 0.4 0.5
L0, unstretched length. Strain
 The mechanical design of spider silks 3299
additional tension that may serve to keep the web taut, and the The A. diadematus MA silk network models are illustrated
increased water content may reduce the level of hysteresis and in Fig. 4, and they have the following features. First, MA silk
alter the strain-rate-dependence described above. contains a crystal-crosslinked and crystal-reinforced polymer
Much remains to be learned, but it is possible that network. That is, the crystals form the intermolecular
supercontraction is simply an unavoidable consequence of the connections between the fibroin molecules, and the crystals are
molecular structure required to create the balance of large enough and abundant enough to act as reinforcing filler
mechanical properties described above. We know that the particles to stiffen and strengthen the network. The amorphous
design of these silks requires a structure that allows chains, which interconnect the crystals, are estimated to be
considerable movement at the molecular level to provide high 16–20 amino acid residues long, and the water absorbed during
levels of toughness and energy dissipation. Perhaps this level supercontraction is associated primarily with these amorphous
of molecular movement could only be achieved with a protein chains. The rubber-like elasticity of the hydrated network
sequence that incorporates a large fraction of polar amino arises from the large-scale extension of these coiled amorphous
acids, thus imparting the hydration-sensitivity that creates chains. The crystals are estimated to have a length/width ratio
supercontraction. Whatever the role or origin of of approximately 5, and they occupy approximately 10–12 %
supercontraction, it is important to note that this tendency of of the volume of the hydrated MA silk (Fig. 4A).
spider MA silk to absorb water, shrink and become rubbery The model for the native state of the MA silk fibre is created
may strongly limit the utility of this design for application to by applying an 80 % stretch to regain the native length, and by
man-made materials. shrinking the model laterally to account for the loss of
approximately 50 % of the volume when the water evaporates
(Fig. 4B). This produces a crystal volume fraction of 20–25 %.
The molecular architecture of spider silks The amorphous chains are extended, and the polymer crystals
X-ray diffraction studies have shown that virtually all silks are rotated during the stretching and shrinking to produce a
contain protein crystals, and the majority of these silks contain strongly preferred molecular orientation parallel to the fibre
β-pleated sheet crystals that form from tandemly repeated axis. The physical state of the extended, amorphous chains
amino acid sequences rich in small amino acid residues. For remains to be determined, but one possibility is that they form
example, textile silk produced by the silk moth Bombyx mori a rigid, extended polymeric glass phase, creating a highly
contains multiple repeats of the hexapeptide motif GAGAGS oriented, fibre-reinforced polymer network. It is likely that this
(where G is glycine, A is alanine and S is serine). Further, it model can explain both the mechanical properties described
is well established that the alternation of glycine with either above and the fibroin sequence designs described below.
alanine or serine causes this sequence spontaneously to form
β-pleated sheet crystals (see Wainwright et al., 1982; Kaplan Molecular genetics of the spider fibroin gene family
et al., 1994). In B. mori silk, these protein crystals occupy Until recently, we knew little about the amino acid sequence
40–50 % of the total volume of the silk fibre (Iizuka, 1965), motifs in spider fibroins, although we knew that each of the
with the remainder being occupied by protein chains having a seven glands produced proteins with unique amino acid
much less ordered, possibly amorphous, structure. Recent compositions (Andersen, 1970). The compositions of most
developments in the study of spider silks are beginning to spider silks are similar to that of the textile silk produced by
reveal the molecular origins of their material properties. the silk moth B. mori, but with some significant differences in
the abundance of the larger and more polar amino acid
Network models for supercontracted MA silk residues. B. mori fibroin is known to contain multiple repeats
One major problem in defining the molecular structure of of a hexapeptide and, as shown in Fig. 5, the hexapeptide
silk fibres has been a lack of information on the structure and GAGAGS occurs in blocks of 8–10 repeats, separated by
volume fraction of the ‘amorphous domains’ that exist between another repeating motif that is somewhat more variable. These
the crystals in silk fibres. Analysis of the mechanical properties two large sequence blocks repeat four or five times between a
of supercontracted MA silk reveals that the amorphous small, approximately 30 residue, section of non-repetitive
domains are a dominant feature of Araneus diadematus MA sequence called the ‘amorphous domain’ (Mita et al., 1994).
silk. Because hydrated MA silk exists in a rubber-like state Thus, B. mori fibroin contains a preponderance of long crystal-
(Gosline et al., 1984), it is possible to use the theory of rubber forming blocks, which establishes a strong potential for crystal
elasticity (Treloar, 1975) to develop a model for the protein formation, and this probably accounts for the high crystal
network in MA silk. In addition, because the crystals are not content of B. mori silk.
altered during supercontraction, it is possible to apply this Recently, sequence data for two MA silk fibroins from the
model to the organization of the native silk fibres. The araneid spider Nephila clavipes (Xu and Lewis, 1990; Hinman
modelling process is based on the tendency of rubber networks and Lewis, 1992) have revealed that the sequence designs of
to become increasingly stiff when the network chains approach spider fibroins are fundamentally different from those of B.
their full extension. Details of this analysis are described mori fibroin. Sequences from two genes expressed in the N.
elsewhere (Gosline et al., 1994, 1995); only the essence of the clavipes MA gland (Nc-MA-1 and Nc-MA-2) reveal repeating
molecular models is described here. sequence motifs approximately 34 and 45 amino acid residues
3300 J. M. GOSLINE AND OTHERS

A
Amorphous
network chains:
16–20 amino acid
residues long

Water: 50% of
total volume
Fig. 4. Models for the molecular architecture of
Araneus diadematus major ampullate (MA) gland
silk. (A) When immersed in water, MA silk β-sheet crystals:
crosslink and reinforce
supercontracts, i.e. it swells, shrinks and becomes
the polymer network
rubbery. Analyses of the mechanical and optical
properties of supercontracted silk provide Supercontracted MA silk:
information on the nature of the amorphous crystal volume fraction is 10–12%
chains and the size and orientation of the β-sheet
crystals in the silk polymer network. (B) Model
of the native MA silk fibre obtained by extending Native MA silk:
and ‘drying’ the model derived for the crystal volume fraction
supercontracted state. is 20–25%

long, respectively (Fig. 5). Within each repeat motif, there is supercontract when they are immersed in water (Work, 1977),
a short poly-alanine block, 8–10 residues in length (highlighted properties that reflect a high crystal content.
yellow), that we now know forms a very short crystal-forming
domain. The remainder of the repeat motif contains a glycine- Major ampullate gland (MA) fibroins
rich domain that is probably very different in its crystal- Nc-MA-1: AGAAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQG
forming potential and may direct the formation of amorphous Nc-MA-2: SAAAAAAAAAGPGGYGPGQQPGGYGPGQQGPGGYGPGQQGPSGPG
domains in the MA silk polymer network. Thus, the repeat Ad-MA-1: ASAAAAAAGGYGPGSGQQGPGQQGPGGQGPYGP
Ad-MA-2: ASAAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPG
motifs contain approximately one-quarter of their sequence in
the form of crystal-forming elements, and these crystal- Minor ampullate gland (MI) fibroins
forming elements are very short. Nc-MI-1: AGAGAGAAAGAGAGGYGGQGGYGAGAGAGAAAAAGAGGAGGYGRG
More recently, Guerette et al. (1996) reported repeating Nc-MI-2: AVAGSGSAAGAGARAGSGGYGGQGGYGAGAGAGAAAGAGAGSAGGYGRG
sequence motifs for fibroins from the spider A. diadematus Ad -MI-1: GAGSGAGAGAAAAAGAGGYGQGY
(Fig. 5), showing that the pattern of small crystal-forming
blocks alternating with larger ‘amorphous’ blocks is a feature Cylindrical gland (CY) fibroin
of spider silk fibroins produced in at least three silk glands, the Ad-CY-1: AAAAAAAAGGQGGQGGYGGLGSQGGAGQGGYGAAGLGGQGG

MA gland, the minor ampullate (MI) gland and the cylindrical

Flagelliform gland (FL) fibroin
(CY) gland. Interestingly, different silk glands express fibroins Nc-FL-1: (GPGGX)N≈50 - (GGX)N≈9 - (Spacer,28 residues long)
with different proportions of crystal-forming and amorphous
sequence elements, imparting different tendencies for the Bombyx mori cocoon silk fibroin
formation of crystals in their silk fibres. For example, the two {(GAGAGS)N=8-10 - [(GA)N=1-3 - GY]N=4-7}N=4-5 - (Spacer)
fibroins found in A. diadematus MA silk (Ad-MA-1 and Ad-
MA-2) have similar proportions to those produced by N. Fig. 5. Amino acid sequence motifs for spider silk fibroins. Crystal-
clavipes, with crystal-forming elements occupying forming motifs are highlighted in yellow; proline residues are
approximately one-quarter of the repeat block. The A. highlighted in purple. The Nc-MA-1 data are taken from Xu and
Lewis (1990), the Nc-MA-2 data are from Hinman and Lewis
diadematus MI fibroin (Ad-MI-1) has a much larger fraction
(1992), the Nc-MI-1 and Nc-MI-2 data are from Colgin and Lewis
of crystal-forming blocks (68 %) and very short amorphous (1998), and the Nc-FL-1 data are from Hayashi and Lewis (1998).
blocks (32 %), a pattern that has been documented recently for All Ad-MA, Ad-MI and Ad-Cy data are taken from Guerette et al.
N. clavipes MI silk (Nc-MI-1 and Nc-MI-2; Colgin and Lewis, (1996). The Bombyx mori heavy-chain fibroin data are taken from
1998). The high crystal-forming content of MI silk fibroins Mita et al. (1994). G, glycine; A, alanine; S, serine; P, proline; Q,
probably accounts for the fact that spider MI silks are more glutamine; Y, tyrosine; L, leucine; V, valine; R, arginine. X can be
birefringent than MA silks and that MI silks do not alanine, serine, valine or tyrosine.
 The mechanical design of spider silks 3301
The repeat motifs for A. diadematus MA fibroins (Ad-MA- the synthesis of genetically engineered silk fibres based on the
1 and Ad-MA-2) provide an opportunity to evaluate the MA fibroin sequences from N. clavipes (Lipkin, 1996) has
network model for supercontracted MA silk shown in Fig. 4. generated an enormous interest in the structure of this material.
The amorphous chains between crystals were predicted to be As a consequence, we know more about N. clavipes MA silk
16–20 amino acid residues long, and the crystals were predicted than any other spider silk. X-ray diffraction indicates that the
to occupy 20–25 % of the total volume in the native fibre. The β-sheet crystals have an inter-sheet spacing of 0.53 nm, a
poly-alanine blocks in the Ad-MA repeats account for 25–30 % spacing characteristic of β-sheet crystals formed from poly-
of the total, and the glycine-rich amorphous blocks are 24–25 alanine (Becker et al., 1994). Detailed analyses of X-ray
residues in length, a reasonable match between the inferred patterns indicate that the crystal dimensions are approximately
polymer network and the fibroin sequences. 2 nm×5 nm×7 nm, a scale consistent with β-sheets formed from
Recently, Hayashi and Lewis (1998) obtained sequence the short poly-alanine blocks seen in the fibroin sequences and
information for a fibroin from the flagelliform (FL) gland having a length/width ratio similar to that predicted in the
(viscid silk core fibre: Nc-FL-1, Fig. 5). This fibroin is almost network models. The crystals are strongly aligned with the
entirely constructed from glycine-rich sequence motifs fibre axis and occupy 10–15 % of the total volume (Grubb and
characteristic of the amorphous blocks in MA fibroins. That is, Jelinsky, 1997; Yang et al., 1997). These conclusions are
there are numerous (40–65) repeats of the pentapeptide confirmed by nuclear magnetic resonance (NMR) studies
GPGGX (where P is proline and X can be alanine, serine, valine (Simmons et al., 1996; Kummerlen et al., 1996); however, it
or tyrosine), and this huge amorphous domain is followed by is not clear whether the fibre contains a simple two-component
a second glycine-rich motif, GGX, that is repeated 6–12 times. (crystal + amorphous) structure. Simmons et al. (1996)
There are no obvious crystal-forming domains that look like observed two populations of alanine residues in their NMR
any found in spider MI, MA or CY silks, and we are left to spectra, only one of which could be considered to be the highly
consider the source of the crosslinking that must certainly exist ordered and oriented crystals depicted in Fig. 4. Apparently,
in viscid silk to explain its exceptional strength. Network only approximately 40 % of the alanine residues are present in
models developed for A. diadematus viscid silk (Gosline et al., the oriented β-sheet crystals, with the remaining 60 % found in
1994, 1995) suggest that the network chains probably contain weakly oriented and unaggregated β-sheets. This would
50–75 amino acid residues between crosslinks, but this number suggest that the total crystal content is higher than the 10–15 %
is considerably smaller than the size of the pentapeptide repeat noted above, and it may explain why the network models
blocks observed in Nc-FL-1 (approximately 240–350 based on mechanical tests (Fig. 4) predict a crystal content
residues). Perhaps the sequence design is different in A. of 20–25 %. It is probably safe to extend these general
diadematus FL fibroins, or perhaps we have much learn about conclusions to the MA silks of A. diadematus since the Ad-
the organization of FL silks. MA fibroins contain poly-alanine blocks of virtually identical
We can now consider the correlation between sequence dimensions to those of N. clavipes, but the details may vary
design and mechanical properties. The network structure of somewhat.
A. diadematus MA silk contains a low volume fraction of The idea that the sequence data could predict the structural
very small crystals separated by rather long amorphous organization of the ‘amorphous’ glycine-rich domains is,
chains, and perhaps this structure is responsible for the fact however, an oversimplification. Kummerlin et al. (1996)
that A. diadematus MA silks are respectably strong but are employed two-dimensional NMR to study N. clavipes MA silk
incredibly tough and viscoelastic. If the amorphous network and discovered strong evidence for an ordered microstructure
chains behave as an extended polymeric glass, then in the glycine-rich domains. They concluded that the simplest
deformation of these chains could account for the high model to fit their experimental data is a 31-helical structure,
extensibility of MA silk and could also explain its high level and they speculate that these 31-helices aggregate to reinforce
of hysteresis. Note also that the glycine-rich motifs in both the highly oriented polymer network present in MA silk. An
Ad-MA-1 and Ad-MA-2 contain numerous polar glutamine alternative hypothesis has been put forward by Thiel et al.
residues (glutamine, Q), and these will explain the tendency (1996) on the basis of electron diffraction and an analysis of
of MA silk to absorb water and supercontract. B. mori silk, the minor peaks in the X-ray diffraction pattern of N. clavipes
with its extremely long crystal sequences and high MA silk. They suggest that, when stretched, the glycine-rich
crystallinity, does not match spider MA silk for stiffness, sequences are able to form large-scale β-sheet crystal
strength or extensibility (Table 1), and the highly crystalline structures that are less perfectly ordered than the poly-alanine
MI silk is no stronger than MA silk (Work, 1977). Clearly, crystals described above. They consider N. clavipes MA silk
crystallinity is not the only important feature of network to be a hierarchical structure in which the poly-alanine crystals
design in spider MA silks. Amorphous chains that allow are surrounded by a ‘non-periodic crystal lattice’ formed in the
deformation and viscoelastic energy dissipation are equally glycine-rich sequences. A key feature of these proposals is that
important features of MA silk designs. the glycine-rich sequences should not contain proline. That is,
the structural proposals for the ‘amorphous’ regions of N.
Physical characterization of the MA silk network clavipes MA silk apply to only one of the MA fibroins, namely
The remarkable success of the biotechnology community in Nc-MA-1, as this is the only fibroin without proline in its
3302 J. M. GOSLINE AND OTHERS
glycine-rich domains. The other three MA fibroins (Nc-MA-2, Alternatively, we could build upon the variation created
Ac-MA-1 and Ad-MA-2) all contain 15–17 % proline (Fig. 5; through millions of years of evolution to develop an
highlighted in purple) in their repeat motifs. understanding of structural designs that already exist in nature
N. clavipes MA silk has a very low total proline content and have been tested in the day-to-day function of spiders. The
(approximately 3 %), whereas A. diadematus MA silk has a comparison between N. clavipes and A. diadematus MA silks
proline content (16 %) that just matches the content of its two outlined here shows that this second approach will work if we
fibroins. This difference in proline content has major have sufficient information about the true function of the silks
consequences for the two MA silks. N. clavipes MA silk is of spiders. Spider fibroin genes belong to a single gene family
apparently made primarily of Nc-MA-1, the proline-free (Guerette et al., 1996; Hayashi and Lewis, 1998), so it should
fibroin, and the proposals for the presence of ordered structures be easy to obtain fibroin sequence information from related
in the glycine-rich domains are reasonable. A. diadematus MA spider species. If our knowledge of functional biomechanics
silk contains fibroins with a high proline content, and this and of silk network structure can keep pace, then the diversity
should prevent the formation of similar ordered structures in that exists in living spiders should help us to develop a full
the A. diadematus silk network. Thus, the simple amorphous understanding of protein-based structural materials.
+ crystal network model for A. diadematus MA silk in Fig. 4
is probably correct, but it probably does not apply to N. This research was supported by grants from the Natural
clavipes MA silk. Sciences and Engineering Research Council of Canada and
If the high extensibility, viscoelasticity and energy from E. I. DuPont de Nemours to J.M.G.
dissipation of A. diadematus MA silk are due to the truly
amorphous organization of the network chains between
crosslinking crystals, then N. clavipes MA silk may have quite References
different mechanical properties. This indeed appears to be the Andersen, S. O. (1970), Amino acid composition of spider silks.
case. Mechanical testing of N. clavipes MA silk (Cunnliff et al., Comp. Biochem. Physiol. 35, 705–714.
1994) indicates that it has a greater initial stiffness Becker, M. A., Mahoney, D. V., Lenhert, P., Eby, R., Kaplan, D.
(Einit=22 GPa) and a lower extensibility (εmax=0.12) but a and Adams, W. (1994). X-ray modulus of silk fibers from Nephila
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report a value for stiffness and extensibility of N. clavipes MA non-silk-like ‘spacer regions’. Protein Sci. 7, 667–672.
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