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Chemical and Biosynthetic MethodsToward Mimicking Nature’s Strong Fiber: Spider Dragline Silk. Maren E. Buck Lynn Group 5/3/2007. Outline. Introduction to spider silks and silk structure Biosynthetic methods to produce silk protein analogs Chemical methods to synthesize silk-like polymers

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chemical and biosynthetic methodstoward mimicking nature s strong fiber spider dragline silk

Chemical and Biosynthetic MethodsToward Mimicking Nature’s Strong Fiber: Spider Dragline Silk

Maren E. Buck

Lynn Group

5/3/2007

slide2

Outline

  • Introduction to spider silks and silk structure
  • Biosynthetic methods to produce silk protein analogs
  • Chemical methods to synthesize silk-like polymers
  • Applications
  • Conclusions
slide3

Large diameter egg

Case fiber (Tubuliform)

Tubuliform

Aggregate

Flagelliform

Minor ampullate

Capture Spiral

(Flagelliform)

Pyriform

Glue coating

(Aggregate silk) (?)

Acini-

form

Major

ampullate

Wrapping and egg case fiber

(aciniform)

Web reinforcement

(Minor ampullate

1 and 2)

Pyriform silk (?)

Dragline (major

ampullate 1 and 2)

Spiders spin 6 different fibers

Vollrath, F. J. Biotechnol. 2000, 74, 67-83.

Hu, X. et al. Cell. Mol. Life Sci.2006, 63, 1986-1999.

slide4

The classic strong synthetic fiber

Kevlar®:

Dupont (1960s)

Uses

- Bulletproof vests and helmets

- Automobile brake pads

- Ropes and cables

- Aerospace components

Fiber

axis

Strength (GPa

)

Energy to break (J/kg

)

Material

Elasticity (%)

5

Dragline Silk

35

4 x 10

1.1

4

Kevlar

5

3 x 10

3.6

4

Rubber

600

8 x 10

0.001

4

Nylon, type 6

200

6 x 10

0.07

Lewis, R. Chem. Rev.2006, 106, 3762-3774. Vollrath, F.; Knight, D.P. Nature2001, 410, 541-548.

Tanner, D.; Fitzgerald, J.A.; Phillips, B.R. Angew. Chem. Int. Ed. Engl. Adv. Mater.1989, 5, 649-654.

Kubik, S. Angew. Chem. Int. Ed.2002, 41, 2721-2723.

slide5

Spider silks have potential in many applications

Biomedical applications

Surgical sutures

Scaffolds for tissue engineering

Technical and industrial applications

High strength

ropes/cables

Ballistics

Parachutes

Fishing line

slide6

Forced silking to obtain silk fibers

Spiders are anesthetized with CO2

and secured ventral side up

Silk is pulled from the spinneret,

attached to a reel, and drawn at a

specified speed

Work, R. W.; Emerson, P. D. J. Arachnol. 1982, 10, 1-10.

Elices, M.; Perez-Rigueiro, J.; Plaza, G. R.; Guinea, G. V. JOM2005, 57.

slide7

Spiders are highly developed fiber “spinners”

Lumen

Duct

Spidroin secretion

Fiber alignment

Spinneret

Tail

Funnel

Duct

1 mm

Lewis, R. Chem. Rev.2006, 106, 3762-3774.

Dicko, C.; Vollrath, F.; Kenney, J.M. Biomacromolecules 2004, 5, 704-710.

slide8

QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGAGQGGRGAGQS

SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGQGGRGAGQN

SQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQS

SQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQS

SQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG

GLGGYGRQGAGGAAAAAAGAGQGGRGAGQS

NQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAGQGGRGAGQN

SQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG

GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQS

SQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG

SSLRSAAAAASAASAGS

Primary structure of spider dragline silk

  • Fibrous protein composed of Spidroin 1 (MaSp1) and Spidroin 2 (MaSp2)
    • - Sequences highly conserved
    • - Repetitive stretches of poly(Ala) and (GlyGlyXaa)n sequences
    • (Xaa = Tyr, Leu, Gln)
    • - MW of MaSp1 ~ 275-320 kDa; Sp1+Sp2 ~ 700-750 kDa

Repeating sequence of MaSp1

Hinman, M.B.; Jones, J. A.; Lewis, R. TIBTECH2000, 18, 374-379. Vollrath, F.; Knight, D. P. Nature2001, 410, 541-548.

Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science1996, 271, 84-87.

slide9

Antiparallel and parallel -sheet structure

C

N

N-terminus

C-terminus

N-terminus

C-terminus

C

N

C

N

C-terminus

N-terminus

Poly(alanine) segment

N-terminus

C-terminus

C

N

Rotondi, K. S.; Gierasch, L. M. Biopolymers2005, 84, 13-22.

Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235-5237.

slide10

Solid state 13C-NMR and FT-IR spectroscopy

1637

0.4050

1612

1666

1691

Absorbance

0.2800

0.1550

1700

1600

1500

Wavenumber (cm-1)

13C-NMR chemical shifts (ppm)

Infrared

wavelengths (cm-1)

-sheet

α-helix

Anti-parallel β-sheet

1630, 1685

Ala C

48.7

52.5

Parallel β-sheet

1630, 1645

Ala C

20.1

15.1

α-helix

1650, 1560

Ala CC=O

171.9

176.5

-carbon

Infrared spectrum of silk from

Nephila clavipes

-carbon

13C-labeled

Alanine

Amide I

(antiparallel

-sheet)

Marcotte, I.; van Beek, J. D.; Meier, B. H. Macromolecules2007, 40, 1995-2001.

Simmons, A.; Ray, E.; Jelinski, L.W. Macromolecules1994, 27, 5235-5237.

Dong, Z.; Lewis, R.; Middaugh, C. R. Arch. Biochem. Biophys.1991, 1, 53-57.

slide11

Proposed secondary structure and mode of elasticity

  • Poly(Ala) modules form anti-parallel β-sheets (~30-40%)
  • Glycine-rich, amorphous regions are thought to be helical

Crystalline region with

-sheet structure

Strain

Disordered

chain region

Kubik, S. Angew. Chem. Int. Ed.2002, 41, 2721-2723.

Van Beek, J. D.; Hess, S.; Vollrath, F. Meier, B. H. Proc. Nat. Acad. Sci. 2002, 99, 10266-10271.

slide12

Synthetic approaches to spider dragline silk

Protein sequences

Biosynthesis

Chemical Synthesis

slide13

Two biosynthetic routes to spidroin proteins

Eukaryotic host

(insect cells)

Nephila clavipes

Reverse transcription

Spider

cDNA

Spider silk protein sequences/mRNA

Protein fibers

Gene design

Synthetic

DNA

Flexibility with

host

Vendrely, C.; Scheibel, T. Macromol. Biosci.2007, 7, 401-409.

Altman, G.H. et al.Biomaterials2003, 24, 401-416.

slide14

protein

synthesis

Protein: MW ~ 60-140 kDa

Fiber diameter ~ 40 μm

Yield ~ 37 mg/L

Expression of spider silk cDNA in mammalian cells

Transformation of vector in mammalian cells

Protein purification,

and characterization

Dragline silk

gene sequence

from A. diadematus

Gene sequence

inserted into

expression vector

Mechanical Properties:

Protein sample

Toughness

(MJ/m3)

Modulus

(GPa)

Elasticity

(%)

Strength

(GPa)

85

13

43.4

0.26

ADF-3

A. diadematus dragline

130

10

30

1.1

Lazaris, A. et al.Science2002, 295, 472-476.

slide15

Recombinant expression of synthetic silk genes

Ligate 8 or 16

DNA fragments

Spidroin 1 analog: DP-1B

[

AGQGGYGGLGSQG--------------------------------------------

AGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGG

AGQG-------GLGSQGA---------- GQGAGAAAAAA----GG

AGQGGYGGLGSQGAGRG-----GQGAGAAAAAA---GG

DNA fragment

]

n=8-16

Hybridize

complementary

strands

Transform in

Escherichia coli

Protein fibers

300 mg/L

Insert gene into

plasmid vector

Protein fibers

1 g/L

DNA duplex

Or transform in

yeast

170 nm diameter fibers

Premature termination with

expression in E. coli

High MW polymers from yeast

Fahnestock, S. R.; Irwin, S. L. Appl. Microbiol. Biotechnol.1997, 47, 23-32.

Stephens, S.J. et al. Mat. Res. Soc. Symp. Proc.2003, 774, 2.3.1-2.3.10.

Fahnestock, S. R.; Bedzyk, L. A. Appl. Microbiol.Biotechnol.1997, 47, 33-39.

O’Brien, J. P.; Fahnestock, S. R.; Termonia, Y.; Gardner, K. H. Adv. Mater.1998, 10, 1185-1195.

slide16

Summary of biosynthetic pathways

Biosynthetic Method

Advantages

Disadvantages

Difficulty with protein

purification (aggregation)

Spider Silk cDNA

Produce proteins most

like native silk

High MW polymers

are readily attainable

Eukaryotic hosts are

expensive

Synthetic DNA

Polymer structure can

be tuned based on

DNA sequence used

Truncated syntheses in

many hosts

Flexibility with

expression host

slide17

Synthetic approaches to spider dragline silk

Protein sequences

Biosynthesis

Chemical Synthesis

slide18

Chemical approaches to synthesizing

silk-like polymers

Poly(Ala) blocks

- PEG linker

- Alkyl linkers

Lego approach

(-sheet template)

- Rigid or short linkers

- Long, flexible linkers

Protein structure

and properties

Living polymerization of peptide monomers

Non-peptide

polymers

slide19

Synthesis of silk-like polymers: “Lego” approach

Linkers

-sheet nucleation center

Peptide sequence (GAGA)

+

A

+

B

Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

slide20

Synthesis of the building blocks

Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

Wagner, G.; Feigel, M. Tetrahedron1993, 49, 10831-10842.

slide21

Spectroscopic evidence for the required

phenoxathiin template

3424 cm-1

3336 cm-1

3407 cm-1

3415 cm-1

3a

3342 cm-1

3b

4

Flexible linear peptide

1

2

Peptides with phenoxathiin template

3

4

Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

slide22

Polymerization of the building blocks

Interfacial Polymerization

“Nylon Rope Trick”

Monomer A

Copolymer AB

Monomer B

Solution Polymerization

22

Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

slide23

Polymerization results

P1

P2

P3

P4

57

50

46

82

% Yield – Interfacial:

60

56

39

67

% Yield – Solution:

19,100

20,600

17,400

20,200

Mn (Solution) (g/mol):

2.08

1.79

1.54

1.79

PDI:

Mn = average molecular weight of sample

PDI = distribution of molecular weights in a sample

Spider silk: Mn = ~ 605,000 g/mol (Sp1+Sp2)

PDI = 1.05

Winningham, M. J.; Sogah, D. Y. Macromolecules1997, 30, 862-876.

slide24

FT-IR characterization of the polymer structure

1:

2:

1645 cm-1

Polymer 1 or 2

Polymer 2

Polymer 1

1645 cm-1

Peptide 1

Peptide 2

Peptide 1 or 2

Winningham, M.J.; Sogah, D.Y. Macromolecules. 1997, 30, 862-876.

slide25

Phenoxathiin template with ethylene glycol linkers

Interfacial:

57% yield

Mn = 22,400

PDI=1.72

Solution:

60% yield

Mn = 14,000

PDI = 2.4

Rathore, O.; Winningham, M. J.; Sogah, D.Y. J. Polym. Sci: Part A, Polym Chem.2000, 38, 352-366.

Dattagupta, N.; U.S. Patent 4,968,602; 1990.

slide26

13C-NMR spectra suggest -sheet structure

Interfacial

polymerization

Solution

polymerization

Total -sheet content:

- Interfacial polymerization: 40%

- Solution polymerization: 80%

Spider silk -sheet content: 30-40%

Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci: Part A, Polym Chem.2000, 38, 352-366.

slide27

Changes in interfacial polymer after annealing above Tg

  • Polymerization procedure affects structure
  • Heating above Tg enhances -sheet content
  • in interfacial polymer

Solution

polymerization

Initial

Post Annealing

1647 cm-1

Raw

1647 cm-1

Raw

1683 cm-1

2nd derivative

2nd derivative

1683 cm-1

1635 cm-1

1633 cm-1

1628 cm-1

Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci: Part A, Polym. Chem.2000, 38, 352-366.

slide28

Poly(Ala) blocks

- PEG linker

- Alkyl linkers

Poly(Ala) blocks

- PEG linker

- Alkyl linkers

Lego approach

(-sheet template)

- Rigid or short linkers

- Long, flexible linkers

Protein structure

and properties

Living polymerization of peptide monomers

Non-peptide

polymers

Chemical approaches to synthesizing silk-like polymers

slide29

QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGQGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGAGQGGRGAGQS

SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGQGGRGAGQN

SQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQS

SQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG

GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQS

SQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG

GLGGYGRQGAGGAAAAAAGAGQGGRGAGQS

NQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG

GLGGYGGQGAGGAAAAAGQGGRGAGQN

SQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG

GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQS

SQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG

SSLRSAAAAASAASAGS

Non-templated polymeric dragline silk mimics

[

]

soft linker

peptide

Generic polymer structure

x ~ 4 or 6

n ~ 13

Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science1996, 271, 84-87.

Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc.2001, 123, 5231-5239.

slide30

Synthesis of triblock copolymers with poly(Ala)

Water-soluble fraction

(46%)

+

Water-insoluble fraction

(54%)

P1: x~4, n~13; 75% yield

P2: x~6, n~13; 69% yield

Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc.2001, 123, 5231-5239.

slide31

Mechanical properties:

Modulus

(MPa)

Tensile strength

(MPa)

Elongation

at break (%)

Polymer

22.9±13.6

P1

410±35

13.0±1.4

750±156

14.2±2.7

P2

5.4±1.7

Silk (N. clavipes)

22,000

1,100

34

Mechanical properties of the polymer fibers

P1: x~4, n~13

P2: x~6, n~13

FT-IR and 13C-NMR indicate formation of anti-parallel -sheets

Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc.2001, 123, 5231-5239.

slide32

Synthesis of silk-like multiblock copolymers containing flexible alkyl linkers

Yield: 70%

MW (viscosity): 44,900

3 cycles

Yao, J. et al. Macromolecules2003, 36, 7508-7512.

slide33

Multiblock copolymers with poly(isoprene) as the

“soft” linker

n = 31 (Mn=2200)

n = 72 (Mn=5000)

Zhou, C. et al. Biomacromolecules2006, 7, 2415-2419.

slide34

P1: n = 31

P2: n = 72

1630

1643

P1

18

P2

1655

48

171

176

Absorbance

52

P1

P2

P1

Chemical shift (ppm)

Wavenumber (cm-1)

Zhou, C. et al. Biomacromolecules2006, 7, 2415-2419.

13C-NMR and FT-IR characterization of the polymers

slide35

Poly(Ala) blocks

- PEG linker

- Alkyl linkers

Lego approach

(-sheet template)

- Rigid or short linkers

- Long, flexible linkers

Protein structure

and properties

Living polymerization of peptide monomers

Living polymerization of peptide monomers

Non-peptide

polymers

Chemical approaches to synthesizing silk-like polymers

slide36

Atom transfer radical polymerization (ATRP) of silk-like triblock copolymers

Mn (GPC): 4.6 kDa

Mn (GPC): 11.5 kDa

PDI: 1.17

PDI: 1.29

Ayres, L. et al. Biomacromolecules2005, 6, 825-831.

slide37

Poly(Ala) blocks

- PEG linker

- Alkyl linkers

Lego approach

(-sheet template)

- Rigid or short linkers

- Long, flexible linkers

Protein structure

and properties

Living polymerization of peptide monomers

Non-peptide

polymers

Non-peptide

polymers

Chemical approaches to synthesizing silk-like polymers

slide38

Silk-like polymers without peptide motifs

Endcapped macrodiol

Macrodiol

% Hard segment:

P1 = 26% P3 = 43%

P2 = 33% P4 = 47%

Soft segment

Hard segment

James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer2006, 47, 3073-3082.

slide39

Modulus (MPa)

Toughness (MJ/m3)

Elongation

at break (%)

Tensile strength (MPa)

Polymer

587

14.9

72.5

65.1

P1

18.1

P2

460

200

59.2

23.6

P3

447

156

77.4

18.2

P4

202

198

31.6

1,100

Spider dragline silk

34

22,000

160

Mechanical properties of poly(urethane) polymers

Soft segment

Hard segment

P1 = 26% P3 = 43%

P2 = 33% P4 = 47%

James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer2006, 47, 3073-3082.

Cuniff, P.M. et al. Polym. Adv. Tech.2003, 5, 401-410.

slide40

Summary of chemical synthetic pathways

Lego approach

(-sheet template)

- Forms -sheets

- Brittle, non-fibrous

Poly(Ala) blocks

- Forms -sheets

- Produces fibers; not

as strong as native silk

Non-peptide polymers

- Self-assembles

into fibers

- High elasticity,

low strength

Living polymerization of peptide monomers

- Forms -sheets

- Control over MW of

peptide blocks

- Low PDI

slide41

Applications for spider dragline silk: Tissue Engineering

Artificial nerve grafts:

- Nerve cells attach and grow on

spider silk fibers

- Nerve construct composed of pig

venules, filled with cells seeded on

silk fibers

Light micrograph of artificial nerve construct

Artificial ligaments:

- Silks promote proliferation of bone marrow

cells

- High tensile strengths could restore

knee function immediately

Allmeling, C.; Jokuszies, A.; Reimers, K.; Kall, S.; Vogt, P. M. J. Cell. Mol. Med.2006, 10. 1-8.

Altman, G. H. et al. Biomaterials 2003, 24, 401-416.

slide42

Spider silks have potential in many applications

Biomedical applications

Surgical sutures

Scaffolds for tissue engineering

Technical and industrial applications

High strength

ropes/cables

Ballistics

Parachutes

Fishing line

slide43

BioSteel®

- Genetically modified goats produce silk in mammary glands

  • Silk is spun from the goats’ milk
      • Extrusion through “spinnerets” produces fibers
  • Aqueous spinning process is environmentally friendly
  • - Anticipated uses:
  • Surgical sutures
  • Adhesives
  • Fishing line
  • Body armor/military applications

Lazaris, A. et al.Science2002, 295, 472-476.

Karatzas, C. N.; Turcotte, C. 2003, PCT Int. Appl. WO03057727.

Karatzas, C. 2001, PCT Int. Appl. WO0156626.

Islam, S. et al. 2004, U.S. Pat. 20040102614.

slide44

Conclusions

- Spiders can spin fibers with exceptional strength, elasticity, and toughness

- Biosynthetic methods have generated fibers with structure and properties

approaching those of native silks

- Chemists can use spider silk as a model to design new fibers and

materials with silk-like properties

- Silk-spinning processes must be optimized in order for commercialization

to occur

slide45

Acknowledgments

Professor David Lynn

Practice talk attendees:

Lauren Boyle

Claire Poppe

Julee Byram

Becca Splain

Alex Clemens

Katherine Traynor

Richard Grant

Matt Windsor

Margie Mattmann

Lynn Group Members:

Jingtao Zhang

Xianghui Liu

Chris Jewell

Nat Fredin

Bin Sun

Mike Kinsinger

Eric Saurer

Ryan Flessner

Shane Bechler