1. Structural characterization of worm and spider silk on cross section surface Weizhen Li Evgeny Klimov Joachim Loos

2. Bombyx mori Bombyx mori worm cocoon Natural Silk Nephila edulis spider silk NATURE 418 (6899): 741-741 AUG 15 2002

3. B. Mori Silkworm fibre A. trifasciata spider silk Sericin coating Engineering Fracture Mechanics 69 (2002) 1035–1048 Proc. R. Soc. Lond. B 263 (1996)147-151

4. Protein conformation – secondary structures

5. Our task:  Vibrational spectroscopic analysis on silk’s cross section  The existence of shell-core structure (Raman mapping, high spectral resolution )

6. Experiment  Embedding fibre into epoxy resin Use microtome to cut sample into slices with thickness of 10-30  m LVSEM

7. AFM AFM images (phase contrast) of the cross section of B. mori (A) and N.edulis (B)

8. XYZ- positioning CCD Andor laser microscope electronic control unit PMT CCD SPM SPM and positioning control electronics SPM positioning optics Laser: He-Ne 632.8 nm XY-resolution: 500 nm Z – resolution: 0.5 - 1  m Spectral resolution: 0.01nm Samples: solids, liquids, bulk, thin films, powder Raman analysis: scanning confocal Raman microscope “Nanofinder”

10. B. mori worm silk Part One

11. Overview spectrum and bands assignment Amide ІAmide Ⅲ random1660-1666 1245-1250 1085 β sheet1665-16801230-1245 αhelical 1675 1645-1658 1264-1310 Surface of degummed wormsilk β sheet J. Raman Spectrosc. 1995 26 901-909 J. Raman Spectrosc. 2001 32 103-107

12. Raman intensity distribution of amide I at 1665 cm -1 High spectral resolution Raman image of silk cross section

13. Core Edge Worm silk spectra with high resolution (After subtraction of epoxy) Sample thickness:30μm amide Ⅰ amide Ⅲ

14. Confocal Raman- high spatial resolution Notch filter Sample Photomultiplier or CCD detector Principle without pinhole with pinhole High spatial resolution

15. Edge and Core area of fibre’s cross section 2  m 30 spots 60-70 nm of one step Average

16. Raman data of edge and core area Core Edge The ratio I(850)/I(830) is a spectral marker of tyrosine hydrogen bonding strength.

17. 850/830 cm -1 Intensity ratio Sample 1Sample 2Sample 3 edgecore edgecoreedge core 850 cm -1 22.7922.2123.68 31.22 20.2422.56 830 cm -1 15.6114.5018.1022.32 12.4 14.30 I(850 cm -1 )/ I(830 cm -1 ) 1.461.531.311.401.631.58 Stable across entire cross section The ratio I(850)/I(830) is reduced going from moderately to strongly hydrogen-bonded tyrosines.

18. Nephila edulis Spider silk Part Two

19. Surface of single fibre —Nephila spider Amide ІAmide Ⅲ random1660-1666 1245-1250 1095 β sheet1665-16801230-1245 αhelical 1675 1645-1658 1264-1310 β sheet Conformation J. Raman Spectrosc. 1995 26 901-909 J. Raman Spectrosc. 2001 32 103-107

20. Raman image Raman intensity distribution of amide I at 1665 cm -1 2  m 30 spots 60-70 nm of one step

21. Core Edge Raman data of edge and core area

22. 850/830 cm -1 Intensity ratio Sample1 core Sample1 edge Sample2 core Sample2 edge 850 cm -1 11.81814.4746.6458.074 830 cm -1 8.235 13.253.2805.982 I(850 cm -1 )/ I(830 cm -1 ) 1.4351.091.721.35 The strength of hydrogen bonds involving the tyrosine residues may influence the forming of core-shell structure of N.edulis. 1.3 times

23. AFM image AFM height (left) and phase contrast (right) images of worm silk (top) and spider silk (bottom) Globular spherical features Diameter: 70-90 nm Less pronounced globular structure multiple nanovoids Multiple 200-300 nm large longitudinal deep voids

24. Conclusion  β-sheet conformation is dominating across entire cross section area in both spider and worm silk fibers.  The comparison of I 850 /I 830 intensity ratio between central and edge area of N. edulis silk displays a higher number of hidden (buried) tyrosine residues in the edge area.  Compared with B. mori wormsilk, cross section of N. edulis fiber reveals less pronounced globular structure with smaller fibrils size containing longitudinal deep voids.

25. Acknowledgement  For sample supply: Ann Terry  For assistance with sample preparation and SEM : Xuejing Zheng  For assistance with AFM: Alexander Alexeev  Edgar