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First published online May 30, 2008
Journal of Experimental Biology 211, 1948-1957 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014225
The role of proline in the elastic mechanism of hydrated spider silks
Department of Zoology, 6270 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada, V6K 1Z4
* Author for corresponding (e-mail: gosline{at}zoology.ubc.ca)
Accepted 5 April 2008
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), and this observation provided the basis for the
proposal that the fibroin network structure of Araneus MA silk
contains amorphous network chains that become fully mobile when hydrated. A
computer model based on this assumption appears to accurately predict the
properties of both dry and hydrated MA silk
(Termonia, 1994). In addition,
rubber-like elasticity appears to be a suitable model for other hydrated
elastomeric proteins such as resilin and elastin
(Weis-Fogh, 1961;
Dorrington and McCrum, 1977).
However, physical characterization of Nephila clavipes (Linnaeus
1767) MA silk indicates that the network chains of this silk are not amorphous
but contain some axially aligned, stable, secondary structures
(Kümmerlen et al., 1996;
Thiel et al., 1997;
van Beek et al., 2002). Thus,
there appears to be a discrepancy between fibroin network models that predict
amorphous network chains and studies based on X-ray and NMR that predict
stable secondary structure. It is worth noting that the amorphous network
models have all been applied to materials that are rich in the amino acid
proline, whereas physical studies identifying the presence of secondary
structure have been based on Nephila silk, which contains little
proline. Sequencing efforts have revealed a domain architecture to MA silk fibroins that resembles a block copolymer. Each fibroin contains multiples of a basic repeat sequence that contains two blocks, a poly-alanine block that is 8–10 amino acids long, followed by a block of 20–30 amino acids that is rich in glycine. Combined with data from a variety of physical studies, a basic MA silk structure is accepted in which the MA fibroin network consists of a `soft' matrix of glycine-rich network chains that are crosslinked and reinforced by alanine-rich, β-sheet crystals. Although the structure of the poly-alanine crystals is well understood, the structure of the network chains is not so clear.
The poly-alanine, crystal-forming blocks are similar in all the araneoid MA
fibroins sequenced thus far, but these fibroins have been grouped as either
spidroin-1 or spidroin-2 based largely on differences in types and
arrangements of amino acids within the glycine-rich network chains
(Gatesy et al., 2001). Perhaps
the most striking difference between the two fibroin groups is the absence of
proline in spidroin-1 fibroins and the abundance of proline in spidroin-2
fibroins. Since Nephila MA silk contains little proline, spidroin-1
is preferentially expressed in the MA glands of Nephila. Conversely,
Araneus MA glands express two spidroin-2 fibroins and contain about
16% proline (Guerette et al.,
1996). It is the differential expression of these two fibroin
types in Nephila and Araneus that may explain the
differences observed in the structure of the glycine-rich network chains in
the MA silks of these spiders. That is, because proline is known to disrupt
secondary structures in proteins, differences in proline content may be
responsible for the observed differences in the structure of the glycine-rich
network chains. Indeed, Rauscher et al.
(Rauscher et al., 2006)
analysed the proline and glycine levels across a range of elastin-like and
amyloidic peptides and found that peptides above a well defined threshold of
glycine and proline content form amorphous, highly hydrated and kinetically
mobile (i.e. elastin-like) aggregates, whereas peptides falling below the
threshold formed amyloid-like structures with significant β-sheet
content. For a given level of glycine, there is a threshold level of proline
that provides elastomeric properties, but below the threshold a protein is
rigid and amyloid-like. Spidroin-1 and spidroin-2 network chains have similar
levels of glycine (
45% and 40%, respectively), but they differ strongly
in proline content (0% and 15–18%, respectively). As a consequence, the
spidroin-2 chains lie well above the threshold, along with the elastomeric
proteins elastin, resilin and abductin, whereas is spidroin-1 chains fall
below the threshold, along with known amyloidogenic sequences.
The accompanying paper (Savage and
Gosline, 2008) clearly documents differences in the mechanical and
optical properties of proline-rich Araneus MA silk and the
proline-deficient Nephila MA silk in their hydrated states. That is,
Nephila MA silk contains a more ordered fibroin network than does
Araneus MA silk, and when hydrated, Nephila silk remains
stiffer and more highly birefringent because the network chains retain much of
their hydrogen-bonded structure. It was hypothesized that this difference in
network structure would mean that Nephila and Araneus MA
silks will exhibit inherently different elastic mechanisms in their hydrated
states, and this hypothesis is tested explicitly in the current study.
Specifically we test the hypothesis that proline-rich silks will be
elastin-like and will exhibit rubber-like elasticity, based on changes in the
conformational entropy of kinetically free protein chains with extension,
whereas proline-deficient silks will have an elastic mechanism consistent with
a crystalline material and hence exhibit bond-energy elasticity.
Unlike the MA silks sequenced so far, the crystal crosslinks in FL silk
have not been clearly identified, although sequence data for FL silks from
several spider species indicate that FL fibroins also have a block copolymer
structure. Each FL fibroin consists of a repeated `spacer' block and a longer
glycine-rich `network-chain' block (Gatesy
et al., 2001). The glycine-rich sequences are similar to those
found in the spidroin-2 MA fibroins, but the presumed network chains in FL
fibroins are 5–15 times longer than those in MA fibroins. However, the
glycine–proline content of FL fibroins is essentially identical to that
of the spidroin-2 MA fibroins, which we hypothesize will exhibit
conformational entropy elasticity. Thus, we hypothesize that hydrated FL silks
will also exhibit conformational entropy elasticity.
In this study we use thermoelastic experiments to test these hypotheses. Our results indicate that proline-rich MA and FL silks from Araneus diadematus exhibit rubber-like, conformational-entropy elasticity, although in the MA silk bond-energy elasticity becomes important as extensions approach 50%. The proline-deficient MA silk from Nephila clavipes exhibits a small component of entropic elasticity at small extensions, but at 10% extension and above the material shows essentially 100% bond-energy elasticity.
Thermoelasticity
The work done to stretch an elastic body is stored as a change in internal
energy (E) and as a change in entropy (S). It as been shown
that, at constant temperature (T), volume (V) and
composition (n), the retractive force developed when an elastic
material is stretched is given as (Flory,
1953; Treloar,
1975):
 | (1) |
S/L)T,V,n=–(F/T)L,V,n
(Flory, 1953), so that
Eqn 1 becomes:
 | (2) |
Rubbery materials are commonly associated with low stiffness and high extensibility, but these attributes do not necessarily classify a material as being a rubber. A rubber must meet two distinguishing criteria: (1) the elastic force in a rubber is due primarily to changes in the conformational entropy of kinetically free polymer chains caused by extension, which means that the force on a rubber at a fixed extension will increase linearly with increasing temperature; (2) the internal energy change associated with bond deformation should contribute to a small fraction of the total force and be nearly constant over a large range of extension and temperature.
However, thermoelastic tests can only be used to determine the changes in
conformational entropy and bond-energy if the tests are carried out at
constant volume and molar composition, as indicated by the subscripts in
Eqn 2. This is because in an open
system, there are other processes such as thermal expansion and
polymer–solvent mixing that can contribute to changes in entropy and to
changes in internal energy. Even in a closed system, where the material cannot
absorb solvent, it is not possible in practice, to perform a test at constant
volume due to the thermal expansion of the material, and so the material is
tested at constant pressure, P, but the force–temperature slope
is corrected for thermal expansion by measuring the force at constant
extension ratio (
). The retractive force, F, becomes
(Flory, 1953):
 | (3) |
F, meeting criterion 1, and
Fe0, meeting criterion 2.
Normalizing Eqn 3 to the
force, F, yields:
 | (4) |
 | (5) |
). In practice, the swelling effect of water on the sample can
vary with temperature, in which case it may not be possible to measure force
at either constant volume or constant composition. It is, however, possible to
measure force, F as it varies with temperature, T, at constant
length, L, and pressure, P, under equilibrium swelling
conditions (eq) (Flory, 1953):
 | (6) |
H is the total enthalpy change of the system,
including changes in enthalpy associated with mixing of solvent and network
chains. Swelling changes, measured as a function of temperature, can be used
to correct
(H/L)T,P,eq,
giving the bond energy component of elastic force,
(E/L)T,V,n.
Where swelling changes with temperature are small,
(H/L)T,P,eq(E/L)T,V,n
and no correction is required. Both resilin
(Weis-Fogh, 1961) and abductin
(Alexander, 1966) show small
changes in swelling with temperature, and thermoelastic experiments yields
results remarkably similar to those described above for lightly crosslinked
rubber. In these cases,
(H/L)T,P,eq
is small relative to the entropic component,
(F/T)T,V,n.
Thus, elastic energy for these two proteins is stored largely as changes in
conformational entropy. Water-swollen elastin, however, exhibits very large
changes in swelling with temperature, and so
(H/L)T,P,eq=(E/L)T,V,n
(Gosline, 1980) because there
are large enthalpy changes associated with changes in solvent–polymer
interactions. When the thermoelastic data are corrected for the
temperature-dependent swelling,
(E/L)T,V,n
is close to zero over a 20% range in extension
(Dorrington and McCrum, 1977).
Thus, elastin also appears to meet the criteria for rubber elasticity. Similar
results were obtained with octopus arterial elastomer
(Shadwick and Gosline, 1985),
and the methods from these two studies have been used in the current
thermoelastic analysis of hydrated spider silks, as described below.
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; Fudge et al.,
2003; Savage et al.,
2004). For these experiments, a sample chamber was constructed on
a large microscope slide (50 mmx75 mm) that was placed on a
temperature-controlled stage with similar dimensions. This assembly was
mounted onto a 3 mm thick aluminium plate with plastic legs, which provided
thermal isolation, and the plate was clamped to the stage of a Wild M-21
microscope at the very outer edge of the microscope stage. Silk samples were
mounted in the sample chamber between a glass beam and a moveable micrometer
mount, as above, and a second, reference glass beam mounted adjacent to the
first was used to help control for vibrations during thermoelastic
experiments. In addition, the reference beam could be used to track the
thermal expansion of the temperature-control stage and attached chamber. The
sample chamber was closed at the top with a large microscope coverslip, which
provided an optically flat surface for tracking the deflection of the glass
beams. The silk was stretched and held at a fixed length at the highest temperature for about 10 min to allow the initial force to stabilize. The temperature was lowered and then raised by switching between two temperature controlled water baths, where one bath was set to the lowest temperature and the other to the highest temperature. The temperature shifts occurred over a time interval of less than 10 min. Movement of the glass beam with changing temperature, relative to the reference beam, was tracked with a video dimension analyzer (VDA; Instruments for Physiology & Medicine, San Diego, CA, USA), and this movement was used to track the change in force with changing temperature. Temperature in the test chamber was measured with a calibrated, thermistor bead that was immersed in the water immediately adjacent to the silk sample. VDA and thermistor outputs were recorded by a PC computer. Following the force–temperature experiment, the silk sample was slackened, and the temperature was again lowered and raised. Movement of the reference beam relative to the optical axis of the microscope provided control data that were used to correct for the thermal expansion of the apparatus.
The voltage output from the VDA was plotted against temperature, and linear
regressions were computed for the experimental and the control
voltage–temperature data. The control regression was subtracted from the
experimental regression to produce a regression for the
voltage–temperature relationship of the silk sample held at fixed length
(Fig. 2). The application of
beam theory with the dimensions of the glass beam allowed the calculated
voltage–temperature curve to be transformed to yield a
force–temperature curve (Fudge et
al., 2003). Since extension of the silk sample also deflects the
glass beam used to measure the force, a correction for the beam deflection was
applied to obtain the constant-length force on the silk sample.
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Swelling experiments
The thermal swelling coefficients for swollen MA silks were measured using
an aluminium, temperature-controlled stage that contained a sample chamber
that was specifically designed to accommodate long silk samples
(Fig. 3). MA silk samples were
mounted in the sample chamber between a glass beam and a moveable micrometer
mount. Water was added to the chamber, and the chamber was covered with a
coverslip. The stage was attached at one end to a lead block that was located
adjacent to a Wild M-21 polarizing microscope, and this assembly was
positioned so that the glass beam at the other end was centred in the field of
view of the microscope. The temperature-controlled stage was not directly
attached to the microscope stage, so that the movement of the glass beam could
be used to track the thermal expansion of the temperature-controlled stage and
attached chamber.
A video camera plus VDA were used to track the deflection of the glass
beam, which allowed us to determine the force-deflection of the silk sample
when it was stretched by the micrometer. Initial lengths of the silk samples
were measured with a vernier calliper to 0.1 mm. The change in length with
temperature was measured as follows. The temperature of the chamber was set
and allowed to equilibrate. The silk was then extended by several percent
while tracking the deflection of the glass beam, and the initial length was
determined by extrapolating the initial force–extension curve to zero
force. This process was repeated four to five times, and an average was taken.
The temperature was then raised, and this process was repeated. The length
change was calculated as the difference in the micrometer reading for initial
length at the two temperatures. This length change was then corrected for the
thermal expansion of the stage, which was measured by tracking the movement of
the unloaded beam over the same temperature range. That is, the movement of
the beam was subtracted from the calculated length change, and this result was
divided by the temperature difference to produce the thermal swelling
coefficient. The thermal swelling coefficient was assumed to be constant
between 283 K and 303 K (Gosline et al.,
1984). The silk samples of Araneus MA silk were
approximately 5.5 cm long, and the Nephila MA silk samples were
approximately 7 cm long.
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H/L)T,P,eq,
then represents the fraction of total force due to enthalpic changes,
Fh/F, at the reference temperature. The
force–temperature plot of this Nephila MA silk sample had an
intercept of about 1.3, indicating that enthalpic changes are responsible for
more than 100% of the total elastic force. The Araneus MA
force–temperature profile has an intercept of about 0.2, indicating that
for this sample approximately 20% of the force is due to changes in enthalpy.
Indeed, these Fh/F values can be negative at
low extensions, as in the case for the FL silk, and this suggests there is
more than enough entropy to explain the elastic force.
The intercepts of the uncorrected, normalized force–temperature
plots, Fh/F, were calculated for a number of
experiments at different extensions, and the values for Araneus and
Nephila MA silk are plotted in
Fig. 5. It is important to note
that the size of the symbols used in this plot is larger than any of the
standard error bars calculated for these data. Thus, the extrapolated
Fh/F values are very precise, but the
results are quite variable. In spite of this variation, the thermoelastic
behaviour of these two silks is clearly different. Nephila MA silk is
dominated by enthalpy for extensions above about 5%, whereas Araneus
MA silk exhibits a large entropic component at all extensions. In
Nephila MA silk an entropic component was only detected at low
extensions, and the enthalpic component increased with increasing extension.
Although the Fh/F values for
Araneus MA silk between extensions of 9% and 20% [taken from the
Gosline et al. study (Gosline et al.,
1984)] were negative or close to zero, there was an indication of
an increase in Fh/F with extension. Several
additional measurements were made to see if this trend continued at higher
extensions, and there appears to be a significant increase in
Fh/F with extension. These initial data are
consistent with the hypotheses stated above, because they seem to suggest a
large enthalpic contribution to the elastic mechanism of Nephila MA
silk and a large entropic contribution to Araneus MA and FL silks.
However, they are not conclusive because they do not account for thermal
swelling changes.
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10°C and at 30°C, and the thermal swelling coefficient over
this range was found to be –0.81x10–4
°C–1 (± –0.31x10–4
°C–1; N=5) for Araneus MA silk. Gosline
et al. (Gosline et al., 1984)
measured the zero force length of Araneus MA silk as a function of
temperature and found a linear decrease in the length of the silk between
10° and 30°C, giving a thermal swelling coefficient over this range of
–1.8x10–4 °C–1 The thermal
swelling coefficient of the Nephila MA silk was found to be
–1.6x10–5 °C–1
(±6.7x10–5 °C–1;
N=5), and given the large error for this measurement, the coefficient
could not be distinguished from zero.
Swelling correction and calculation of Fe/F
Since the thermal swelling coefficient for Nephila MA silk was too
low to be detected, we concluded that
Fh/FFe/F for this
silk. Araneus MA silk, however, required a correction because a
significant thermal swelling coefficient was obtained. Swelling corrections
were applied as follows. The negative thermal swelling coefficient for
Araneus MA silk indicates that the length of an unstretched
Araneus MA fibre decreases with increasing temperature. Thus, when a
stretched fibre sample is held at constant length while the temperature is
increased, the fibre extension will increase as a result of the
temperature-dependent swelling. To obtain force–temperature curves at
constant extension, a force–length isotherm was generated according to
the following process (Shadwick and
Gosline, 1985). Force–length isotherms in
Fig. 6 were plotted from the
regression lines generated from the force–temperature data from a
typical experiment for Araneus MA silk at 27% extension. The force
measured at this extension was plotted at the highest and lowest temperatures,
and the relative lengths at zero force were plotted for each temperature based
on the thermal swelling coefficient. Fig.
6A shows a plot of these points from a typical
force–temperature experiment using a thermal swelling coefficient of
–1x10–4 °C–1. Owing to the
small value of the swelling coefficient, the uncorrected and corrected
isotherms are difficult to distinguish, and
Fig. 6B shows the same data,
but with an exaggerated thermal swelling coefficient of
–1x10–2 °C–1. Increasing
temperature decreases the initial length of the silk, and therefore the zero
force length at 298 K is shorter than that at 283 K. Thus, the silk fibre is
at a larger extension at 298 K because of this swelling phenomenon. Based on
the new dimensions of the silk at 298 K, the force at constant extension can
be calculated. The slope of the force–temperature curve is reduced by
eliminating the force increase with temperature due to the decrease in linear
dimensions of the silk from solvent de-swelling. The corrected slope for
Araneus MA silk, based on a thermal swelling coefficient of
–1x10–4 °C–1, is shown in
Fig. 7. Note that the effect of
the swelling correction is to raise the magnitude of the intercept; in this
case, the intercept is about 0.3, or approximately 30% of the total force.
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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),
that the presence of proline in the glycine-rich network chains plays a key
role in determining the structure of the fibroin networks in MA silks.
Araneus diadematus MA glands are unique among those studied thus far,
in having two spidroin-2 type genes expressed
(Guerette et al., 1996). A
spidroin-1 type fibroin is present in the Araneus genome (Gatsey et
al., 2001), but was found to be expressed only in the cylindrical gland and
was below detection limits in the MA gland
(Guerette et al., 1996). Thus,
the predominant fibroins expressed in the Araneus MA gland contain
about 16% proline, and this silk contains amorphous network chains and
exhibits rubber-like elasticity when hydrated. By contrast, Nephila
MA silk, which contains primarily spidroin-1 fibroins and has low proline
content, has semi-ordered network chains and exhibits bond-energy elasticity
when hydrated. The uncorrected thermodynamics (Fig. 5) indicate that the enthalpic component of the elastic force, Fh/F, is small in Araneus MA silk. Indeed Fh/F is negative in some samples, which suggests that the entropic component is more than sufficient to account for the elasticity of this material. The situation is dramatically different for Nephila MA silk. The enthalpic component of the elastic force is dominant at all extensions, and an entropic component of the elastic force is only seen at the smallest extensions. Thus, it appears that hydrated Araneus MA silk contains a network of kinetically-free protein chains and is truly an entropic rubber. By contrast, hydrated Nephila MA silk exhibits bond-energy elasticity associated with the presence of stable secondary structures within its network chains. However, before we accept these conclusions, it is important to consider the thermal swelling and its effect on the thermodynamics of these systems.
Our estimate of the thermal swelling coefficient for Araneus MA
silk, –1x10–4 °C–1, is
similar to that recorded previously
(Gosline et al., 1984) and
allowed us to estimate the bond-energy component of the elastic force,
Fe/F, which turned out to be very small, particularly
at small extensions (Fig. 8).
Hence, our data are entirely consistent with the interpretation that hydrated
Araneus MA silk is an entropic rubber. Our attempts to measure the
thermal swelling coefficient for Nephila were largely unsuccessful
because our method was not precise enough to actually quantify the swelling of
this material. The uncertainty of our measurement,
–1.6x10–5°C–1
(±6.7x10–5°C–1), is so
large relative to the mean that we cannot even determine if the coefficient is
positive or negative. We chose, therefore, to treat the coefficient as zero,
and we present the Fh/F as giving a best
estimate of the bond-energy component of the elastic force
Fe/F. The data for Nephila MA silk in Figs
5 and
7 suggest that by an extension
of 10%, Fe/F=1.4, meaning that there is 40% more
bond-energy available than is needed to account for the elastic force. This is
almost certainly not true, and it leads to the conclusion that if we could
measure the thermal swelling coefficient of hydrated Nephila MA silk,
it would likely be positive and have a magnitude of approximately
+3x10–5 °C–1. That is, this level
of positive swelling would be just sufficient to shift the highest
Fh/F to an Fe/F
value of 1.0, indicating that all of the elastic force at 10% extension is due
to bond-energy elasticity. It remains to be seen if this is true.
Interestingly, it is not necessarily true that an improved method would
actually improve the precision of this experiment. Recall that, from the
previous study of the mechanical and optical properties of hydrated
Araneus and Nephila MA silks, these silks are highly
variable in their properties [see Figs
4,
5,
6,
7 in Savage and Gosline
(Savage and Gosline, 2008)].
Thus, the variation in the thermal swelling coefficient is likely to arise
from sample variation as opposed to measurement error. To truly resolve this
issue with Nephila silk, it may be necessary to conduct thermoelastic
experiments and measure the thermal swelling coefficient on the same sample,
which we were unable to do in this study.
One interesting feature of the thermoelasticity of both MA silks is that
the bond-energy component of the elastic force, Fe/F,
seemed to increase with extension, a feature not usually seen for lightly
cross-linked natural rubbers, or for the protein rubbers elastin and resilin.
This very likely reflects the presence of crystalline structures in the
networks of the MA silks, which become more prominent as extension proceeds.
By contrast, natural rubber, resilin and elastin are crosslinked covalently by
structures that are much smaller than the β-sheet crystals that crosslink
and reinforce the networks in the spider MA silks. We therefore carried out
our thermoelastic experiments over a range of extensions to evaluate the
extent of this shift from entropic to bond-energy elasticity. The trends for
the MA silks are clear, but there is considerable scatter because of the
enormous variation seen in the structure and properties of these silks
(Savage and Gosline,
2008).
In the case of Araneus MA, there was a modest rise in the
bond-energy component of the elastic force at extensions above about 20%, and
by 50% extension Fe/F0.5. This rise may indicate
that the relatively large, poly-alanine, β-sheet crystals are
constraining the network chains in a way that causes this rise in bond-energy
elasticity at higher extensions. Indeed, Termonia
(Termonia, 1994) developed a
model for the structure and mechanical properties of MA silks that was based
on the thermoelastic properties of hydrated Araneus MA silk
(Gosline et al., 1984). That
is, he assumed that the glycine-rich network chains were amorphous and behaved
as kinetically-free random-coils in the hydrated state. He found that it was
only possible to predict the properties for wet and dry MA silk if he included
a layer of greater stiffness in the network chains just outside the
β-sheet crystals. He assumed a 5 nm thick layer with a sixfold increase
in stiffness, and his model successfully predicted the stress–strain
properties of both hydrated and dry Araneus MA silk. This layer of
increased stiffness was attributed to the physical constraint of network
chains emerging from the β-sheet crystals in dense clusters, and hence
with restricted mobility. The rise in Fe/F between
20% and 50% extension probably reflects a rising contribution to the
elasticity of hydrated Araneus MA silk from the deformation of these
conformationally constrained regions of the network chains.
The situation for Nephila MA silk is clearly quite different.
Fe/F0.5 at 5% extension, and it rises rapidly to
essentially 1 by 10% extension. This clearly indicates a much more dominant
role for bond-energy elasticity in the Nephila silk. Thus, it is
appropriate to conclude that stable, secondary structures are present in the
network chains of Nephila MA silk, and that entropic processes are
only important at very low extensions where these secondary structures
reorient before they are actually strained. By about 10% extension, therefore,
the deformation of stable secondary structures becomes the dominant feature of
this silk. The dashed arrow in Fig.
8 indicates the trend that we would expect to see for
Nephila silk at higher extensions, namely that bond-energy elasticity
will be maintained at this high level. Unfortunately, we could not confirm
this because the stiffness of the silk was so great at higher extensions that
we could not carry out thermoelastic tests with the apparatus we had
constructed.
The experiments with Araneus FL silk reinforces the conclusion that proline plays a key role in creating a network of kinetically-free, amorphous chains in hydrated spider silks. The thermoelastic data in Figs 9 and 10 clearly indicate that changes in conformational entropy are responsible for the storage of elastic energy in this silk. Most values of Fh/F were close to zero or slightly negative, and the application of a thermal swelling coefficient of –4x10–4°C–1 brings the predicted Fe/F values into full agreement with a conformational entropy system. It is important to note that this swelling coefficient is just a guess, and the validity of this conclusion rides on the reasonableness of the guess. We assumed that the thermal swelling coefficient for FL silk would be negative, like that of Araneus MA silk, because the glycine-rich chains in both silks are very similar, both in the relative contents of glycine and proline and in the arrangement of these and the other residues. Based on this assumption, there are two good reasons why the magnitude of the thermal swelling coefficient for FL silk should be larger than that for MA silk.
First, Araneus FL silk network chains are much longer (115
residues) than those in Araneus MA silk (25 residues), which
means that the stiffness of FL silk will be lower. This is actually true, as
supercontracted Araneus MA silk has an average initial stiffness of
10 MPa (Gosline et al., 1994;
Savage and Gosline, 2008), and
water-swollen Araneus FL silk has an average initial stiffness of 1
MPa (Pollak, 1991;
Gosline et al., 1994). Thus,
temperature effects on the chain chemistry should have a much larger effect on
the swollen volume of the softer network, as predicted by the
Flory–Rehner Theory for network swelling
(Flory, 1953).
In addition, the MA silk has a higher content of β-sheet crystals,
again because of its sequence design, and when the MA silk supercontracts, the
abundant crystals and short network chains prevent the material from relaxing
to a completely isotropic state. Thus, supercontracted Araneus MA
silk has a birefringence of 6x10–3, which indicates
some residual orientation of crystals parallel to the fibre axis. Hydrated
Araneus FL silk has a birefringence of 9x10–4
(Gosline et al., 1995), and
this sevenfold lower birefringence indicates that the properties of FL silk
are more nearly isotropic than those of the MA silk. Thus, even if thermal
changes in swelling cause equal increases the volumes of the two silks, the FL
silk will expand more along its fibre axis, giving it a higher thermal
swelling coefficient. Thus, our guess of
–4x10–4°C–1 for the thermal
swelling coefficient is reasonable, if not exact.
Interestingly, five of the FL samples had extremely negative
Fh/F values that remained large and negative
after correction for swelling. This is not surprising, given the extreme
variability that has been observed for the mechanical properties of hydrated
FL silks (Pollak, 1991;
Gosline et al., 1994). This
variation in mechanical properties would lead to variation in swelling
behaviour, and it is not surprising that some samples could not be adequately
corrected with a single, assumed value for the thermal swelling coefficient.
As with the results for Nephila MA silk, if we were able to measure
the thermal swelling coefficient of each sample subjected to thermoelastic
analysis, we would discover that these exceptional samples would show the same
behaviour as indicated in Fig.
10 for the majority of FL silk samples.
The structural state of the network chains in MA and FL silks
Our results show that the glycine-rich network chains are very different in
Araneus and Nephila MA silks, and we believe that this
difference is due to the difference in proline levels in the fibroins that
make up these two silks. Proline is known to disrupt secondary structure in
glycine-rich peptide sequences (Rauscher
et al., 2006), and the high levels of proline in the network
chains of Araneus MA silk are almost certainly responsible for the
mobility of the network chains in hydrated Araneus MA silk.
Interestingly, amino acid sequences from this silk indicate that the majority
of network chains contain a proline within three to five residues upon exiting
β-sheet crystals. Presumably, the effect of this proline is to increase
flexibility and loosen the constrained clusters as the chains emerge from the
β-sheet crystals. Although we believe that the thermoelastic data
presented here provide the strongest demonstration of the amorphous and
kinetically-free structure of the network chains in Araneus MA silk,
Shao et al. (Shao et al.,
1999) used single-fibre Raman spectroscopy to probe the effect of
solvent on its structure, and these data provide additional evidence to
support this conclusion. Differences between the Raman spectra taken parallel
to and perpendicular to the fibre reveal a high degree of order in dry
Araneus MA silk, a finding that is consistent with a spinning process
that would orient the β-sheet crystals and all or most of the network
chains in the dry fibre. However, this study could not quantify the
conformational components that would indicate what, if any, secondary
structure stabilized this alignment in the network chains of the dry thread.
With supercontraction, the peaks in the Raman spectra associated with
random-coil and β-sheet, increased and decreased, respectively, upon
supercontraction. The spectra indicated that the β-sheets remain in the
supercontracted state but lose much of their alignment, while the network
chains appear to become random-coils.
The situation for Nephila MA silk is very different. The dominance
of bond-energy elasticity in this silk in its hydrated state clearly suggests
the presence of stable secondary structures in the glycine-rich network
chains, and the prevalence of the proline-free spidroin-1 in this silk
suggests that the lack of proline may enable this structure. Although the
thermoelastic evidence presented here is consistent with this conclusion, it
does not provide information to distinguish between the secondary structures
that have been proposed for the glycine-rich sequences in Nephila MA
silk. Thus, it remains to be determined if the non-periodic lattice
β-sheet crystal structure (Thiel et
al., 1997) or the 31 helix structure
(Kümmerlen et al., 1996;
van Beek et al., 2002) is most
appropriate. Either should be quite rigid and could explain the bond-energy
nature of the elastic mechanism in Nephila MA silk. That is, as the
fibre is stretched, the inter-chain hydrogen bonds in these ordered regions
are deformed and could play a major role in the elastic recoil of the fibre.
More recently Eles and Michal (Eles and
Michal, 2004a) have shown through NMR of glycine-labelled, dry
Nephila MA silk that 47% of glycines are well oriented to the fibre
axis and 53% are poorly oriented (Eles and
Michal, 2004a). They concluded that the orientation of ordered
glycine-rich network chains is consistent with 31 helices, but this
is not direct evidence for the presence of such structure. Eles and Michal
(Eles and Michal, 2004b) used
NMR to probe the network structure of dry, hydrated but partially restrained,
and fully supercontracted Nephila MA silk. Again, the network chains
in dry Nephila MA silk are in highly extended conformations; however,
these chains became more mobile as the hydrated silk was allowed to shorten.
The authors concluded that network chains acted as entropic springs in
supercontracted silk and that the molecular mechanism of the dry silk was
based on "latent entropic springs" that are axially aligned by the
spinning process and stabilized by hydrogen bonding in the absence of
water.
In summary, our thermoelastic analysis of the MA and FL silks from Araneus diadematus and the MA silk Nephila clavipes confirm the conclusion of our previous study that proline residues present in the glycine-rich network chains of spider silk fibroin networks will destabilize secondary structures and will favour a more amorphous network structure. This leads to an entirely new avenue of enquiry into the functional significance of proline-rich and proline-deficient fibroin networks. We suspect that the difference in fibroin expression levels between species may reflect differences in the physical environment in which these species live, allowing them to adjust material properties to suit the key function or functions of their MA silks. In addition, it will be interesting to see if changes in fibroin expression occur during ontogeny or with environmental changes. There is clearly much left to learn about the interface between the polymer physics and mechanics of spider silks and the biology of these amazing animals.
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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