Structural characterization of worm and spider silk on cross section surface

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Structural characterization of worm and spider
silk on cross section surface
Weizhen Li
Evgeny Klimov Joachim Loos
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Natural Silk
Bombyx mori worm cocoon
Nephila edulis spider silk
NATURE 418 (6899) 741-741 AUG 15 2002
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B. Mori Silkworm fibre A. trifasciata
spider silk
Sericin coating
Engineering Fracture Mechanics 69 (2002)
10351048 Proc. R. Soc. Lond. B 263 (1996)147-151

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Protein conformation secondary structures
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Our task

• Vibrational spectroscopic analysis on silks
  cross section
• The existence of shell-core structure
• (Raman mapping, high spectral resolution )

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Experiment

• Embedding fibre into epoxy resin

LVSEM
Use microtome to cut sample into slices with
thickness of 10-30 ?m
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AFM
AFM images (phase contrast) of the cross section
of B. mori (A) and N.edulis (B)
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Raman analysis scanning confocal Raman
microscope Nanofinder
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
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Part One

• B. mori worm silk

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Overview spectrum and bands assignment
Surface of degummed wormsilk
ß sheet
J. Raman Spectrosc. 1995 26 901-909 J. Raman
Spectrosc. 2001 32 103-107
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Raman image of silk cross section
Raman intensity distribution of amide I at 1665
cm-1
High spectral resolution
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Worm silk spectra with high resolution (After
subtraction of epoxy)
amide ?
amide?
Core
Sample thickness
30µm
Edge
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Confocal Raman-high spatial resolution
without pinhole
Principle
with pinhole
High spatial resolution
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Edge and Core area of fibres cross section
Average
2 ?m 30 spots 60-70 nm of one step
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Raman data of edge and core area
Core
Edge
The ratio I(850)/I(830) is a spectral marker of
tyrosine hydrogen bonding strength.
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850/830 cm-1 Intensity ratio
Stable across entire cross section
The ratio I(850)/I(830) is reduced going from
moderately to strongly hydrogen-bonded tyrosines.
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Part Two

• Nephila edulis Spider silk

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Surface of single fibreNephila spider
ß sheet Conformation
J. Raman Spectrosc. 1995 26 901-909 J. Raman
Spectrosc. 2001 32 103-107
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Raman image
Raman intensity distribution of amide I at 1665
cm-1
2 ?m 30 spots 60-70 nm of one step
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Raman data of edge and core area
Core
Edge
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850/830 cm-1 Intensity ratio
1.3 times
1.3 times
The strength of hydrogen bonds involving the
tyrosine residues may influence the forming of
core-shell structure of N.edulis.
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AFM image
Globular spherical features Diameter 70-90 nm
multiple nanovoids
Less pronounced globular structure
Multiple 200-300 nm large longitudinal deep voids
AFM height (left) and phase contrast
(right) images of worm silk (top) and spider silk
(bottom)
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Conclusion

• ß-sheet conformation is dominating across entire
  cross section area in both spider and worm silk
  fibers.
• The comparison of I850/I830 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.

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Acknowledgement

• For sample supply Ann Terry
• For assistance with sample preparation and SEM
  Xuejing Zheng
• For assistance with AFM Alexander Alexeev
• Edgar

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• Thanks for attention!