The natural silk spinning process - A nucleation-dependent aggregation mechanism?
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Eur. J. Biochem. 268, 6600–6606 (2001) q FEBS 2001
The natural silk spinning process
A nucleation-dependent aggregation mechanism?
Guiyang Li1, Ping Zhou1, Zhengzhong Shao1, Xun Xie1, Xin Chen1, Honghai Wang2, Lijuan Chunyu2 and
Tongyin Yu1
1
The Key Laboratory of Molecular Engineering of Polymers, Ministry of Education, Macromolecular Science Department and
2
Institute of Genetics, Fudan University, Shanghai, China
The spinning mechanism of natural silk has been an open thermodynamically unfavorable association of b sheet unit,
issue. In this study, both the conformation transition from i.e. the formation of a nucleus or seed; (b) once the nucleus
random coil to b sheet and the b sheet aggregation growth of forms, further growth of the b sheet unit becomes
silk fibroin are identified in the B. mori regenerated silk thermodynamically favorable, resulting a rapid extension
fibroin aqueous solution by circular dichroism (CD) spectro- of b sheet aggregation. The aggregation growth follows a
scopy. A nucleation-dependent aggregation mechanism, first order kinetic process with respect to the random coil
similar to that found in prion protein, amyloid b (Ab) fibroin concentration. The increase of temperature accel-
protein, and a-synuclein protein with the conformation erates the b sheet aggregation growth if the b sheet seed is
transition from a soluble protein to a neurotoxic, insoluble b introduced into the random coil fibroin solution. This work
sheet containing aggregate, is a novel suggestion for the enhances our understanding of the natural silk spinning
silk spinning process. We present evidence that two steps process in vivo.
are involved in this mechanism: (a) nucleation, a rate-
limiting step involving the conversion of the soluble random Keywords: silk fibroin; spinning mechanism; conformation
coil to insoluble b sheet and subsequently a series of transition; nucleation-dependent; CD spectroscopy.
A number of studies have been reported silk processing formation by extensional flow [14]. But the mechanism
techniques [1 –11], including those of spider and silkworm. involved in the conversion of a hydrogel of silk fibroin in the
However, the controlling factors that determine the silk I state into the silk II state remains unresolved.
efficiency of silk spinning at ambient temperature and Recently, a nucleation-dependent aggregation (polymer-
normal pressure remains unclear [6 –8]. The silk fibroin in ization or crystallization) model has been proposed to
the silkworm gland possesses relatively low viscosity in a account for the formation of insoluble fibers of b proteins in
concentrated solution as a result of its storage in a liquid a range of neurodegenerative diseases. These involve the
crystalline state before spinning [7 –9]. This is coupled with conversion of the endogenous prion PrPc into the pathogenic
a low critical shear rate for inducing crystallization in PrPSc [15], Ab amyloid fibril formation from amyloid b
aqueous solution [10], as well as the low draw ratios for the (Ab) protein [16], and a-synuclein fibrillogenesis [17]. It
production of uniaxially aligned fibrous structures [11]. has been shown in these studies that the formation of an
Theoretical elucidation of the mechanism for the natural silk insoluble fiber is a two-phase process with an initial
spinning process has implications for material science, nucleation phase in which the formation of nuclei is limited
particularly in the design of engineering polymers. by the time taken for the protein to undergo conformation
The primary sequence of silk fibroin of the B. mori changes as well as the association of converted protein, and
silkworm predominantly consists of the -(Gly-Ala-Gly-Ala- then an aggregation growth phase following first order
Gly-Ser)8- motif [12]. It has been demonstrated that two kinetics with respect to the protein concentration once
types of conformations exist in the silk fibroin: silk I, a nuclei are formed. This model has a number of interesting
mainly coiled chain conformation in the present silk gland, features. Firstly, the aggregation process is a nucleation-
and silk II which is formed by regularly aligned crystalline b dependent process that could be induced by the preformed
sheet in the silk fibers [7]. Previous studies have suggested aggregates or nuclei. Secondly, in whole propagation the
that the b sheet conformation can be induced in silkworm nucleation of orderly aligned nuclei is a rate-limiting step.
fibroin by a stretching force [13], and the formation of spider Thirdly, the aggregation growth follows a first order
dragline silk also involves a stress-induced b sheet kinetics. Lastly, the conformation adapted by the protein
after conversion is less stable thermodynamically than its
precursor, but the formation of more stable aggregates
Correspondence to P. Zhou, The Key Laboratory of Molecular compensates the energy required by the conversion and the
Engineering of Polymers, Ministry of Education, Macromolecular nucleation process [15]. In this work, we suggest that the
Science Department and Institute of Genetics, Fudan University, spinning process of natural silk, a structural protein, may
Shanghai 200433, China. Fax: þ 86 21 65640293, also adopt such a process.
Tel: þ 86 21 65642866, Email: pingzhou@fudan.edu.cn As the regenerated silk fibroin (prepared in Experimental
(Received 4 July 2001, revised 5 October 2001, accepted 19 October procedures) in aqueous solution has a similar structure to the
2001) concentrated silk fibroin in the gland of silkworm [18], theq FEBS 2001 Viewing into silk fibroin conformation transition (Eur. J. Biochem. 268) 6601
regenerated silk fibroin is adopted in this study to serve as an of 0.1 mg·mL21 silk fibroin solution by repeatedly injecting
ideal in vitro model system for elucidating the details of the it through the aperture of an 0.8-mm diameter tube,
silk spinning process in vivo. The conformation transitions mimicking the duct in the natural silk spinning process. The
of the silk fibroin were investigated by CD spectroscopy, resulting solution was shown contain a mixture of random
which is an effective tool in the characterization of protein coil and b sheet conformations (Fig. 1, curve d). Both
conformation transition [19,20]. The effect of temperature methods produce clear seed solutions and neither crystals
on the conformation transition was also investigated. nor precipitates formed in all measured solutions during the
CD experiments.
E X P E R I M E N TA L P R O C E D U R E S
Sample preparation Circular dichroism measurement
Raw silk was extracted from B. mori cocoon and degummed The experiments were conducted using a Jasco J-715 spectro-
in boiling aqueous Na2CO3 (0.5%, w/v) solution for 0.5 h. polarimeter equipped with RTE bath/circulator (NESLAB
The regenerated silk fibroin solution was prepared by RTE-111) and purged with N2 gas at a flow-rate of
dissolving the degummed silk into a 9.3 M LiBr solution for 3 –5 mL·min21. The spectra were recorded from 190 to
1 h at room temperature (22 ^ 1 8C). This solution was 250 nm with a resolution of 0.2 nm and accumulation of six
dialyzed against deionized water for 3 days to remove LiBr scans. The scan speed was 100 nm·min21 and the response
and insoluble material was removed with a medium rate time was 0.25 s. Samples of the 0.1 mg·mL21 and
qualitative filter paper in a triangle funnel. The resulting 0.01 mg·mL21 solutions were stored in 0.1-cm and 1-cm
solution has a concentration of6602 G. Li et al. (Eur. J. Biochem. 268) q FEBS 2001
at < 195 nm, confirming the predominance of the random increase of both seed and silk fibroin concentrations
coil conformation [22]. With increasing time, the Cotton increases the rate of the conversion of random coil to b
effect is gradually replaced by a positive one at 197 nm, sheet and accelerates markedly the formation of b sheet
while a negative peak at 217 nm increases simultaneously. aggregates.
Both of the Cotton effects at positive 197 nm and negative
217 nm are characteristics of a b sheet [22]. In short,
Fig. 2A,B shows that a transition from random coil to b
sheet takes place and the transition is much faster in seeded First order aggregation growth mechanism
solution than that in seed-free solution, for instance, the Because the aggregation growth process involves transition
seed-free solution takes 74 h (curve 4 in Fig. 2A) to reach of one conformation to another once nucleation is
the same level by 20% seeded solution within 6 h (curve completed, the process can be expressed as:
20 in Fig. 2B). Note that leaving the freshly prepared seed-
free solution (0.1 mg·mL21) to stand for a long period of k
Acoil ÿ! Ab ð2Þ
time (over 1 month) results in the formation of precipitates
that were shown to have b sheet conformation by Raman in which the reactant is in a random coil conformation and
spectroscopy (Fig. 3). The peaks at 1087, 1234, 1266 the product is in a b sheet conformation. For first order
and 1665 cm21 in Fig. 3 arise from the silk II (b sheet kinetics:
conformation), and the weak absorption at 1109 cm21 d½Acoil
implies the existence of small amount of silk I (random coil ¼ 2k½Acoil ð3Þ
or a helical conformation) [23]. The peak at 1444 cm21 dt
[d(CH2) scissoring] is independent of silk fibroin confor- and [A]/[A]0 takes the form
mation [24].
In our CD spectroscopy, the ellipticity is normalized ½A/½A0 ¼ ½Acoil /ð½Acoil þ ½Ab Þ ð4Þ
using Eqn (1): where [A]coil and [A]b are the concentration of the random
coil and b sheet conformation, respectively, the logarithmic
½Qnor obs 0 max 0
217 ¼ ð½Q217 ÿ ½Q217 Þ=ð½Q217 ÿ ½Q217 Þ ð1Þ form of (Eqn 3) becomes
where ½Q0217 is the original ellipticity at 217 nm during the
ln ½Acoil /ð½Acoil þ ½Ab Þ ¼ 2kt ð5Þ
measurement in the random coil solution, ½Qobs 217 is the
observed ellipticity and ½Qmax 217 is the maximum ellipticity From the molar ellipticity shown in Fig. 1, the random coil
observed. Therefore, the correlation between normalized has a weak absorption at 217 nm, whereas the b sheet
ellipticities at 217 nm and the time course for the seed-free conformation has a strong absorption. Because the ellipticity
silk fibroin solution and various seeded solutions are shown at 217 nm is proportional to the concentration of the b sheet
in Fig. 4. Note that a lag time exists in seed-free solution. conformation:
During this lag time no b sheet conformation is observed
(Fig. 4A, inset, B). Small but undetectable amounts of b ½Ab =ð½Acoil þ ½Ab Þ
sheet (as nuclei) may be built up during this period.
However, this slow nucleation step can be bypassed by the ¼ ð½Qobs 0 max 0
217 2 ½Q217 Þ=ð½Q217 2 ½Q217 Þ
introduction of exogenous nucleus or seeds. As a conse-
¼ ½Qnor
217 ð6Þ
quence of seeding, the lag time is eliminated (W curve in
Fig. 4A). The half-life time (t1/2) for the disappearance of Thus, a linear relationship exists between 2ln(1 2 ½Qnor
217 )
random coil maintains at 64, 32 and 9 h for 0, 5 and 20% and t:
seeded solutions, respectively. It is shown (Fig. 4B) that the
2 lnð1 2 ½Qnor
217 Þ ¼ kt ð7Þ
Because ½Qnor
217 is available from the CD experiment for a
series of fibroin solution, we put these ½Qnor217 values into
Eqn (7) and obtained the curves of –ln(1 2 ½Qnor 217 ) vs. t.
Figure 5 clearly shows a linear relationship between
2 ln(1 2 ½Qnor217 ) and t, demonstrating that the b sheet
aggregation growth process is first-order with respect to the
random coil concentration for both the seed-free and seeded
solutions. For these plots, the determined slopes are
approximately identical, indicating that the reaction
constant k remains the same at a given temperature after
nucleation, and the observed differences in the intercepts are
attributed to the original differences in seed addition.
The effect of temperature on the conformation transition
process was also probed and the results are shown in Fig. 6.
From CD spectra shown in Fig. 6A, only a small change was
observed in seed-free solutions despite increasing tempera-
ture up to 95 8C, but the transition process sped up
Fig. 3. Raman spectrum of the aggregate precipitate of silk considerably once the solution was seeded under the same
fibroin. temperature conditions.q FEBS 2001 Viewing into silk fibroin conformation transition (Eur. J. Biochem. 268) 6603
Fig. 4. Change in normalized ellipticity [u] at 217 nm with time. The inserted illustrations in (A) show the early stage of seed-free (B) and 20%
seeded (W) solutions in the 0.1 mg·mL21 silk fibroin solution. (A) show the effect of seeding on the b sheet formation speed (B, seed-free, P, 5%
seeded, W, 20% seeded). (B) reveals a slower b sheet formation speed in a lower silk fibroin concentration with 20% seed addition (O, 0.01 mg·mL21
silk fibroin solution, W, 0.1 mg·mL21 silk fibroin)
DISCUSSION We suggest that either a very small quantity of b sheet
aggregates are formed but remain highly unstable or the
The results presented above suggest that silk fibroin amounts of b sheet aggregates formed during this time are
solutions show a typical nucleation-dependent aggregation too small to be detected. Thus, the bulk silk fibroin mainly
process with the following features: the transition of silk remains in the random coil conformation. We suggest that
fibroin conformation from coiled chain to b sheet nuclei is only when b sheet aggregates exceed a given critical size
initiated by addition of b sheet seeds. In the absence of after a lag time period or upon addition of exogenous b sheet
added seed, a lag time exists because the spontaneous seeds, does further growth become spontaneous, resulting in
conversion of coiled chain to b sheet is slow. The b sheet the formation of large amounts of b sheets and finally the
aggregation growth process follows first order kinetics once appearance of the insoluble material after long time
nuclei have formed, and increasing temperature favors the aggregation. In other words, the nucleation step involves
conformation transition once seeded solutions have been the transition of random coil to b strand and the formation of
added. These features are similar to the characteristics of ordered b sheet association in the silk fibroin solution,
prions, amyloid b (Ab) protein, and a-synuclein protein whereas b sheet growth is based on the b sheet nucleus
[17,25–32]. formation. It has been reported that the dependence of molar
As seen from the inserted illustration in Fig. 4A, the ellipticity on the protein concentration serves as an indicator
conformation transition in seed-free silk fibroin solution of the extent of intermolecular aggregation [33], otherwise
cannot be detected by CD at the initial stage (within < 8 h). it suggests an intramolecular aggregation if the molar
Fig. 5. Linear plots of 2ln(1 2 [u]217) vs. time
course for 0.1 mg·mL21 silk fibroin solutions
with different seed-additions. The ellipticities
were normalized and earlier parts were cut off
before 10 h because of the nucleation process. (A)
Seed-free, (B) 5% seeded, (C) 20% seeded, (D) a
repeatedly injected solution. The values in the
brackets are the errors and R is the regression
factor.6604 G. Li et al. (Eur. J. Biochem. 268) q FEBS 2001
Fig. 7. CD spectra of different silk fibroin concentration with 20%
seed addition after 233 h incubation. (a) 0.1 mg·mL21 silk fibroin
solution; (b) 0.01 mg·mL21 silk fibroin solution.
217 nm (see Fig. 7). Thereby this concentration-dependent
molar ellipticity indicates that intermolecular aggregation
occurs in the aggregation growth of silk fibroin.
It has been shown that the rate of formation of a b sheet
nucleus, or seed, is slow because of unfavorable association
equilibrium rather than the intrinsically slow association
rates [23]. Therefore, it is necessary to understand the
energy difference between the reactant and product. We
calculated the energies of random coil and b strand (a
fragment of (Gly-Ala-Gly-Ala-Gly-Ser)8 was adopted)
using SYBYL software. The calculated formation energy is
5.8 and 8.2 kJ·mol21 for the random coil and b strand,
respectively, after energy minimization. The results reveal
that the precursor, i.e. random coil, is more stable than the
converted form, b extended strand, and the random coil
requires 2.4 kJ·mol21 to complete this transition. In the
study of the thermodynamics of model prions, Harrison et al.
[35] demonstrated that proteins that are relatively less stable
as monomers are more susceptible to forming alternative
native states as homodimers. Fossey [36] calculated the
energies of both the single b strand and the triply stacked b
sheet for the B. mori silk protein; the results indicate that the
later is more stable. Accordingly, we suggest that for silk
fibroin the conversion of random coil to b strand is thermo-
dynamically unfavorable for a single strand. Although the
stacking of b strands may compensate the energy needed to
convert random coil to b sheet, the ordered nucleus
formation in the beginning requires a series of association
steps that may also be thermodynamically unfavorable
Fig. 6. Temperature effect on the conformation transition of owing to the loss of entropy by association [37]. Once a b
random coil to b sheet in 0.1 mg·mL21 silk fibroin solution. (A), sheet nucleus is formed, further addition of the random coil
(B) and (C) reflects different transition speed in 0, 5, 20% seeded renders the growth of aggregates thermodynamically
solutions, respectively, at different temperatures. CD spectra were favorable because the contacts between random coil chains
recorded at 20 8C (curves 1, 4, 7), 60 8C (curves 2, 5, 8) and 95 8C with growing aggregates are established at multiple sites
(curves 3, 6, 9). All samples were incubated for half an hour at a [37]. As a result, the energy gained by this multiple site
predesignated temperature. interaction outweighs the losses of both energy and entropy
during the aggregate growth. Further evidence supporting
this conjecture may be derived from a consideration of IR
ellipticity is protein-concentration-independent [34]. We evidence of silk fibroin gel [38]. The silk fibroin gel is
therefore compared the molar ellipticity of a 0.1 mg·mL21 composed of the b sheet conformation formed from the
20% seeded silk fibroin solution with that of a regenerated silk fibroin aggregation. From FTIR spectrum
0.01 mg·mL21 20% seeded silk fibroin solution after a [38], it has been concluded that strong hydrogen bonding
233-h incubation, and found that the former had a larger interactions exist in this silk fibroin gel, stabilizing the b
molar ellipticity by < 1 £ 103 degree·cm2·dmol21 at sheet aggregates.q FEBS 2001 Viewing into silk fibroin conformation transition (Eur. J. Biochem. 268) 6605 Fig. 8. Schematic illustration of natural silk spinning process. (a) Nucleation, which includes a transition of random coil to b strand as well as a formation of ordered b sheet aggregates (nuclei); (b) aggregation growth, which involves coiled chain changing its conformation on the preformed b sheet nuclei, followed by formation of the larger b sheet aggregation. In addition, it has been long considered that the protein slow spinning speed of silk is readily rationalized. This slow folding is hydrophobic interaction dependent. The high- speed becomes necessary for the conformation transition energy molecular collision is important to the enthalpy, process, leading to the formation of well-oriented b sheet entropy and heat capacity during the hydrophobic interaction nuclei and liquid state to sustain the aggregation growth. based on the potential of mean force perspective [39]. Spek et al. [42] studied the role of the alanine sequence in Obviously, the temperature increasing accelerates the forming b sheet of spider-like polypeptide and demon- molecular movement therefore leading to the molecular strated experimentally that a buildup of the polypeptide collision increase, consequently, speeding up the b sheet strand by accretion of alanine domains with different lengths aggregation. was nucleated by the domains with seven alanine residues, To obtain the further insights into the natural silk spinning i.e. a b sheet core. Therefore, it is plausible that a nucleation- mechanism of B. mori silkworm in vivo, we carried out the dependent aggregation process exists in the formation of experiments mimicking the spinning force of silkworm. It is spider silk. It is also noted that the tensile properties of spider well known that the shearing force can induce b sheet silk are affected by the spinning speed, i.e. fast spinning leads formation from the coiled chain [2 –5]. We find that the to a higher modulus but a lower draw ratio [1]. It indicates shearing force induced by injecting the silk fibroin solution that the natural silk conformation conversion is controllable. through an aperture, like a spinneret of silkworm, can lead to Spider could control the stress force under given spinning the b sheet formation (Fig. 1, curve d). The resultant b sheet rate to control the formation of b sheet components that can also promote the aggregation growth similar to the determine the fibril tensile properties. behavior of seeds produced by shaking the solution (see Fig. 5D). We therefore propose that natural silk spinning mechanism of silkworm is similar to that of a b sheet CONCLUSION aggregation process in the regenerated silk fibroin. As the In this study, the coiled-chain to b sheet conformation silk fibroin in the middle section of the silkworm gland is change of silk fibroin is suggested to be a nucleation- predominantly composed of the coiled chain, when the silk dependent aggregation process. This mechanism may help fibroin approaches the spinneret, the hydrogel-like silk to explain the natural silk spinning process. The nucleus fibroin is sheared by the silk press in the silkworm duct, or formation, involving coil chain to b strand conversion stressed by extensional flow, or simply stretched during the followed by a b sheet aggregation, is a rate-limited step. The spinning [11]; the molecular chain is thus stretched into b sheet nucleation step is crucial for a smooth transition of the b strand, followed by orderly alignment through self- the random coil in the silkworm gland to the b sheet in the assembly into a b sheet aggregate, or nucleus. This step is crystalline form. Once nuclei are formed, the b sheet rate-limited. Once b sheet nuclei are formed, the b sheet aggregation growth process may obey a first order kinetic aggregation growth occurs as the coiled chain associates mechanism in vivo as in vitro. with the preformed nuclei. The ordered b sheet fibrils are formed when drawn by the head of the silkworm throughout the spinneret into open air. The uniaxial orientation of the ACKNOWLEDGEMENTS ordered b sheet aggregate is along the direction of drawing This work was supported by NSFC (no. 29974004, China); NSF or shearing force [40]. The b sheet aggregation growth may (no. 99ZA14001, Shanghai), the Laboratory of MRAMP (no. 991504, follow a first order kinetic process with respect to the silk China) and the Foundation for University Key Teacher by the Ministry fibroin concentration in the duct of silkworm. Figure 8 of Education. Authors wish to acknowledge Wuhan Institute of Physics schematically illustrates the proposed mechanism of the and Mathematics of the Chinese Academy of Science for supplying the natural silk spinning process. We believe that defects shown SYBYL software for the protein energy calculation. Helpful discussions in many morphology pictures of the crystalline silk reported with Prof. Steve C. F. Au-Yeung and Prof. S. L. Lam of the Chinese elsewhere [41] arise from the nonordered alignment during University of Hong Kong are also gratefully acknowledged. the aggregation. The liquid crystalline phase exists in the silkworm gland [40] where the concentration of silk fibroin may be under control for the requirement of a given rate of REFERENCES the b sheet aggregation growth when it goes through the 1. Vollrath, F. & Knight, D.P. (2001) Liquid crystalline spinning of spinneret. spider silk. Nature 410, 541 –548. Silkworm silk spinning is processed at a relatively slow 2. Sirichaisit, J., Young, R.J. & Vollrath, F. (2000) Molecular speed (6 –8 mm·s21) under ambient conditions [40]. Within deformation in spider dragline silk subjected to stress. Polymer 41, the framework of the nucleation-dependent aggregation, the 1223–1227.
6606 G. Li et al. (Eur. J. Biochem. 268) q FEBS 2001
3. Pérez-Rigueiro, J., Viney, C., Llorca, J. & Elices, M. (2000) 24. Edwards, H.G.M. & Farwell, D.W. (1995) Raman spectropic
Mechanical properties of silkworm silk in liquid media. Polymer studies of silk. J. Raman Spectrosc. 26, 901 –909.
41, 8433–8439. 25. Scott, M., Foster, D., Mirenda, C., Serban, D., Coufal, F.,
4. Seidel, A., Liivak, O., Calve, S. et al. (2000) Regenerated spider Walchli, M., Torchla, M., Groth, D., Carlson, G., DeArmond, S.J.,
silk: Processing, properties, and structure. Macromolecules 33, Westaway, D. & Prusiner, S.B. (1989) Transgenic mice expressing
775–780. and references therein.. hamster prion protein produce species-specific scrapie infectivity
5. Liivak, O., Blye, A. & Shah, N. (1998) A microfabricated wet- and amyloid plaques. Cell 59, 847–857.
spinning apparatus to spin fibers of silk proteins. Structure-property 26. Mclaurin, J., Franklin, T., Zhang, X.Q., Deng, J.P. & Fraser, P.E.
correlations. Macromolecules 31, 2947–2951. (1999) Interaction of Alzheimer amyloid-beta peptides with
6. Magoshi, J., Magoshi, Y. & Nakamura, S. (1985) Liquid crystal and glycosaminoglycans-Effects on fibril nucleation and growth. Eur.
fiber formation of silk. J. Appl. Polym. Sci., Appl. Polym. Symp 41, J. Biochem. 263, 1101–1110.
187–204. 27. Kapurniotu, A., Bernhagen, J., Greenfield, N., Al-Abed, Y.,
7. Li, G.-X. & Yu, T.-Y. (1989) Investigation of the liquid-crystal state Teichberg, S., Frank, R.W., Voelter, W. & Bucala, R. (1998)
in silk fibroin. Makromol. Chem. Rapid Commun. 10, 387 –389. Contribution of advanced glycosylation to the amyloidogenicity of
8. Kerkam, K., Viney, C. & Kaplan, D. (1991) Liquid crystallinity of islet amyloid polypeptide. Eur. J. Biochem. 251, 208–216.
natural silk secretions. Nature 349, 596– 598. 28. Come, J.H., Fraser, P.E. & Lansbury, P.T. Jr (1993) A kinetic model
9. Vollrath, F. & Knight, D.P. (1999) Structure and function of the silk for amyloid formation in the prion diseases: importance of seeding.
production pathway in the Spider nephila edulis. Int. J. Biol. Proc. Natl Acad. Sci. USA 90, 5959–5963.
Macromolecules 24, 243–249. 29. Jarret, J.T. & Lansbury, P.T. Jr (1993) Seeding ‘one-dimensional
10. Iizuka, E. (1985) Silk thread-mechanism of the spinning and the crystallization’ of amyloid: a pathogenic mechanism in Alzheimer’s
physical properties. J. Appl. Polym. Sci., Appl. Polym. Symp. 41, disease and scrapie? Cell 73, 1055–1058.
173–185. 30. Glover, J.R., Kowai, A.S., Schirmer, E.C., Patino, M.M., Liu, J.J. &
11. Willcox, P.J., Gido, S.P., Muller, W. & Kaplan, D.L. (1996) Lindquist, S. (1997) Self-seeded fibers formed by Sup35, the
Evidence of a cholesteric liquid crystalline phase in natural silk protein determinant of [PSIþ], a heritable prion-like factor of
spinning peocesses. Macromolecules 29, 5106–5110. S. cerevisiae. Cell 89, 811– 819.
12. Mita, K., Ichimura, S., Zama, M. & James, T.C. (1988) Specific 31. Sherzinger, E., Sittler, A., Schweiger, K., Heiser, V., Lurz, R.,
condon usage pattern and its implications on the secondary
Hasenbank, R., Bates, G.P., Lehrach, H. & Wanker, E.E. (1999)
structure of silk fibroin mRNA. J. Mol. Biol. 203, 917–925.
Self-assembly of polyglutamine-containing huntingtin fragments
13. Zheng, S.D., Li, G.X., Yao, W.H. & Yu, T.Y. (1989) Raman
into amyloid-like fibrils: Implications for Huntington’s disease
spectroscopic investigation of the denaturation process of silk
pathology. Proc. Natl Acad. Sci. USA 96, 4604–4609.
fibtoin. Appl. Spectrosc. 43, 1269–1272.
32. Gursky, O. & Aleshkov, S. (2000) Temperature-dependent b-sheet
14. Knight, D.P., Knight, M.M. & Vollrath, F. (2000) Beta transition
formation in b-amyloid Ab1240 peptide in water: uncoupling
and stress-induced phase separation in the spinning of spider
b-structure folding from aggregation. Biochim. Biophys. Acta.
dragline silk. Int. J. Biol. Macromolecules 27, 205 –210.
1476, 93 –102.
15. Morrissey, M.P. & Shakhnovich, E.I. (1999) Evidence for the role
33. Ho, S.P. & DeGrado, W.F. (1987) Design of 4-helix bundle protein:
of PrPc helix 1 in the hydrophilic seeding of prion aggregates. Proc.
synthesis of peptides which self-associate into a helical protein.
Natl Acad. Sci. USA 96, 11293– 11298.
J. Am. Chem. Soc. 109, 6751– 6758.
16. Harper, J.D. & Lansbury, P.T. Jr (1997) Models of amyloid seeding
34. Lyu, P.C., Liff, M.I. & Marry, L.A. (1990) Side-chain contributions
in Alzheimier’s disease and scrapie: Mechanistic truths and
to the stability of alpha-helical structure in peptides. Science 250,
physiological consequences of the time-dependent solubility of
amyloid proteins. Annu. Rev. Biochem. 66, 385–407. 669–673.
17. Wood, S.J., Wypych, J., Steavenson, S., Louis, J.C., Citrom, M. & 35. Harrison, P.M., Chan, H.S. & Prusiner, S.B. (1999) Thermo-
Biere, A.L. (1999) a-Synuclein fibrillogenesis is nucleation- dynamics of model prions and its implications for the problem of
dependent. J. Biol. Chem. 274, 19509–19512. prion protein folding. J. Mol. Biol. 286, 593–606.
18. Asakura, T., Watanabe, Y. & Uchida, A. (1984) NMR of silk 36. Fossey, S.A., Nemethy, G., Gibson, K.D. & Scheraga, H.A. (1991)
fibroin. 2. 13C NMR study of the chain dynamics and solution Conformational energy studies of beta sheets of model silk fibroin
structure of Bombyx mori silk fibroin. Macromolecules 17, peptides. I. sheets of Poly (Ala-Gly) chains. Biopolymers 31,
1075–1081. 1529–1541.
19. Ky Hirschberg, J.H.K., Brunsveld, L., Ramzi, A., Vekemans, J.J., 37. Chothia, C. & Janin, J. (1975) Principles of protein-protein
Sijbesma, R.P. & Meijer, E.W. (2000) Helical self-assembled recognition. Nature 256, 705 –708.
polymers from cooperative stacking of hydrogen-bonded pairs. 38. Ayub, Z.H., Arai, M. & Hirabayashi, K. (1994) Quantitative
Nature 407, 167–170. structural analysis and physical properties of silk fibroin hydrogels.
20. Yang, D.S., Yip, C.M., Jackson Huang, T.H., Chakrabartty, A. & Polymer 35, 2197–2204.
Fraser, P.E. (1999) Manipulating the amyloid-beta aggregation 39. Shimizu, S. & Chan, H.S. (2000) Temperature dependence of
pathway with chemical chaperones. J. Biol. Chem. 274, hydrophobic interactions: a mean force perspective, effects of
32970– 32974. water density, and nonadditivity of thermodynamic signatures.
21. Canetti, M., Seves, A., Secundo, F. & Vecchio, G. (1989) CD and J. Chem. Phys. 113, 4683– 4700.
small-angle X-ray scattering of silk fibroin in solution. Biopolymers 40. Magoshi, J., Magoshi, Y. & Nakamura, S. (1994) Mechanism of
28, 1613–1624. fiber formation of silkwoum. ACS Sym. Series 544, 292 –310.
22. Iizuka, E. & Yang, J.T. (1966) Optical rotatory dispersion and 41. Shen, Y., Johnson, M.A. & Martin, D.C. (1998) Microstructural
circular dichroism of the beta form of silk fibroin in solutions. Proc. characterization of Bombyx mori silk fibers. Macromolecules 31,
Natl Acad. Sci. USA 55, 1175–1181. 8857–8864.
23. Monti, P., Freddi, G., Bertoluzza, A., Kasai, N. & Tsukada, M. 42. Spek, E.J., Wu, H.-C. & Kallenbach, N.R. (1997) The role of
(1998) Raman spectroscopic studies of silk fibroin from Bombyx alanine sequences in forming beta-sheets of spider dragline silk.
mori. J. Raman Spectrosc. 29, 297–304. J. Am. Chem. Soc. 119, 5053– 5054.You can also read
