Variation of Mechanical Properties with Amino Acid ... - ACS Publications

Biomacromolecules 2004, 5, 732-738

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Variation of Mechanical Properties with Amino Acid Content in the Silk of Nephila Clavipes David B. Zax,* Daniel E. Armanios,† Sally Horak,‡ Chris Malowniak, and Zhitong Yang Department of Chemistry and Chemical Biology, Baker Laboratory, and Cornell Center for Materials Research, Cornell University, Ithaca, New York 14853-1301 Received August 20, 2003; Revised Manuscript Received February 20, 2004

In this paper, we explore the impact of dietary deprivation, where spiders are provided diets missing one or more of the amino acids, on the properties of the spider dragline silk spun after one month on the diet. Cohorts of female N. claVipes spiders were selected for diets deprived of alanine (Ala) and glycine (Gly), arginine (Arg), leucine (Leu), or tyrosine (Tyr), and their silk was harvested twice weekly during the onemonth course of the diet. Significant mechanical differences are observed after as little as 6 days on the diet. Utilizing conventional tensile testing methods, single fibers were strained to break so as to study the influence of diet on the stress/strain properties. Diets deprived of Ala and Gly appear to most directly impact the load-bearing foundation of dragline silk. Diets deprived of Arg, Tyr, and possibly Leu reduce the strength of the silk, and diets missing Tyr and Leu reduce the strain-to-failure. Observations obtained from ESEM photos of the fracture interfaces after tensile testing illustrate the fracture mechanics of spider silk. Both solid-state NMR and amino acid analysis of the digested protein suggest, however, that the relationship between diet and amino acid incorporation into the silk fiber is not straightforward. Introduction Spider silk, a natural polymer based on repeating amino acid motifs, fascinates the materials world because of its high toughness and strain to failure. Spiders make different types of silk, and their number, properties, and purposes vary among different spiders, though, for many silks, the primary protein sequence has been determined and certain broad trends have been identified. Work and Young have observed that significant variations of amino acid composition are found, even within a single species and for a single type of silk, and even from the same spider silked at different times.1 Ko et al. have studied the tensile strength and shearing mechanisms of N. claVipes dragline silk and compared its properties to those of silk obtained from the spider A. aurentia.2 Shao and Vollrath also tested single fibers and demonstrated the importance of solution media in determining the mechanical properties of spider silk.3 Although efforts to identify the fundamental building blocks, the highly conserved sequences of amino acid residues in the silks, and their role in these multiphase materials, relatively less effort has been applied to understanding the role individual amino acid residues play in determining bulk properties. With the recent demonstration that substantial quantities of spinnable protein solution can be produced outside of the spiders’ silk glands,4 however, the idea that modifications of properties might be effected by modifications of sequence becomes * To whom correspondence should be addressed. E-mail: dbz1@ cornell.edu. Phone: 607 255 3646. Fax: 607 255 4137. † Current address: Department of Mechanical Engineering, University of Pittsburgh. Benedum Hall, Pittsburgh, PA 15261. ‡ Current address: Cortland Junior-Senior High School, 8 Valley View Drive, Cortland, NY 13045.

more relevant. On the other hand, absent a convincing postproduction processing regimen which can reproduce the desirable mechanical properties, such “unnatural” synthetic routes cannot be usefully tested, as the isolation of “chemical” effects from processing is difficult. Nonetheless, within certain limits, the natural processes by which silk is synthesized in the spider itself can be modified. It is well-known that differences in diet can have significant influence on the quality of the final silk product,5 and we thought to exploit this observation in a somewhat more systematic fashion than has appeared elsewhere. In this paper, we describe some of our efforts to understand the contributions of specific amino acid residues to the bulk properties which make the dragline silk of N. claVipes spiders admired as a high-performance fiber. Our hope is that by focusing more attention on the individual amino residues, and by studying how mechanical properties vary with changes in the relative abundances of these amino acid residues, we can provide further input to models which seek to explain mechanical behavior, as, for example in the work of Termonia.6 Although the primary sequence of amino acids which comprise the dragline silk is known,7 there is still debate as to how individual molecules of silk are assembled into the silk fiber. We have previously presented a model based on dynamical NMR measurements. Illustrated in Figure 1 is the partial consensus sequence of the dragline silk of N. claVipes. The figure provides our correlation between primary structure and dynamically distinct regions of the silk fiber.8 Both the role of individual amino acids in determining the different phases in the fiber and the issue of the stacking of molecules so as to form fibrils remain unclear. Due to the multiple

10.1021/bm034309x CCC: $27.50 © 2004 American Chemical Society Published on Web 04/03/2004

Mechanical Properties in Amino Acid Deprived Silks

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Figure 1. Amino acid structure of N. clavipes dragline silk. The repeating amino acid units are represented according to their dynamical classification; white letters on black background represent the β-sheet region, immobilized amino acid units; open letters represent the amorphous region which goes floppy when wet, and the dark letters represent the transitional region which is partially mobile when wet.

levels of complexity presented in this polymer and the large numbers of monomers which comprise the polymer, it seems unlikely that any of the theories which explain how properties are modified by the introduction of occasional “odd” monomers will prove predictive. In Figure 1, we note that only a relatively small number of amino acids appear, and their appearance is limited to a small number of recurring motifs. Within these motifs, we have identified five amino acid residues as targets for this study: alanine (A), glycine (G), leucine (L), arginine (R), and tyrosine (Y). We have little understanding of the biochemical processes which take place in the spider’s metabolism; although some of these amino acids (R and L) have been identified as “essential” in the silkworm’s diet,9 there is no similarly detailed understanding of the role of essential and nonessential amino acids in our spiders. Alanine and glycine are the primary contributors to the crystalline region, arginine and tyrosine appear almost uniquely at the ends of the amorphous zones, and leucine appears both in the transitional zones between the amorphous and crystalline zones and well into the middle of the amorphous linkers. However, the role each of these amino acids plays in the mechanical and chemical mechanisms of spider dragline silk is still unknown. Furthermore, it is also still unknown how spider silk fractures, which would provide more clues on how spider silk responds under various loading conditions. For this work, the effects of these five key amino acids in the formation of N. claVipes dragline silk on the mechanical properties of the silk as well as the fracture mechanics of spider silk were investigated. Materials and Procedures 1. Sample Preparation. Spider silk was obtained from a group of female N. claVipes spiders; the spiders were divided

Figure 2. Specimen layout used to place dragline silk fiber in tensile testing apparatus. Black dots represent where the fibers were initially glued; the dashed line shows how the fibers were placed over the holes.

into different cohorts based on the diets provided to the spider. One group was provided a diet of crickets several times weekly; their silk was taken to be the control silk representative of the native material. Other groups of spiders were deprived of specific amino acids in a diet which consisted of an amino acid mixture, DMEM (Delbucco’s Modified Eagle’s Medium) and glucose, but missing one or more amino acidssarginine, tyrosine, leucine, or both alanine and glycine. The diets were maintained for one month, during which time the dragline silk was harvested by the forced silking method of Work and Emerson10 twice weekly. The collected silk was then stored in airtight containers for subsequent analyses. In preparation for mechanical testing on the Instron Tensile Testing Platform, individual silk fibers were placed on paper tabs as shown in Figure 2. Holes were punched in the middle of the paper, as shown, before placing the fibers on the specimen layout. Surgical scissors and tweezers were used

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Table 1. Mechanical Properties of Fibers from Dietarily Deprived Spiders amino acid deprived # fibers tested % strain-to-failure force (N) displacement (mm) ultimate stress (GPa)

control

Tyr

Arg

Ala/Gly

Leu

4 18.5 0.0163 1.88 1.41

6 8.35 0.0120 0.849 0.943

4 12.4 0.0486 1.26 0.868

4 8.97 0.0476 0.912 0.779

5 8.74 0.00650 0.883 1.23

to manipulate the fibers so that each was straight and exposed through the hole. Additional paper tabs were placed atop each fiber and fastened in place with all-purpose plastic cement. Diameters of each fiber were measured under a polarized light microscope (Olympus CH series) at 400× utilizing a stage micrometer. Samples presented in this work represent silks harvested from spiders fed on the amino acid deprived diets for one month. 2. Tensile Loading. Tensile testing was performed on an Instron model 1125 retrofitted with model 4400R electronics, in a room maintained at 50% relative humidity and at 23 °C. The fibers were placed in the tensile stage with grips placed at the initial gluing points of the paper tabs, so that the grip and gauge lengths were equal. The initial gauge length was maintained at a constant of 1 cm. The paper sides along the hole were cut to ensure that only the fiber would experience tensile loading. Fibers were then stretched at a constant rate of 0.5 mm/min using a 500-gram load cell. In preparation for each day’s measurement, the load cell was calibrated. Fractured pieces were removed from the grips and saved for subsequent study via the environmental scanning electron microscope (ESEM), a special SEM which required no “sputter” coating. Samples were mounted on a Pelltier stage using carbon conductive tape, which itself was placed in an electro-scan ESEM chamber (Ernest B. Fullam, Inc., Albany, NY). The chamber environment was carefully maintained under a vacuum at 6-7 Torr, maintained through constant flooding of the chamber with water. Utilizing a beam gun with a voltage of 10.0 kV, photos were taken of the fracture interfaces at 245×, 645×, or 1100×. 3. NMR Measurements. Any model for the impact of dietary deprivation on the bulk properties of silks requires an assessment of the impact specific amino acids the sequence have on the different bulk properties. We have attempted to assess this by 13C NMR spectroscopy on both natural silks and samples of silks taken from the dietarily deprived spiders, at the same 30 day period after initiation of the amino acid deprived diets. Spectra were taken on a home-built NMR spectrometer operating at 383 MHz for 1H, under high-resolution solid-state conditions. Samples were packed into a 4 mm magic angle spinning (MAS) rotor spun at 15 kHz, and the natural-abundance 13C signals were enhanced using cross-polarization (CP). The native silk NMR spectrum corresponded to 10.95 mg of silk; for each of the dietarily deprived samples, only 1-2 mg were available for spectroscopic analysis. 4. Amino Acid Analyses. From each dietary cohort, fiber pieces were chosen and an amino acid analysis was performed. From 50 to 350 µg of the protein fiber was sent to the W. M. Keck Biotechnology Resource Laboratory at

Table 2. Percent Abundances of Amino Acids Found in Dragline Silk Fibers, as a Function of Dieta amino acid deprived Gly Ala Glx Leu Tyr Ser Pro Arg Asx Val Thr Ileu Phe Lys sample mass (µg)

control

Tyr

Arg

43.5 29.0 10.8 4.3 2.9 2.7 2.0 1.6 1.0 0.8 0.5 0.4 0.3 0.2 8.62

44.1 28.2 10.9 4.5 3.4 2.8 1.2 1.7 1.0 0.8 0.5 0.4 0.3 0.2 5.871

44.4 28.2 10.9 4.3 3.4 2.8 1.0 1.7 1.1 0.7 0.5 0.4 0.2 0.2 2.117

Ala-Gly

Leu

45.0 28.9 11.1 4.4 3.4 3.2

45.1 29.1 10.6 4.5 3.4 3.1

1.7 1.3

1.9 1.4

0.4 0.5

0.5 0.5

0.322

0.349

a

Amino acids are listed in descending order of abundance in the silk fiber from the control populations of cricket-fed spiders. Glx and Asx are combined abundances for glutamic acid/glutamine and aspartic acid/ aspartamine, respectively. Amino acids detected at 0.1% or below are not listed.

Yale University. There the fiber was hydrolyzed for 16 h at 115 °C in 100 µL of 6 N HCl, 0.2% phenol that also contains 2 nmol norleucine which served as an internal concentration standard. The resulting solution was dried and redissolved in 100 µL Beckman sample buffer and finally transferred to a Beckman model 7300 ion-exchange instrument for analysis of the amino acid composition. Results for each of the five fibers tested are provided in Table 2, except that the method provides only the sum of aspartate and aspartic acid and of glutamine and of glutamic acid, which are listed in the table as Asx and Glx, respectively. Results 1. Supercontraction of Silk in Water. Samples taken in the first few days from the different cohorts show few differences in properties; but by approximately one week into the amino acid deprived diets, significant differences are observed. In Figure 3 we demonstrate this effect with a measure of the extent of supercontraction found for the different silk samples, as a function of diet after 4 and after 6 days. In the early samples, differences in the unconstrained length when wet to that when dry vary only minimally from one another, but by the sixth day, significant differences are found. In particular, the Arg- and Ala/Gly deprived spiders produced silks which supercontract much more than the silk from Leu- or Tyr-deprived spiders or the spiders on “normal” diets. These observations suggested that a more detailed study was necessary. We describe below the more extensive tensile


Figure 3. Measurements of extent of supercontraction of N. clavipes dragline silk as a function of time on amino acid deprived diet. Measurements are the relative lengths of fibers when wet and dry, after 4 and 6 days on the diet. Diet label denotes the amino acid that was deprived from the spiders’ feeding, except that the control (CTRL) group was fed crickets. Little difference is measured after 4 days, but after 6 days, the different dietary cohorts demonstrate very different supercontraction behavior.


Figure 5. Stress-strain curves of dragline silk from Leu-deprived spiders. Only polynomial fits to the stress-strain data are shown, so as to clarify the different fiber measurements. Natural variability between different fibers is shown, though the fibers within one dietary cohort resemble one another more than fibers from other cohorts.

Figure 4. Force-displacement curves of dragline silk from Argdeprived spiders. Four fiber measurements are shown; the light background corresponds to the raw data obtained under tensile testing, whereas the heavy lines correspond to the illustrative polynomial fits to the data. Some natural variability in the different fibers is expected.

Figure 6. Stress-strain curves of individual fibers of dragline silk from all cohorts of dietarily deprived spiders, as well as from the control grouping. Only polynomial fits to the stress-strain data are shown, so as to clarify the different fiber measurements, and for only a single fiber representative (i.e., with neither unusually large or small stress or strain-to-failure) of the cohort is shown. Natural variability between different fibers within a cohort is seen, though these differences are less pronounced than those observed between different cohorts.

testing, and some of the solid-state NMR measurements, which provide some enlightenment as to the effect of diet on properties. 2. Mechanical Properties. Figures 4-6 represent the data observed in tensile testing. Each curve represents a single fiber measurement, though multiple fibers (4-6) were tested from each cohort of spiders. Figure 4 represents the forcedisplacement data derived directly from the tensile measurements on a group of fibers derived from the Arg-deprived spider cohort. Each measurement consists of many hundreds of data pairs. As there was considerable scatter in the raw data, we have superposed on each set of measurements a smoothed curve representing a polynomial regression (typically 6 or 8 coefficients) through the raw data. The polynomial regression provides a substantial reduction in the measurement noise, though we associate no particular physical meaning to the coefficients thus derived. Figures 5 and 6 represent, instead, the stress-strain curves derived from the force-displacement data sets. The stress-strain curves are more representative of the fiber properties, as the differences in fiber diameters contribute strongly to the force required to break any individual fiber but are irrelevant to

the stress-strain measurements. Each single fiber of spider silk was assumed cylindrical with constant diameter throughout its length, and we further assumed that the change in gauge length was identical to the change in the grip length, as it would be if each fiber is securely held in place. In Figure 4 ,we present instead the polynomial fits to the stress-strain data for four different fibers from the Leu-deprived cohort and in Figure 5 representative fibers (i.e., fibers with neither maximum or minimum stress to break) from each of the five dietary cohorts. Numerical averages from multiple fibers (between 4 and 6) are list in Table 1 for each group. The values for normal silk differ somewhat from those of Ko et al.; many reasons that might contribute to this difference are unclear but many factors contribute to the differences in mechanical strength, and the samples we have tested were from spiders who had been in captivity for relatively long periods. Furthermore, the samples were stored in airtight bottles nearly one year before the mechanical testing was attempted. We focus therefore on the differences between cohorts, all of which were treated comparably in our laboratory. Where we take the properties of the cricketfed spiders’ silk as “normal”, we find that each silk produced under dietary stress showed reduced stress and strain to

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Figure 7. ESEM micrographs of a fiber of N. clavipes dragline silk after tensile testing, at magnifications as indicated in the figures. (a) Splitting occurring along the fiber axis of a single fiber. (b) Splitting of a single fiber across its axis. (c) Evidence for microfibrils within a fiber.

failure. Nonetheless, the differences between cohorts were large, as is demonstrated in Figure 5. Fibers from spiders deprived of Ala and Gly failed at roughly half the stress and strain as the normal fibers; the Arg-deprived fibers showed reductions of roughly one-third in each property. In Tyrdeprived spiders, fibers showed reductions in ultimate stress to failure of one-third and in strain to failure of greater than one-half. Finally, the fibers from leucine-deprived spiders showed minimal reductions of stress to failure (only oneeighth), yet the percent strain to failure fell by half. The same trends are, of course, seen in the force-displacement curves. ESEM photos demonstrate that spider silk failure mode involves splitting along the fiber axis, as seen in Figure 7a, though splitting across the fiber axis is also observed (Figure 7b). Once delamination has occurred, even smaller microfibrils can also be seen (Figure 7c). 3. Solid State 13C NMR Spectra. The 13C spectrum represents the distribution of amino acids found in the sample, as each 13C is nearly equally likely to appear in each of the C positions in each of the amino acids. The spectrum of the native silk at the bottom of Figure 8 demonstrates a typical 13C spectrum of the silk protein. In the vicinity of shifts 170-180 ppm are found all of the carbonyl resonances, largely overlapping with one another. The region from 110 to 160 ppm, generally representative of aromatic and double bonded carbons, is relatively unpopulated in proteins; it corresponds to the carbon resonances of side chains containing phenyl rings in Tyr and Phe (little of which appears in silks) and the guanidinyl group in Arg. The CR resonances of all of the amino acids (other than Gly, at 45) appear between 52 and 59 ppm, the Cβ cluster primarily in the region 29-42 (though outlying peaks include the Ala resonance at 20 ppm, and Ser at 64 ppm). The longer alkyl chains show further resonances at well-characterized peaks.11,12

Figure 8. 13C solid-state CP/MAS NMR spectrum of native silk (bottom) and the difference spectra of silk from dietary stressed spiders, representing the difference between the (scaled) spectra of the dietarily deprived cohorts and the normal spider’s silk. Positive peaks reflect sites in excess in the native silk; peaks plotted with negative intensity are in relative excess in the dietarily deprived silk. Many of the shifts are associated with only one possible amino acid residue, or reflect the local amino acid conformation, and can therefore be used to provide evidence as to the local structure of the silk protein molecule. Assignments are suggested in the text.

To evaluate the NMR spectra, we focus on the differences between native silk spectrum at bottom, and the spectra observed for the four dietarily deprived species. Because of the substantial differences in signal intensities observed, as we have substantially more of the native silk available for study, the spectral intensities were normalized based on the assumption that, other than the small number of carbonyl sites found in the relatively rare amino acids Glu, Gln, Asn, or Asp, the carbonyl resonances appear one per amino acid


residue. As a result, the spectra of the dietarily deprived samples were scaled so that the areas under the carbonyl resonances of the native and special fibers were equal and then were subtracted from the native spectrum. Although this leaves some intensity, both positive and negative, in the region of the carbonyls, representing the slight variations in resonance shifts with chemical substitutions and changes in local conformation, the more diagnostic region of the spectrum is found in the region of smaller shifts where peaks plotted with positive intensity represent resonances missing in the deprived silk, whereas peaks plotted with negative intensity are in relative excess in the dietarily deprived silk. For example, in the Ala/Gly deprived diets, silk produced seems to have significantly less of the strong spectral peaks at 43 and 50 ppm, which is precisely the signatures associated with Ala and Gly in β-sheet regions of a protein, and what might be an excess of Tyr (negative peaks near 138 and 176). Thus, dietary deprivation in this case leads directly to less incorporation of Ala and Gly into the silk. Similarly, in the Leu-deprived diet, we see significant increases in the fraction of 13C resonances, and thus amino acids, corresponding to the Ala and Gly sites in those same β-sheet sites. (As none of the Leu resonances are well-removed from the general background, it is difficult to see in these spectra whether there has been a simultaneous depletion of the Leu from the final product.) Results for the other two samples are even less intuitive. In the Tyr-deprived silk, there appears to be an overall shift to greater R-helical and less β-sheet content and to a slight decrease in the Ala/Gly fraction. Finally, in the Arg-deprived silk, there appears to be less β-sheet Ala and Gly and perhaps lower Gln signal (though we are unable to identify the change in Arg as, similar to the Leu work, no easily identified peaks are unique to the Arg). 4. Amino Acid Analysis. Surprisingly, the amino acid analyses proved less useful than was the evidence provided from solid state NMR. Although we expected significant variations between fibers, and in particular that as dietary influence had a systematically significant influence on mechanical properties it should similarly inpact the incorporation of amino acids into the fiber, in practice, the amino acid counts reported to us via HPLC/MS varied surprisingly little between the different dietary cohorts. No systematically significant differences could be observed in the group of amino acids that appear at greater than 1% of all residues in the control group of silk fibers, except that all dietarily deprived cohorts incorporated less proline (P) into the silk fiber than the cricket-fed spider, and all of the dietarily deprived cohorts incorporate more of the arginine; otherwise, only the most abundant residues (G, A, and the combined glutamine/glutamic acid count) seemed to vary noticeably. Whether this reflects the impact of changes in abundances too small to be statistically significant, or an indication that the effect we are seeking is not simply reflected in the abundances of amion acids, and instead would require an understanding, for example, of their sequence in the fiber, remains unclear. Discussion Our goal was to study the effect of chemistry on silk properties. The data presented in Figure 6 and Table 1


suggest experimentally that the deprivation of Ala and Gly reduces strength, which is consistent with the models of Gosline13 and Termonia6 as well as the general observation that Ala and Gly form the bulk of the residues found in the load-bearing structural foundation of spider dragline silk as well as most other silk-like fibers found in nature. Loss of strength in the Arg and Tyr-deprived silk fibers would appear similarly associated with the loss of Ala and Gly from β-sheet regions of the protein. Leucine’s role is more difficult to assess, other than the surprising observation of an increase in the Ala and Gly found in the silk. As a result the stress to failure seems to change little; on the other hand, the strain to failure, which presumably is more closely associated with the contributions from the amorphous regions of the fiber, changes dramatically. Conclusion The complex molecular structure of spider dragline silk leaves many questions about the role of design parameters in function. In this paper we have initiated an investigation into the roles of specific amino acids in determining tensile properties of the dragline silk from N. ClaVipes. Dietarily deprived spiders were silked and the fibers harvested in the process have been subjected to mechanical testing and solidstate NMR analyses. These studies confirm the key role played by Ala and Gly in establishing the basic load-bearing structure. The roles of other amino acids are less clear, as the relationship between dietary deprivation and incorporation into the silk fiber is complex, and in many cases the deprivation of Arg, Leu, and/or Tyr appears to be strongly correlated to a disruption in the Ala/Gly concentration and/ or conformation in the fiber. Further studies by MS which will determine the amino acid distributions in the differing fiber cohorts,and by ESEM which will provide a snapshot of the failure mode in broken fibers should help clarify the microscopic and macroscopic details. From initial ESEM pictures, splitting fractures are the most prevalent form of failure as it is seen both along and across the polymer’s fiber axis. If Figure 7c proves to be correct, then lamina or some form of microfibrils would be existent in spider silk. This would mean that, across the fiber axis, splitting would be evidence of delamination. Since tensile tests pull along the silk’s fiber axis, overcoming internal shearing along the fiber axis would be necessary to commence delamination across the fiber axis. Acknowledgment. This work would not have been possible without the many hours of aid and support from facility managers John Sinnott and John Hunt in the material testing and ESEM facilities, respectively, at the Cornell Center of Material Research. The technical advice and expertise of Drs. John P. Holmes and D. Stefan Dancila from the Georgia Instiute of Technology is gratefully acknowledged, as is support provided through the National Science Foundation Materials Research Science and Engineering Centers (MRSEC) Program (DMR-0079992) and the REUSite program Research Experience for Teachers programs (DMR-0097494) of the National Science Foundation. Additional funding is provided by Cornell University.

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References and Notes (1) Work, R. W.; Young, C. T. J. Arachonol. 1987, 15, 65-80. (2) Ko, F. K.; Kawabata, S.; Inoue, M.; Niwa, M.; Fossey, S.; Song, J. Materials Research Society 2001 Fall Meeting Proceedings, Symposium U1.4. (3) Shao, Z.; Vollrath, F. Polymer 1999, 40, 1799-1806. (4) Lazaris, A.; Arcidiacono, S.; Huang, Y.; Zhou, J.-F.; Duguay, F.; Chretien, N.; Welsh, E. A.; Soares, J. W.; Karatzas, C. N. Science 2002, 295, 472-477. (5) Craig, C. L.; Riekel, C.; Herberstein, M. E.; Weber, R. S.; Kaplan, D.; Pierce, N. E. Mol. Biol. EVol. 2000, 17, 1904-1913. (6) Termonia, Y. Macromolecules 1994, 27, 7378-7381.

Zax et al. (7) Xu, M.; Lewis, R. V. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 71207124. (8) Yang, Z.; Liivak, O.; Seidel, A.; LaVerde, G.; Zax, D. B.; Jelinski, L. W. J. Am. Chem. Soc. 2000, 122, 9019-9025. (9) Arai, N.; Ito, T. Bull. Sericul.: Exp. Sta. 1967, 21, 373-384. (10) Work, R. W.; Emerson, P. D. J. Arachnol. 1982, 10, 1-10. (11) Howarth, O. W.; Lilley, D. M. Prog. NMR Spectrosc. 1978, 12, 1-40. (12) Saito, H. Magn. Reson. Chem. 1986, 24, 835-852. (13) Gosline, J. M.; DeMont, M. E.; Denny, M. W. EndeaVour 1986, 10, 37-43.

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