Toward Single-Fiber Diffraction of Spider Dragline Silk from

Chapter 16 Toward Single-Fiber Diffraction of Spider Dragline Silk from Nephila clavipes 1

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S. G. McNamee , C. K. Ober , L. W. Jelinski , E. Ray , Y. Xia , and D. T. Grubb 1

Downloaded by CORNELL UNIV on July 26, 2016 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch016

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Department of Materials Science and Engineering and Biotechnology Program, Cornell University, Ithaca, NY 14853

The results of a preliminary synchrotron X-ray study of the spider dragline silk from Nephila clavipes are presented. Comparisons of two different detector systems are made. The diffraction from 100-fiber bundles was observed as a function of environment and load on the samples. While the introduction of a dehydrating helium environment affected the tension of the samples, no concurrent change in the scattering behavior was observed. Mechanical loading of the bundles resulted in an increase in the orientation of the scattering centers within the fibers. Ideas for potential experimental protocols for single fiber diffraction are discussed. The dragline silk from the major ampullate gland of the golden orb weaver spider, Nephila clavipes, has excellent structural and mechanical properties. For example, the specific modulus of dragline silk is greater than that of steel and the ability of the silk to absorb energy is superior to that of Kevlar (1). Although research on this biomaterial has been ongoing for many years, it has generated renewed interest in view of the potential for modern biotechnology to produce engineered proteins. The physical and mechanical properties of silks in general continue to intrigue numerous investigators, as demonstrated by the breadth of topics covered at this workshop. For a good introduction to the subject, the reader who is not familiar with this biomaterial or the arachnid that produces it is referred to references 14 and the references contained therein, as well as other chapters of this volume. The fact that spider silk fiber is drawn from a liquid crystalline solution under mild conditions (5) is of great interest to those who wish

0097-6156/94/0544-0176$06.00/0 © 1994 American Chemical Society Kaplan et al.; Silk Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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to spin high-performance fibers from a liquid crystalline melt or solution. There are numerous factors (rheological, chemical, and thermal) that combine in the processing of spider silk to produce practically defect-free fibers. It is known that the nascent silk in the gland is transformed from an isotropic liquid to a liquid crystalline solution (5) as water is removed, but the role that water plays in the processing and in the material's structure and performance after the fiber exits the spinneret is not clear. If water is lost or gained as the fiber is spun from the spider, we may expect the microstructure of the fiber, that is the combination of β-sheet and amorphous domains, to change as the silk travels from the spider during the spinning process. Oriented fibers, crystalline domains and liquid crystalline phases all possess distinct X-ray diffraction characteristics that we would like to use to monitor microstructural changes upon spinning. The manner in which the microstructure of the fibers responds to post-spinning mechanical loading is also of interest, since the natural fiber's properties are set without the need for any post-spinning draw, in contrast to conventional synthetic polymerfiberprocessing. The use of in situ, time resolved, single fiber X-ray diffraction can help us to accomplish the goals of better understanding the processing and resultant microstructures of silk fibers drawn from the liquid crystalline state. This study is particularly timely in view of recent uncertainties in the molecular mechanism of spider silk elasticity (6,7). Fourier transform infrared (FTIR) techniques have been used to follow changes in the helical content of the silk as it is stretched (6). It is anticipated that X-ray diffraction can provide complementary results, with the added advantage of in situ measurements. This paper describes our preliminary work on spider silk diffraction using a synchrotron X-ray source. While these studies were carried out on multifiber bundles, it is our ultimate goal to investigate a single fiber as it is being spun from the spider and to determine the effect of process conditions on the resulting fiber. This effort to obtain X-ray diffraction information from a single thread of dragline silk is intended to complement the magnetic resonance imaging (MRI) and solid state nuclear magnetic resonance (NMR) work being done on the in vivo chemical processing of the fibers. The diffraction experiments provide information primarily about the crystalline components, whereas the magnetic resonance experiments will provide information about the molecular dynamics of the amorphous regions. More generally, the techniques developed will also be useful in ongoing research into synthetic high performance fibers and liquid crystalline polymers. Experimental Fiber samples of dragline silk were collected from the spiders of the species Nephila clavipes originally obtained in Panama and

Kaplan et al.; Silk Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

maintained in individual humidified cages. The spiders were hand-fed crickets three times a week. Fiber samples were obtained by the motor method at a draw rate of 24 cm/sec. Bundles of 100fiberswere mounted on card stock under just enough tension to avoid tangling of the fibers. The samples were mounted in a tensile apparatus developed for work at the Cornell High Energy Synchrotron Source (CHESS), which is described elsewhere (8). Care was taken to clamp the ends of the bundles as securely as possible, using adhesive tape and closed cell foam rubber grips on the tensile clamps. The distance between the clamps was changed by driving a stepping motor until the fibers were held steady. The piezoelectric load cell was then set to zero. The entire stretching device was mounted on a sensitive, steppingmotor-driven, uniaxial slide to allow micron-scale translation of the sample within the X-ray beam. Two separate detectors were used. The first was a storage phosphor image plate and Kodak scanner system which allowed a very large area of detection with 100 μπι resolution. The second was a direct coupled 80 mm diameter image-intensified detector of similar resolution that had a smaller active detector area but a much more easily used data-manipulation system. The interested reader is directed to references 9 and 10, which describe the two detectors that led to the one that was used in this experiment, and also to reference 11, which compares various quantitative X-ray imagers. The monochromated beam (hutch A l , CHESS, λ = 1.565 Â) was collimated to a diameter of 300 μιη when using the 80 mm detector and 500 μπι when using the storage phosphor. In the initial experiment using the storage phosphor, a poly(ethylene) bag was used to enclose the sample and the image plate in a helium atmosphere. Relative humidity within the bag was monitored as was the change in the tension of the fiber sample while the helium environment was being introduced. For the second experiment using the 80 mm detector, a stream of helium was passed over thefibersample while the change in the tension of the sample was monitored. Exposure times were about 5 minutes with a sample to detector distance of 58 mm using the 80 mm detector, and 10 minutes with a sample to detector distance of 102 mm using the storage phosphors. No visual evidence of beam damage to the fibers was observed after any of the scattering experiments. Results and Discussion During the initial experiment using the storage phosphor detector, it was observed that the tension on the fiber bundle increased when the sample was placed in a helium atmosphere, where the relative humidity was 30%. The helium atmosphere was introduced in an effort to reduce the air scatter that was fogging the image plate and obscuring the diffracted image. The air scatter was only slightly



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diminished and the use of the bag was discontinued when subsequent images were not improved sufficiently to warrant the inconvenience of the bag. It is important to note that careful background subtraction was necessary for observation of thefiberdiffraction pattern. Figure 1 shows equatorial scans through the diffracted images as captured by the two detectors employed in this study. The measured d spacing of the 120/210 reflections agree with the literature (12). The S/N for the storage phosphor is clearly better than that of the image intensifier and the area of the storage phosphor was much greater than the 80 mm detector. However, the data handling and manipulation was much more facile with the smaller area detector and allowed for much faster data collection. Further discussion of the data is limited to that which was taken using the image intensifier. The slight asymmetry of the image intensifier data is due to a slight skew in the face of the detector at the time of the experiment.

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2Θ (°) Figure 1. Comparison of the equatorial scans through the primary layer reflections as monitored by both detectors. 100-Fiber bundles under comparable loads of ca. 2 GPa. Figure 2 shows that no structural change could be detected as a result of the flow of helium over the bundle despite the 46% increase in stress. Prior to exposing the bundle to the helium, the load on the bundle corresponded to a calculated stress of 0.358 GPa. The stress increased to 0.524 GPa after the fiber tried to shrink as a result of the dehydrating flow of helium but was constrained by the tensile device. Scanning parallel to the fiber direction through the layer lines shows again no significant change in the crystalline scattering, as seen in Figure 3. In Figure 3, ξ is defined as arctan(L /a) where L is the n


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2Θ (°) Figure 2. Effect of helium (He) environment on the equatorial scattering from the fiber bundle. Data captured using image intensifier.

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distance from the center of the 120/210 reflections and a is the sample to detector distance. The right side of the image is profiled as it was the more intense side due to the aforementioned skew of the detector. Taken together, the data in Figures 1, 2 and 3 suggest that essentially no change occurs in the crystalline regions of silk upon dehydration and under low amounts of stress. However, the situation differs at greater levels of stress. Table I shows the stress to which the bundle was subject for each of the X-ray images captured on the 80 mm detector after the bundle had been exposed to the helium and then left to equilibrate in the ambient humidity. The increase in stress for exposure A was a result of the attempted shrinkage of the fiber after application of the helium. Subsequent increases in the stress on the sample were a result of mechanically stretching thefiberbundle. The diameter of a single fiber was approximately 4 μιη; a 100-fiber bundle would have a crosssectional area of 1257 μιη . This value was used for calculating the stress on the bundles while under tension. Strain calculations were not made, given the uncertainty of the differences in the modulus of the fiber before and after being subject to the helium environment. Experiments to compare the effects of environment on the single fiber and bundle moduli are presently underway. 2

Table I: Mechanical loading of the sample within the X-ray beam Exposure reference

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Stress(GPa)

A Β C D

67.3 159.1 246.3 321.3

0.524 1.24 1.92 2.51

% increase in stress 46 136 55 30

One can see in Figure 4 that the intensity of the equatorial reflections increases slightly with increasing load, but that the position of the peaks remains constant. Analysis of the diffraction peak shapes showed a slight decrease in the FWHM with increasing load. This indicates an increase in the orientation of the scattering centers. Our results show very little change in the crystalline component under stress, suggesting that the amorphous component must participate in the distribution of load in the fiber. The data are in qualitative agreement with FTIR studies (6), and would further suggest that spider silk's excellent mechanical properties depend greatly on the amorphous regions' response to stresses. A slight rotation of the 120/210 reflections about the axis of the X-ray beam is due to a straigh-



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tening out of the fiber as it is loaded, a problem that the next generation of tensile tester will address.

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15 20 25 2Θ (°) Figure 4. Effect of stress on equatorial scatter from thefiberbundle. Only the right-hand reflection is shown. Although we have previously been successful in scattering from single fiber synthetic polymer samples (S), we were unable to capture a signal from a single fiber of dragline silk. We attribute this to several factors. Because the volume of material was small and the inherent cross-sectional scattering areas of the atoms that make up the silk are small as well, sufficient exposure times for the X-ray fluxes would have been prohibitively long. Furthermore, the S/N from a single fiber was simply too small for the detectors that were available. By using a bundle of 100 fibers, the S/N problem was overcome in the present study, but that, of course, precluded probing the silk as it was being spun from the spider. However, the data reported here enable us to calculate the feasibility of in situ scattering from a single fiber. Our calculations suggest that it will be necessary to either develop new detectors or increase the flux of X-rays scattering from the fiber. Work on the former is progressing, but it is the increase in flux that holds particular promise at this time. The sample of interest is only a few microns wide, so much of the 500 μπι-wide X-ray beam is wasted. Very precise X-ray slits may be used, but the problem of aligning the fiber sample to the beam becomes more difficult. Furthermore, in order to probe the fiber at different points along its length, the slits would have to be so short that the primary beam would be attenuated to the same extent as with present



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cylindrical collimators. A solution to this dilemma may emerge from the research being done at CHESS in the area of tapered glass capillaries to funnel a 500 μπι beam of X-rays to a submicron diameter beam of very high brightness (23). Using tapered capillaries, one can obtain an increase in flux on the order of 1000-fold. Development of such a system for use with single polymer fibers, including dragline silk, is currently underway. This work has shown the feasibility of probing the microstructure of spider silk fiber bundles under various conditions of stress and environment with a sufficiently bright X-ray source. As the technology of micron-sized X-ray beams is developed, the possibility of scattering from a single fiber of spider silk becomes greater. Within such an experimental protocol, one may scan across a single fiber to profile the domains of β-sheet packing vs. other regions in the material. It may be possible to follow the transition of the ηοη-β-sheet regions as they transform due to water adsorption or absorption, due to applied mechanical strain, or due to other changes in the environment. Should the development of thefiberfroma liquid crystalline solution happen as the silk emerges from the spinneret, it may be possible to follow the concurrent change in the microstructure as the single fiber is being drawn from the spider, profiling the fiber at different draw rates or at different distancesfromthe spinneret. Acknowledgments The authors wish to thank E. Eikenberry and S. Gruner for the very generous use of their 80 mm detector. Funding by the NSF and the ACS/PRF is gratefully acknowledged. The authors appreciate the use of the facilities of CHESS and Cornell's Materials Science Center. S. Mc. thanks the Department of Education for a fellowship. Other people who have aided in this study include M. Went, C. Trojan, and Z. Li. Literature Cited 1. 2. 3. 4. 5. 6.

Gosline, J . M.; DeMont, M. E.; Denny, M. W. Endeavour. 1986, 10, 37-43. Vollrath, F. Scientific American, 1992, Mar.,70-76. Lewis, R. V. Accounts of Chemical Research, 1992, 25, 392-398. Kaplan, D. L.; Lombardi, S. J.; Muller, W. S.; and Fossey, S. A. In Biomaterials: Novel Materials from Biological Sources; Byrom, D., Ed.; Stockton Press: NY, NY, 1991; pp 3-53. Kerkam, K.; Kaplan, D.; Lombardi, S.; Viney, C. Materials Synthesis Based on Biological Processes. MRS Proceedings, 1991, Vol. 218; pp 239-244. Dong, Z.; Lewis, R.V.; Middaugh, C.R. Arch. of Biochem. and Biophys. 1991, 284, 53-57.


184 7. 8. 9. 10.


11. 12. 13.


Hinman, M.B.; Lewis, R.V. J. Biol. Chem. 1992, 267,19320-19324. Prasad, K.; Grubb, D. J. of Polym. Sci.: Part B: Polym. Phys. 1990 28, 2199-2212. Templer, R.H.; Gruner, S.M.; Eikenberry, E.F. Advances in Electronics and Electron Physics Academic Press: London, 1988; pp 275-283. Eikenberry, E.F.; Tate, M.W.; Belmonte, Α.; Lowrance, J . ; Bilderback, D.; Gruner, S.M. IEEE Trans. Nucl. Sci., 1991, 38, 110-118. Eikenberry, E.F.; Tate, M.W.; Bilderback, D.H.; Gruner, S.M. In Photoelectronic Image Devices, 1991; Morgan, B.L., Ed.; Institute of Physics; Bristol, 1992; pp 273-280. Work, R.W.; Morosoff, N. Text. Res. Jour. 1982, 52, 349-356. Hoffman, S.A.; Thiel, D.J.; Bilderback, D.H. In Optics for HighBrightness Synchrotron Radiation Beamlines; SPIE: 1992;Vol. 1740, pp 252-257.

R E C E I V E D May 4, 1993


Toward Single-Fiber Diffraction of Spider Dragline Silk from

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