Structural characterization of worm and spider silk on cross section surface Weizhen Li Evgeny...
-
Upload
reginald-lamb -
Category
Documents
-
view
218 -
download
2
Transcript of Structural characterization of worm and spider silk on cross section surface Weizhen Li Evgeny...
Structural characterization of worm and spider silk on cross section surface
Weizhen Li
Evgeny KlimovJoachim Loos
Bombyx moriBombyx mori worm cocoon
Natural Silk
Nephila edulis spider silk
NATURE 418 (6899): 741-741 AUG 15 2002
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
Helices Antiparallel - sheet Parallel - sheet
Turns Others (traditionally Random Coil)
?Helices Antiparallel - sheet Parallel - sheet
Turns Others (traditionally Random Coil)
?
Protein conformation – secondary structures
Our task:Our task:
Vibrational spectroscopic analysis on silk’s cross section
The existence of shell-core structure
(Raman mapping, high spectral resolution )
Experiment
Embedding fibre into epoxy resin
Use microtome to cut sample into slices with thickness of 10-30 m
LVSEM
AFM
AFM images (phase contrast) of the cross section of B. mori (A) and N.edulis (B)
XYZ-
positioning
CCDAndor
lase
r
mic
rosc
ope
electroniccontrol unit
PM
T CCD
SPM
SPM and positioning
control electronics
SPM positioning optics
Laser: He-Ne 632.8 nmXY-resolution: 500 nm Z – resolution: 0.5 - 1 m Spectral resolution: 0.01nmSamples: solids, liquids, bulk, thin films, powder
Raman analysis: scanning confocal Raman microscope “Nanofinder”
B. mori worm silk
Part One
Overview spectrum and bands assignment
Amide І Amide Ⅲ
random 1660-16661245-1250
1085
β sheet 1665-1680 1230-1245
αhelical1675
1645-16581264-1310
Surface of degummed wormsilk
β sheet
600 800 1000 1200 1400 1600 18000
50
100
150
(C-C)
Tyr
Tyr
(C-H)
Amide III (C-N)
Amide I (C=O)
Ram
an in
tens
tiy
Raman shift (cm-1)
J. Raman Spectrosc. 1995 26 901-909 J. Raman Spectrosc. 2001 32 103-107
Raman intensity distribution of amide I at 1665 cm-1
High spectral resolution
Raman image of silk cross section
600 900 1200 1500 1800
4000
5000
6000
7000
8000
Ram
an in
tens
ity
Raman shift (cm-1)
1400 1500 1600 1700
0
750
1500
2250
Ra
ma
n in
ten
sity
Raman shift (cm-1)
Core
Edge
Worm silk spectra with high resolution(After subtraction of epoxy)
Sample thickness: 30μm
amideⅠ amide Ⅲ
1000 1100 1200 13002000
4000
6000
8000
10000
Ram
an in
tens
ity
Raman shift (cm-1)
1400 1500 1600 17000
3000
6000
9000
12000
Ram
an in
tens
ity
Raman shift (cm-1)
Confocal Raman-high spatial resolutio
n
Notch filter
Sample
Photomultiplier or CCD detector
Principle
without pinhole
with pinholeHigh spatial resolution
1500 1600 1700 1800 1900
6000
8000
10000
12000
Ra
ma
n in
ten
sity
Raman shift (cm-1)
Core
Edge
1667 cm-1
1400 1500 1600 1700
0
750
1500
2250
Ra
ma
n in
ten
sity
Raman shift (cm-1)
Edge and Core area of fibre’s cross section
600 800 1000 1200 1400 1600 1800
-60
-40
-20
Inte
nsity
Wavenumbers(cm-1)
800 1000 1200 1400 1600 1800
20
40
Inte
nsi
ty
Wavenumbers(cm-1)
2 m 30 spots60-70 nm of one step
600 800 1000 1200 1400 1600 1800
-60
-40
-20
Inte
nsi
ty
Wavenumbers(cm-1)
Average
Raman data of edge and core area
Core
Edge
800 850 900 950 10000
15
30
45
60 edge core
Tyr
833
858
Ram
an in
tens
ity
Raman shift (cm-1)
800 1000 1200 1400 1600 18000
15
30
45
60
(C-C)
(C-H)
Amide I
Amide III
edge core
Tyr
833858
1085
Ram
an in
tens
ity
Raman shift (cm-1)
The ratio I(850)/I(830) is a spectral marker of tyrosine hydrogen bonding strength.
850/830 cm-1 Intensity ratio
Sample 1 Sample 2 Sample 3
edge core edge core edge core
850 cm-1 22.79 22.21 23.68 31.22 20.24 22.56
830 cm-1 15.61 14.50 18.10 22.32 12.4 14.30
I(850 cm-1)/
I(830 cm-1)
1.46 1.53 1.31 1.40 1.63 1.58
Stable across entire cross section
The ratio I(850)/I(830) is reduced going from moderately to strongly hydrogen-bonded tyrosines.
Nephila edulis Spider silk
Part Two
Surface of single fibre—Nephila spider
Amide І Amide Ⅲ
random 1660-16661245-1250
1095
β sheet 1665-1680 1230-1245
αhelical1675
1645-16581264-1310
β sheet Conformation
600 800 1000 1200 1400 1600 18000
1000
2000
3000
(C-H)
TyrTyr
Amide III (C-N)
Amide I (C=O)
Ra
ma
n in
ten
sity
Raman shift (cm-1)
(C-N)(C-C)
J. Raman Spectrosc. 1995 26 901-909 J. Raman Spectrosc. 2001 32 103-107
Raman image
Raman intensity distribution of amide I at 1665 cm-1
2 m 30 spots 60-70 nm of one step
800 850 900 950 10000
15
30
45
60 edge core
Tyr
831
855
Ram
an in
tens
ity
Raman shift (cm-1)
905
800 1000 1200 1400 1600 18000
15
30
45
60
(C-N)(C-C)
edge core
Amide III
(C-H)
Amide I
(C-C)Tyr
831855
Ram
an in
tens
ity
Raman shift (cm-1)
1095905
Core
Edge
Raman data of edge and core area
850/830 cm-1 Intensity ratio
Sample1
core
Sample1
edge
Sample2
core
Sample2
edge
850 cm-1 11.818 14.474 6.645 8.074
830 cm-1 8.235 13.25 3.280 5.982
I(850 cm-1)/
I(830 cm-1)
1.435 1.09 1.72 1.35
The strength of hydrogen bonds involving the tyrosine residues may influence the forming of core-shell structure of N.edulis.
1.3 times 1.3 times
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
Conclusion
β-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.
Acknowledgement
For sample supply: Ann Terry
For assistance with sample preparation and SEM : Xuejing Zheng
For assistance with AFM: Alexander Alexeev
Edgar