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First published online May 30, 2008
Journal of Experimental Biology 211, 1937-1947 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014217
The effect of proline on the network structure of major ampullate silks as inferred from their mechanical and optical properties
Department of Zoology, 6270 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada, V6K 1Z4
* Author for correspondence (e-mail: gosline{at}zoology.ubc.ca)
Accepted 5 April 2008
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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MA silk is a strong material with a failure stress (
1 GPa) comparable
to that of high tensile steel (Gosline et
al., 1999
) or to the aramid fibre, Kevlar
(Hayashi and Lewis, 1998).
Unlike these materials, MA silk is also highly extensible, with a failure
strain of about 0.30. This combination of high strength and extensibility
confers a degree of toughness unmatched by industrially produced materials.
The molecular mechanisms that result in these remarkable properties have
remained elusive. Understanding the relationship between amino acid sequence,
protein secondary structure, fibroin network structure and the mechanical
properties of MA silk in its functional states could lead to insights into how
these remarkable properties are achieved.
In the web, MA silk functions in both the dry and the hydrated states. In
the dry state, MA silk is much stiffer than most elastomeric proteins;
however, it is worth noting that these proteins also function in the hydrated
state. A more apt comparison would be between elastomeric proteins and MA silk
in the hydrated state. Work (Work,
1977) found that when immersed in water, MA silk supercontracts,
shrinking in length and increasing in volume. Supercontraction is associated
with a change in mechanical properties and a decrease in birefringence
(Work, 1977). The mechanics of
supercontraction have been documented for the MA silk from a variety of spider
species (Work, 1977;
Work, 1981;
Work, 1982;
Work, 1985;
Fornes et al., 1983).
Birefringence studies have been used as a measure of molecular order within
dry and supercontracted fibres and associated with the mechanical properties
of MA silk. In these studies higher birefringence (higher molecular order)
values are associated with stiffer, stronger fibres. The function of
supercontraction is unknown; however, since the web is tethered to rigid
structures, and MA silk is laid down in tension, supercontraction generates
stresses of about 50 MPa (Savage et al.,
2004; Work and Young,
1987) that could act to help tension the web.
At present, partial sequences are available for 21 fibroins expressed in
the MA glands of ten species of spiders from six genera
(Gatesy et al., 2001). In each
case, the structure of the MA proteins is highly conserved, consisting of two
distinct and repeated sequence blocks: (1) a poly-alanine block, 5–10
amino acids long and (2) a glycine-rich sequence block of about 20–30
amino acids long. MA silk fibroins have been grouped as either MA spidroin-1
or MA spidroin-2 according to the variation and frequency of stable repeating
amino acid motifs within the glycine-rich sequence blocks. These motifs
include GA, GGX and GPG(X)n, where X represents a small subset of
amino acids.
The most apparent difference between spidroin-1 and 2 is that spidroin-1
fibroins are deficient in the amino acid proline and spidroin-2 fibroins are
proline rich. Thus, spidroin-1 and 2 probably have different propensities for
secondary structure based on this difference in proline content. Molecular
dynamics simulations of model peptides indicate that only those sequences
containing proline exhibit elastin-like properties, and only sequences devoid
of proline had a propensity to aggregate into amyloid-like fibrils
(Rauscher et al., 2006).
Indeed, an analysis of the proline and glycine content of elastomeric proteins
and amyloids indicate that elastin-like behaviour occurs above a threshold
level of combined proline and glycine content
(Rauscher et al., 2006). That
study identified proline as the primary determinant of elastin-like behaviour,
and noted that spidroin-1 fibroins are below the proline and glycine
threshold, and so are likely amyloidogenic, whereas spidroin-2 fibroins are
above the threshold, and hence should be elastin-like.
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).
Gatsey et al. (Gatsey et al., 2001) classified a third fibroin (ADF-2 isolated
by Guerette et al.) as a MA-spidroin-1 type fibroin, but this fibroin was
isolated from a cDNA library derived from the cylindrical gland, which
produces cocoon silk. Using northern analysis ADF-2 was found to be expressed
in the cylindrical gland and was below detection limits in the MA gland. Thus,
the predominant fibroins expressed in the Araneus MA gland are
elastin-like and contain about 16% proline, and the MA gland contains about
16% proline (Andersen, 1970).
The Araneus fibroins are, at first glance, analogous in structure to
elastin, containing both a poly-alanine sequence block and a glycine and
proline-rich sequence block. Nephila clavipes, however, expresses one
gene from each group, Nc-MA-1, encoding a spidroin-1 fibroin, and
Nc-MA-2 encoding the elastin-like spidroin-2 fibroin
(Fig. 1). Since the
Nephila MA gland contains approximately 3.5% proline
(Work and Young, 1987) and the
Nc-MA-2 gene product contains about 18% proline, we estimate that Nc-MA-1
makes up about 80% of the content of the gland.
Studies into the structure of spider silks have focused mainly on the MA
silks because of the requirement of large samples to obtain diffraction
patterns with X-ray crystallography. X-ray diffraction of MA silks has
revealed an ordered phase of anti-parallel β-sheet crystals embedded in a
softer, or `amorphous' phase (Warwicker,
1960). A more detailed analysis of the ordered phase has revealed
an inter-sheet spacing of 0.53–0.55 nm for several spider species within
the Araneus and Nephila genera
(Becker et al., 1994;
Grubb and Jelinski, 1997;
Sheu et al., 2004;
Warwicker, 1960;
Work, 1982). This spacing is
consistent with that found in β-sheet crystals of poly-alanine or alanine
alternated with glycine or serine (Fraser
and MacRae, 1973). Grubb and Jelinski
(Grubb and Jelinski, 1997)
reported the mean crystal dimensions to be 2 nmx5 nmx7 nm, a size
consistent with the scale of the poly-alanine blocks found in the fibroins
discovered to-date.
These crystals have been shown to be strongly aligned with the fibre axis
and to occupy about 10–15% of the total volume of the fibre
(Fornes et al., 1983;
Grubb and Jelinski, 1997;
Yang et al., 1997). These
conclusions have been subsequently confirmed by nuclear magnetic resonance
(NMR) (Simmons et al., 1996;
Kummerlen et al., 1996) and
Raman spectroscopy studies (Shoa et al., 1999; Edwards et al., 1995). The
study by Simmons et al. (Simmons et al.,
1996) found that in addition to the highly oriented β-sheet
crystals, a second population of alanine residues existed as weakly
orientated, unaggregated β-sheet. In fact, 40% of the alanine present
existed as highly oriented β-sheet while the remaining 60% existed as the
more poorly aggregated β-sheet, implying that the crystal volume fraction
is actually 20–25%, a value higher than that presented by X-ray
crystallography. X-ray diffraction of bundles of MA silk
(Grubb and Jelinski, 1997) and
of single fibres (Riekel et al.,
1999) has confirmed the presence of two populations of
β-sheets.
Since supercontracted Araneus MA silk behaves like a filled rubber
(Gosline et al., 1984), and
exhibits rubber-like elasticity, the theory of rubber elasticity can be used
to develop a model of fibroin network structure. The application of this model
requires two major assumptions: (1) that the individual fibroin molecules are
crosslinked to form a mechanically continuous fibroin network, and (2) the
fibroin network chains between crosslinks adopt an amorphous, kinetically
free, random-coil state. When this random-coil network is stretched, the
conformational entropy of the network chains falls, and this change in entropy
provides the rubber-like, entropic elastic mechanism. The modelling process is
based on the increasing stiffness of the network as the amorphous random-coil
network chains are straightened. This increase in stiffness associated with
straightening network chains of finite length is compared to the Gaussian
ideal of network chains of infinite length. The rise in stiffness of the
fibroin network from the Gaussian ideal reveals information as to the length
and stiffness of the network chains. Such analysis predicts network chains of
15–20 amino acids with a length of 8–10 amino acids required for
the chain to behave as a single `ideal' link in the random chain
(Gosline et al., 1994). Thus,
the model predicts network chains that are 10–15 amino acids shorter
than the elastin-like network chains found in the spidroin-2 fibroins
expressed in Araneus. However, the model predicts the length of
network chains based on the assumption that all bonds in the peptide backbone
have equal rotational freedom. If this is not the case, the model will
underestimate the number of amino acids in the network chains.
Using the same assumptions of the two-phase model, Termonia
(Termonia, 1994) created a
three-phase molecular model that accurately predicted the mechanical
properties of both wet and dry MA silk. This model has the added refinement
that the network chains leaving the crystals are constrained and have a higher
stiffness than that in the bulk of the network chains. This restriction on the
mobility of network chains may account for the difference in the length of
network chains seen in the Araneus fibroins and the length of network
chains predicted by the network model described above. The model prediction
that network chains are shorter than those indicated by sequence data
indicates that five to seven amino acids are required to relieve the
constraining effect of the crystals before the network chains are fully able
to adopt amorphous conformations. The actual sequence data for the Araneus
fibroins (Fig. 1) indicate that
proline occurs within two to five residues on either end of the poly-alanine
sequence blocks. Thus, the conformational `kink' imposed by proline should
disrupt the structure of the network chains as they emerge from the
poly-alanine β-sheet crystals. This will make more of the network chains
available to adopt amorphous conformations.
Although fibroin network models for Araneus MA silk assume
amorphous network chains, physical studies based on X-ray diffraction and NMR
have provided alternative hypotheses for the structure of the network chains
in other MA silks. In the case of Nephila, spidroin-1
(Fig. 1; Nc-MA-1) is
preferentially expressed, and the lack of proline means that the majority of
the network chains will be highly constrained as they exit the poly-alanine
β-sheet crystals, and this could facilitate the transition to stable
secondary structures. Thiel et al. (Thiel
et al., 1997) proposed a model for Nephila MA silk, which
stated that the poly-alanine regions would form the β-sheet crystals, and
the glycine-rich sequence blocks would also form a non-periodic crystal
lattice with varying degrees of perfection depending on their ability to
associate with their nearest neighbours. A helical conformation has also been
proposed as the stable secondary structure for the network chains, based on
NMR of Nephila MA silk (Kummerlen
et al., 1996). This study found the best fit to be axially
aligned, 31 helices stabilized by inter-chain hydrogen bonding. The
stiffness and high strength of MA silks was attributed to this inter-chain
hydrogen bonding. van Beek et al. (van
Beek et al., 2002) proposed a hierarchical model of silk structure
in which fibrillar substructures make a core that is covered by a hard skin,
or outer core. In turn the fibrillar substructures are composed of a network
in which β-sheet crystallites reinforce a network of 31
helices (van Beek et al.,
2002). An NMR study on dry, hydrated, restrained and
supercontracted Nephila MA silk also showed local structure in the
dry state, possibly 31 helices
(Eles and Michal, 2004a).
To date, fibroin network models have been based on mechanical data from
Araneus diadematus, and proposed secondary structures have been based
on X-ray or NMR of Nephila species. The presence of stable secondary
structure in Nephila means that current network models of
Araneus might not apply to Nephila. However, if the
secondary structure is not stable and `melts' with hydration, as suggested by
NMR data, a `latent' rubber-like mechanism may also exist in this silk
(Eles and Michal, 2004b). The
validity of a rubber-like model for Nephila silk would then depend on
the propensity of hydrated network chains to re-form the stable secondary
structures seen in the dry state, as the silk is extended. Mechanical tests on
hydrated Nephila silk may provide insight into the validity of a
rubber-like elastic mechanism.
Thus, Araneus and Nephila MA silks provide a system for testing the consequences of having a proline-deficient network versus a proline-rich network on the properties of MA silks. This is based on the ability of the network to form strong, stable hydrogen bonds depending on the propensity of the network chains to form secondary structures. Fig. 2 is a conceptual model depicting hypothetical distributions of bond energies, or bond strengths, of the hydrogen bonds contained within either proline-rich (+Pro) or proline-deficient (–Pro) networks. In both cases the network is held together by poly-alanine β-sheet crystal crosslinks. The regular repeating pattern within the poly-alanine crystals provides the ideal bond length and angles for hydrogen bonding, and consequently the hydrogen bonds are strong and stable, even in the presence of a polar solvent such as water. Conversely, the peak representing the distribution of bond energies within the proline-rich network chains is broader and is shifted left to lower energy levels. This indicates that, while extended during silk spinning, the network chains are essentially random-coils that have been aligned to the fibre axis and stabilized by hydrogen bonds, and are therefore well below their glass transition temperature, Tg. Since there is no particular secondary structure to these straightened random-coils, the hydrogen bonds are not necessarily at an ideal length or angle and thus are at lower energy levels than those in the stable, regular poly-alanine crystals.
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The distribution of bond energies within the proline-deficient network is represented by a bimodal distribution. This means that even in the proline-deficient network, not all the chains have a secondary structure as regular and as stable as the poly-alanine β-sheet crystals. A small population of hydrogen bonds is at the bond energy associated with the proline-rich network, but the majority of bonds are associated with the secondary structures proposed to exist within the proline-deficient network chains of Nephila silk. These hydrogen bonds are at higher energy levels and fall somewhere between that proline-rich peak and the peak representing the poly-alanine crystals. Of course, exactly where the proline-deficient peak falls between the proline-rich peak and the poly-alanine peak is unknown. If the distributions of hydrogen bond strength were similar between the proline-rich and proline-deficient networks, as is depicted in Fig. 2A, then we would expect the dry mechanics of Araneus and Nephila MA silks to be similar. However, if the secondary structures associated with the proline-deficient network chains are particularly stable, there may be a significant difference in the bond strength between proline-rich and proline-deficient networks (Fig. 2B). If this were the case, then we would expect a measurable difference in the mechanical properties of dry MA silks.
Dry MA silks can be considered as composite-like materials consisting of strong, stiff poly-alanine crystal crosslinks that reinforce a softer, glycine-rich matrix. The initial modulus of the silk is a result of the crystals and the matrix acting in series. If the matrix is stiffer because of the strength of the stabilizing hydrogen bonds, then the initial modulus will be higher. The yield points represent the strain at which stress is sufficiently high to break the hydrogen bonds, allowing the network chains to be extended. Thus, if there were significant differences in the bond energy distributions between proline-rich and proline-deficient networks, we would specifically predict measurable differences in the yield points and in the initial modulus.
Fig. 2C shows the effect of
hydration on hydrogen bond energies within MA silk networks. In both cases, it
is known that the poly-alanine β-sheet crystals are relatively unaffected
by water (Work, 1982), and so
this peak remains well away from kT (the energy associated with Brownian
motion) upon hydration. Of course, it is unknown if the poly-alanine
β-sheet crystals are completely unaffected by water, or if the crystals
soften, becoming less rigid upon supercontraction. In
Fig. 2C we have chosen,
somewhat arbitrarily, to shift the poly-alanine peak to a lower stability upon
supercontraction, but it remains well away from kT, as these crystals clearly
do not `melt' in water. Based on thermoelastic experiments on hydrated
Araneus MA silk (Gosline et al.,
1984), we know that a hydrated, proline-rich network becomes
rubber-like, exhibiting entropic elasticity. Consequently, the proline-rich
peak has been shifted to a lower energy, at or near kT. Thus, the hydrogen
bonds within the network chains are easily broken and reformed, and the
network chains behave, on average, as kinetically free, random-coils.
Likewise, one would expect the same shift in bond energy for those portions of
the proline-deficient network not contained within stable secondary structure.
However, it is not clear where on this spectrum the peak representing
proline-deficient secondary structure will fall. Yet, if the peak falls
anywhere on the scale above kT, as indicated in
Fig. 2C, then even if hydrated,
the proline-deficient network chains will maintain some secondary structure.
If this is the case, then we expect there to be measurable differences in the
mechanical properties of the hydrated MA silks. Specifically, the
proline-deficient network (Nephila) will not shorten and swell as
much as the proline-rich MA silk (Araneus). In addition, the hydrated
Nephila MA silk will remain stiffer and contain more ordered
structures as indicated by birefringence measurements.
Studies on spider silks are difficult because of the small diameter of the fibres, and some variability exists in the literature depending on the criteria used to define diameters by individual researchers. This study quantifies supercontraction properties, wet and dry mechanical data, and wet and dry birefringence values for Araneus and Nephila MA silk. The same researcher using the same criteria for difficult measurements such as diameter and birefringence produced the data provided here. Based on the physical data reviewed above for Araneus and Nephila MA silk, it is hypothesized that there will be significant differences in the mechanical and optical properties of these two silks. The results clearly confirm this prediction.
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MATERIALS AND METHODS |
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), were fed and
given water regularly, and were producing normal webs. Silk was collected by
allowing the spiders to roam freely over a level, black surface. The MA silk
left behind as a safety line was carefully collected onto paper frames cut
from 10 cmx15 cm recipe cards so that it remained at its natural length.
Each sample used in the experiments was from a different individual
spider.
Supercontraction properties
When submersed in water, MA silk supercontracts, shrinking in length and
increasing in cross sectional area. Length contraction was measured by placing
an approximately 3 cm long piece of dry silk fibre on a moveable micrometer
mount described below. The micrometer was moved under x50 magnification
until the slack in the fibre was just taken up. Water was then added and the
micrometer was moved until the slack in the wetted fibre was just taken up.
The wet to dry ratio was the ratio of the wet and dry lengths as measured to
the nearest 0.05 mm by a pair of vernier calipers.
The ratio of wet to dry diameter was measured under a x100 oil immersion lens on a Leitz Orthoplan polarizing microscope using a x15 filar micrometer eyepiece, for a total magnification of x1500. The system was calibrated using a 10 µm calibration slide (Bausch and Lomb, Rochester, NY, USA). Dry fibres were mounted with the slack taken up on a slide, placed in immersion oil, covered with a cover slip and observed through immersion oil. For supercontracted diameters, fibres were mounted as dry fibres then wetted, and one end was cut allowing the fibre to recoil. The fibre was then covered with a cover slip and observed through immersion oil.
Mechanical testing
The mechanics of dry dragline silk were tested on an Instron (Norwood, MA,
USA) universal testing machine (model 5500) with a custom built load cell that
could be used at a sensitivity of 1 g force full-scale. Silk was fastened to a
13 cmx26 cm card with the centre cut out to produce a frame. The
specimen was fastened to the frame by Scotch 810D `Magic Tape' and the free
ends were glued down with Devcon five minute epoxy. Once the card was fastened
to the Instron by means of two screw-tightened clips, the sides of the frame
were cut away. The crosshead was moved to adjust the length so the silk was
just slack. The crosshead speed was 10 mm min–1, which for
samples of approximately 5 cm gave a strain rate of approximately 20% per
minute (0.003 s–1).
Supercontracted silk was stretched under equilibrium conditions on a
microscope-based, glass micro-beam test apparatus, which has been previously
described (Gosline et al.,
1995; Fudge et al.,
2003; Savage et al.,
2004). A video dimension analyzer (VDA) tracked the movement of
the glass beam, and this movement was translated into force. In order to
obtain stress, the cross-sectional area of the silk was determined on the test
frame by measuring the diameter of the silk using a x20, polarizing
objective and a x15 filar micrometer eyepiece for a total magnification
of x300.
Material properties were calculated by converting force into engineering
stress (
) based on initial cross-sectional area
(=F/A0; where F is force and
A is the initial cross-sectional area of the silk fibre), and
deformation was converted into engineering strain
(
=
L/L0; where L is
the change in fibre length and L0 is the initial fibres
length). In all cases, the initial dimensions (A0 and
L0) correspond to the dimensions of the silk samples in
their dry or wet states, as appropriate.
Birefringence measurements
Birefringence measurements were taken on dry, wetted but restrained, and
supercontracted dragline fibres using a Wild M-21 polarizing microscope. Dry
fibres were mounted in oil and measured with a x40 objective, a
x1.25 extension, and a x15 filar micrometer eyepiece for a total
magnification of x750. Wet, restrained fibres were measured with the
same magnification setup but under water.
The birefringence of supercontracted dragline was measured when at slack
length and at two to four percent increments of strain. The apparatus used for
strain measurements of supercontracted fibres was similar to that used for
mechanical tests, with the exception of a stationary mount instead of the
glass micro-beam. The thickness of this device prevented the use of high
magnification. As a consequence diameter measurements were taken at a
magnification of x300. All birefringence measurements were taken using a
Wild Senarmont 546 nm compensator with a 546 nm interference filter. The
specimen was placed at 45° to crossed polarizers (point of maximum
brightness), and the analyzer was rotated until the specimen reached
extinction. The retardation, 
, caused by the sample is equal to 3.03 nm
multiplied by the analyzer angle in degrees. The path length was measured as
the diameter, d, of the fibre taken at or near the location of the
birefringence reading, and the birefringence,
B=/d.
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RESULTS |
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f, of about 1 GPa, which is consistent with values published
for Araneus sericatus (Denny,
1976). Neither the mean Eo nor mean
f were statistically different based on a Student's
t-test (Eo: t-statistic= –1.199,
P=0.238; f: t-statistic=0.445,
P=0.659). The yield stress for Araneus and Nephila
MA silks were y=157 MPa and y=174 MPa,
respectively, and they were not statistically different (Mann–Whitney
rank sum test, T=209, P=0.1). The yield strain for
Araneus and Nephila MA silks were y=0.017
and 0.019, respectively, and were not statistically different
(t-statistic=–1.170, P=0.251).
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The failure strain of Nephila MA silk is 0.27, similar to that
published previously for Nephila
(Cunnliff et al., 1994) and
also similar to the failure strain for Araneus sericatus
(Denny, 1976). The failure
strain of Araneus MA silk was f=0.23, which is the
same as the value reported for Araneus gemmoides
(Stauffer et al., 1994) and
within the range of values reported for Araneus sericatus
(Denny, 1976). However, the
failure strain of Nephila MA silk was statistically higher than the
failure strain of Araneus MA silk (t-statistic=–3.607,
P=0.001), although the difference is quite small.
Hydrated mechanics
Table 2 summarizes the
parameters of supercontraction for Araneus and Nephila MA
silk. Araneus contracts in length by about 50% and increases in
cross-sectional area by almost a factor of 5, for a total volume-swelling
ratio from wet to dry of 2.42. This is higher than the swelling of
Nephila MA silk, which shrinks in length by 34% and increases in
cross-sectional area by 2.56 times, for a total volume-swelling ratio of 1.69.
Our values for Araneus MA silk can be compared to data acquired by
Work (Work, 1977), who
recorded a wet to dry length ratio of 0.55 and a wet to dry cross-sectional
area ratio of 3.90 for dragline silk samples. Our length contraction ratio is
quite similar, but our cross-sectional area ratio is about 25% higher. This
seems acceptable given the difficulty in obtaining diameter measurements from
such small fibres.
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The mechanical properties of supercontracted MA silks are shown in Figs 4, 5 and 6. Fig. 4A shows individual test results from 12 samples of Araneus MA silk, and Fig. 5A shows individual test results from 24 samples of Nephila MA silk. Since the data exhibit a high degree of variation, an averaged curve has been generated for each data set and is shown in Fig. 4B and Fig. 5B for Araneus and Nephila, respectively. Each individual test in the Araneus data set was fitted to a second or third order polynomial, and the Nephila data were fitted to either a third or fourth order polynomial. The equations were solved at 5% increments as explained below. Since the individual samples were not tested to failure, but rather to the largest strain allowed by the experimental setup, each equation was solved only to the largest strain achieved for that respective test. The predicted values at each 5% increment were averaged from every curve for which a value was available and plotted with standard deviations. For the purposes of direct comparison, the data for Araneus and Nephila are shown together in Fig. 6. There is a large degree of variation within both data sets; however, there is clearly minimal overlap between the two data sets, indicating that supercontracted Nephila MA silk is initially much stiffer than Araneus silk.
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Fig. 7 shows how the stiffness of Araneus and Nephila MA silk increases with strain, and these lines are obtained by taking the first derivative of the regression lines in Fig. 4B and Fig. 5B. The data derived from the regression analysis for Araneus MA silk exhibited a nearly tenfold increase in the stiffness by a strain of 0.1. Since none of the individual tests (Fig. 4A) exhibited such a large increase in stiffness at low strains, it was assumed that the average MA silk properties derived from the regression analysis did not accurately portray the silk behaviour at low strains. Thus, in Fig. 7, the modulus data are plotted between the strains of 0.1 and 0.65. In order to clarify the shape of the stiffness curves in Fig. 7, the average of initial modulus values taken from the individual mechanical tests are also plotted for Araneus and Nephila MA silk at zero strain, with error bars that indicate one standard error of the mean. Fig. 7B plots the data from Fig. 7A on a log scale and clearly shows the difference in stiffness between Nephila and Araneus silks with increasing extension.
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Birefringence
Birefringence data is shown in Figs
8,
9,
10 and
Table 3.
Table 3 shows the effect of
hydration on the birefringence of silk fibres. Birefringence values are
clearly higher for Nephila in all experiments: dry,
wetted/restrained, supercontracted and supercontracted with strain. Values for
Araneus are generally lower than those reported by Work
(Work, 1977); however, the
trends are the same, birefringence being highest for dry fibres and lowest for
supercontracted fibres. In each case, for Araneus and
Nephila MA silk the trends in birefringence shown in
Table 3 are the same; however,
the magnitude of the changes from dry to supercontracted are larger for the
Araneus MA silk. The birefringence of both silks falls by roughly
10–15% when hydrated but restrained; however, when supercontracted the
birefringence falls by a factor of four in Araneus but only by a
factor of about two in Nephila.
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This difference is reflected in the birefringence-strain behaviour of these two silks. Fig. 8A shows individual test results from nine samples of hydrated Araneus MA silk. The individual samples show a high degree of variability; however, the average initial slope of the nine birefringence-strain tests is 0.0122. Fig. 9A shows individual test results from seven tests of hydrated Nephila MA silk. Again, the individual tests show a high degree of variability, but the average slope of all the tests is 0.0456. Thus, Nephila MA silk has approximately three times the birefringence of Araneus silk when just slack, and in addition, the birefringence readings increase with increasing strain, and the slope is four times higher in Nephila than is in Araneus. Because of the high degree of variability in both data sets, an averaged curve has been generated as above (using second or third order polynomials) for both Araneus and Nephila (Fig. 8B and Fig. 9B, respectively). For the purposes of direct comparison, the data for Araneus and Nephila are shown together in Fig. 10, which clearly shows that there is no overlap between the two data sets.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Thus,
it is tempting to conclude from the dry mechanical properties that there is no
difference in the strength of the hydrogen bonds that stabilize the network
chains in these two dry MA silks.
It is unwise, however, to jump to this conclusion, because the exceptional
variability of spider silks may simply have masked real differences that might
exist if more uniform silk samples were tested. We now know that spiders have
a friction brake, located within the spinning apparatus, that they can use to
apply a force to the silk threads as they are drawn from the spinneret.
Spiders can control this force to levels that range from 0.1 to 4 times the
spiders' body weight, and at its highest levels this force can create stresses
in excess of 50% of the silk's tensile strength
(Ortlepp and Gosline, 2004).
This friction brake, therefore, will have important implications for the
amount of draw-induced alignment within the MA silk network and hence the
properties of the silks formed. Indeed, Perez-Rigueiro et al.
(Perez-Rigueiro et al., 2005)
were able to measure the silking forces and the tensile properties of
individual MA silk fibres, and they were able to show a strong correlation
between the silking stress during spinning and the tensile properties of that
silk. It was found that as the silking stress increased, the silk fibres
became both stiffer and less extensible. This study also quantified a range of
properties present in silk naturally spun by the spiders; presumably the large
variability seen in naturally spun silk is a result of the spiders' ability to
vary the force on the silk as it is produced. Since the silk samples used in
this study were obtained from the draglines left by freely walking spiders, we
do not know if the samples used were spun under the same conditions. Thus, it
is possible that the lack of difference in dry mechanical properties is simply
due to the variability of the samples used, which means that the dry data may
not be sufficient to determine if there are differences in hydrogen bonding in
the network chains of these two silks. The birefringence data for the dry
silks may, however, provide a better indication of this difference.
Dry Nephila silk has roughly 50% higher birefringence than dry Araneus MA silk (Table 3, P<0.001), and since both silks have similar amounts of poly-alanine, β-sheet crystals, it is likely that there are real differences in the network structure of these two silks. Apparently the regular repeating pattern of bond angles within the protein backbone and the regular pattern of hydrogen bonding that can occur in the stable secondary structures of the network chains in Nephila MA silk create a molecular alignment that is greater than that created by the extension of the amorphous chains in Araneus. So it is still possible that there are significant differences in the strength of hydrogen bonding between these two silks in the dry state, even if sample variability prevented us from observing this in the dry mechanical tests. The supercontraction behaviour may help resolve this issue.
If the secondary structures in the proline-deficient network of Nephila MA silk do not `melt' in water, it is an indication that water is not able to penetrate and disrupt the hydrogen bonds that stabilize these structures. Alternatively, if the hydrogen bonds in the proline-rich network of Araneus MA silk are disrupted by water, then these bonds must be weaker than those in Nephila silk, and the transition to the supercontracted state might be more dramatic for Araneus than Nephila silk. Our results show that this is indeed the case. Araneus MA silk swells to a much larger volume than Nephila silk, indicating that water is more able to penetrate the network of the Araneus silk. In addition, Araneus MA silk contracts to a shorter relative length upon hydration, suggesting that more of its structure `melts' in water (Table 2). Presumably, the greater swelling and contraction seen for Araneus silk is due to the disruption of the hydrogen bonds that stabilize the extended glass structure of the network chains in Araneus silk, which collapse from the drawn state into an amorphous network when hydrated. Nephila apparently maintains some structure that does not `melt', and the 34% decrease in length (Table 2) is probably due to the reorientation of large-scale structures. Thus, there appear to be clear differences in the stability of the hydrogen bonds that exist in the network chains of these two silks in their dry state. Comparison of the changes in birefringence with supercontraction provides additional insights into this process.
The data in Table 3 indicate
that the birefringence of Nephila MA silk falls by about 50% when the
silk is supercontracted in water, whereas Araneus MA silk's
birefringence falls by 75%. As a result, the difference in birefringence of
these two silks in their supercontracted states is approximately threefold. We
interpret these changes as follows. Because the poly-alanine, β-sheet
crystals in MA silks do not melt when these silks supercontract in water
(Fornes et al., 1983), we know
that the changes in birefringence arise from changes in the state of the
network chains with hydration, and from the re-orientation of the β-sheet
crystals and network chains with contraction. In Nephila silk, the
network chains retain their ordered structure, and the modest fall in
birefringence is due to the partial reorientation of stable secondary
structures. In Araneus silk, the dramatic drop in birefringence
reflects the melting of extended, amorphous network chains, which allows a
reorientation of the β-sheet crystals and also allows the network chains
to relax to an essentially isotropic state. These dramatic changes in network
structure with supercontraction must have important consequences, and the
mechanical tests clearly reveal dramatic differences in material
properties.
Fig. 6 clearly shows that there is essentially no overlap in values between the two populations of hydrated silk fibres. Thus, in spite of the enormous variability of the silk samples (Figs 4 and 5), the difference in the hydration-induced changes of hydrogen bonding within the network chains is so large that the two silks become entirely different in their mechanical properties. The initial modulus of Nephila MA silk dropped by a factor of 113 when supercontracted, but that of Araneus MA silk fell by a factor of 1010. This eightfold difference in stiffness change between Nephila and Araneus implies stiffer, more ordered network chains in Nephila MA silk, and any of the proposed secondary structures could account for this difference. Proline is a known β-sheet breaker and could act to disrupt the formation of crystalline structure in the network, and Araneus MA silk, with its high proline content, should thus have a more amorphous network. On contact with water, the Araneus MA silk would swell to a greater degree, and the modulus would be lower once supercontracted because the network chains become kinetically free.
Fig. 7A clearly shows how stiffness varies with strain for both silks. Nephila MA silk retains a higher fraction of its dry stiffness, whereas the stiffness of Araneus MA silk increases much more with strain. The log stiffness versus strain plot shown in Fig. 7B more clearly documents these changes in stiffness. Initially, hydrated Nephila silk is 10 times stiffer than hydrated Araneus MA silk. At a strain of 0.65, the stiffness of the Araneus silk has increased, and the difference in stiffness between Nephila and Araneus is reduced to 1.66. The fact that Nephila MA silk retains more of its initial dry stiffness indicates the presence of a more ordered fibroin network, and provides evidence for the existence of ordered structure in the glycine-rich network chains. The rise in stiffness in Nephila silk is probably due primarily to the reorientation of both the poly-alanine crystals and the ordered structures within the network chains.
Araneus MA silk exhibits a much more pronounced drop in stiffness upon hydration, and the low stiffness seen at lower strains likely reflects the fact that the network chains are kinetically free. As the network chains are oriented with strain, the stiffness of the Araneus fibre rapidly approaches that of the Nephila silk, probably because of the non-Gaussian behaviour of the glycine-proline-rich network chains. It is, therefore, expected that at a strain of this level the Araneus silk may still be dominated by entropic elasticity.
The differences in network structure indicated by the mechanical properties
of the supercontracted silks are also reflected by the change of birefringence
with strain (Figs 8,
9,
10). Supercontracted
Nephila MA silk retains more molecular order than does
Araneus, and therefore it was hypothesized that the birefringence of
the Nephila silk would be both higher and exhibit a higher increase
with strain than the Araneus silk. Indeed, this was found to be the
case; the initial hydrated birefringence of Nephila MA silk was three
times higher than that of Araneus
(Table 3). In addition, while
there was a high degree of variability in the birefringence-strain data,
Fig. 10 clearly shows that
there was no overlap between the two data sets. The rapid rise in
birefringence with extension seen in Nephila MA silk may be due to
existing structures in ordered network chains re-orientating with strain. In
the case of Araneus, the smaller rise in birefringence is likely due
to the amorphous network chains unwinding with strain. In fact, similar
birefringence-strain data has previously been published in a study that
attempted to attribute changes in birefringence to aspects of a two-phase
model for a silk network (Gosline et al.,
1995). In that study, the predicted birefringence for a random
polymer network in which network chains occupy 88% of the network volume was
calculated, and subtracted from the measured values of birefringence. The
result suggests that the poly-alanine crystals re-orientate with the first 30%
of strain, after which they are essentially fully oriented to the fibre
axis.
There are two basic models for the structure of the glycine-rich network
chains: (1) the network chains are kinetically-free, random-coils, and they
sample many possible conformations over a small time scale; (2) the network
chains form stable secondary structures, which have been proposed to be
non-periodic lattice crystals, 31 helices, or β-sheets. The
first model is based on the fact that supercontracted Araneus MA
silks are entropic (Gosline et al.,
1984), and their properties can be predicted from the theory of
rubber elasticity. The second model is based on NMR and X-ray data that have
been used to propose non-periodic lattice crystals and/or 31
helices as possible stable secondary structures. In this study we have shown
that in the dry state, the properties of both Araneus and
Nephila MA silks are dominated by the stabilizing effect of inter and
intra-chain hydrogen bonds, both within the poly-alanine β-sheets and
within the glycine-rich network chains. These hydrogen bonded structures are
responsible for the high initial stiffness (10 GPa) of both silks. However,
once hydrated, MA silks swell, shrinking in length and increasing in volume in
a process termed supercontraction. This phenomenon is associated with a
dramatic drop in the initial modulus for both silks, but the results discussed
above provide strong evidence that there are dramatic differences in the
nature and extent of the structural organization of the glycine-rich network
chains of these two silks. These differences are almost certainly associated
with differences in the strength of the hydrogen bonds that form in the
proline-deficient and proline-rich network chains.
In Fig. 2C we presented a hypothetical distribution of hydrogen-bond energy, or bond strength that we created to illustrate our hypotheses for the state of the proline-rich (+Pro) or proline-deficient (–Pro) silk networks, both in the dry and in the hydrated states. The key feature of our model was that the peak representing the distribution of hydrogen bond energies of proline-rich network chains is shifted left to lower energies and is much broader than the distribution of hydrogen bond energies of proline-deficient network chains. These bond energy distributions represent our hypothesis that the proline-rich network chains are essentially random-coils, and that the proline-deficient sequences form stable secondary structures. The figure shows distributions for these hydrogen-bonds for silk fibres in water, and the key features were that the energy distribution for the proline-rich network chains shift to much lower energies, such that thermal energy, kT, is sufficient to induce extensive mobility, whereas the distribution for proline-deficient network chains shift to lower energies but not low enough to induce wide-spread mobility. The mechanical and optical data presented in this study are entirely consistent with this hypothetical energy distribution, and it likely, therefore, forms a useful starting basis for understanding the structure and dynamics of the protein networks in these two types of MA silk. However, in spite of the fact that there is a good fit of our data to this bond energy scheme, it is clear that the relationship is entirely qualitative and much remains to be learned about the structure and dynamics of spider silk networks. In particular, the mechanical and optical properties of intact fibres presented in this paper measure the average properties, but do not take into account any hierarchal substructures within silk fibres.
Indeed, using a combination of light microscopy and scanning electron
microscopy, Vollrath et al. (Vollrath et
al., 1996) observed that Nephila clavipes MA silk has a
distinct skin–core structure when supercontracted in urea. van Beek et
al. (van Beek et al., 2002)
further refined the idea of a skin–core structure based on NMR data of
Nephila edulis MA silk. They proposed that this silk is composed of
axially aligned fibrillar substructures covered by a hard skin; each fibril is
composed of a matrix of 31-helices crosslinked by poly-alanine
β-sheet crystals. Sponner et al.
(Sponner et al., 2005) stained
cross-sections of Nephila clavipes MA silk fibres with antibodies to
both spidroin-1 and spidroin-2, and immuno-electron microscopy confirmed the
presence of a skin–core structure. The presence of a mixture of
spidroin-1 and 2 seems to promote a hierarchical structure in MA silk on a
larger scale than the nano-scale composite of β-sheet crystals and
network chains seen by X-ray or NMR studies. This skin–core structure is
composed of a thin skin, roughly 0.5 µm thick of spidroin-1, and a core
region containing both spidroin-1 and 2. The spinning process apparently
induces a polymer phase separation that aggregates spidroin-2 and promotes the
formation of fibrils (200 nm diameter) within the core. To date, similar
structural studies using X-ray crystallography, NMR and immuno-staining have
not been done on Araneus MA silk, in which only spidroin-2 fibroins
are expressed. It remains to be seen if such studies on proline-rich silks
will yield additional insights into the relationship between the molecular
structure of the polymer network, hierarchical skin–core structures, and
the material properties of MA silks.
Conclusions
Araneus MA silk contains essentially amorphous network chains,
which become highly mobile when hydrated. It exhibits rubber-like elasticity
(Gosline et al., 1984), and
random-network models based on rubber elasticity appear to accurately portray
the behaviour of this silk. Thus, the decrease in conformational entropy that
occurs by stretching a hydrated Araneus MA silk fibre provides the
energy necessary to drive the elastic recoil when the stretching force is
removed. Conversely, random-network models do not appear to provide an
appropriate model for the network chains in Nephila MA silk.
Comparison of the mechanical and optical properties of hydrated
Nephila and Araneus MA silk clearly indicate that the
network chains in Nephila silk are quite stiff and ordered in both
the dry and hydrated states. Thus with the presence of stable secondary
structures in the hydrated Nephila MA silk network, the elastic
mechanism in this silk is likely to be dominated by changes in bond energy
associated with the deformation of rigid or semi-rigid structures. Thus, our
results indicate that these two silks have fundamentally different network
structures, and if this is correct, then a study to examine the thermodynamics
of their elasticity should clearly reveal these differences. Thermoelastic
experiments to determine the nature of their elastic mechanism are presented
in another paper (Savage and Gosline,
2008).
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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