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