1. Biological Polymers: A Focus on Dragline Spider Silk Spidroin Protein
SEM of an ion etched silk fibroin fiber
Picture courtesy of: Chang et. Al., Polymer 46: 7909 (2005)

2. General Functions of Biological Polymers–
3 Main classes of biological polymers:
Nucleic acid polymers
Linear informational polymers comprised of 4 nucleotide monomers
Polysaccharides
Branching storage/structural polymers comprised of one of a few select monosaccharide monomers
Proteins and peptides
Linear informational polymers comprised of 20 standard amino acid monomers
Nucleic acids and proteins are considered informational because the sequence of monomers in the polymers is:
Nonrandom
Significant to function
A similar argument can be made for branched carbohydrates comprised of different monomers.
General Functions of Biological Polymers–
Nucleic Acids
Information storage (genome)
Translational molecules (mRNA & tRNA)
Biological catalysts (RNA ribozymes)
Carbohydrates
Energy storage (glycogen)
Structural (cellulose cell walls or chitin exoskeletons)
Recognition (carbohydrates of glycoproteins and glycolipids)
Proteins
Structural (fibrous proteins)
Biological catalysts (enzymes)
Recognition (immunoglobulins)

3. Biopolymer Synthesis Via Condensation–
Implies that monomers must have hydrogen-bearing and hydroxyl moieties.
Directed polymerization is accomplished by chemically activating monomers via:
Direct activation using ATP or Coenzyme A
The use of a carrier molecule (i.e. tRNA)
Polymerization dictates that biological polymers have chemically distinct ends.
Scheme of biopolymer macromolecular assembly
Figure 2-17: Becker et. Al., World of the Cell 6th Ed.

4. Biopolymers Utilize a Variety of Functional Groups for Polymerization by Condensation–
Common functional groups
employed for biopolymer formation
Nucleic acid structure highlighting
chemically distinct ends
Candidate functional groups for condensation polymerization must either act as a nucleophile or electrophile
Left– Figure 1-2: Voet et. Al., Fundamentals of Biochemistry. Right– Figure 3-6a: Voet et. Al., Fundamentals of Biochemistry.

5. Efficiency of Biopolymer Synthesis–
Scheme of amino acid
polymerization by condensation
Biopolymer condensation is spontaneous and relatively rapid at moderate temperatures in aqueous environments.
Chemical initiators are not required.
The use of biological catalysts (enzymes) and activating molecules:
Improves efficiency to favor polymerization over depolymerization (hydrolysis) by moving the reaction away from equilibrium
Makes biopolymer formation kinetically competent to support life
Scheme of ribosomal (catalytic)
protein synthesis
Top– Figure 4-3: Voet et. Al., Fundamentals of Biochemistry. Right– Figure 26-28: Voet et. Al., Fundamentals of Biochemistry.

6. 3-D Structure is Intimately Related to Function–
Three-dimensional arrangements of biological polymers are more important for function than the chemical nature and composition of the monomers.
Examples:
The tertiary structure of proteins is largely responsible for biological activity.
The double helical structure of DNA is responsible for stability, replication efficiency, and packing in small cellular volumes.
The 3-D arrays of complex carbohydrates determines optimal intracellular storage conditions and recognition properties.

7. The Hierarchal Structure of Proteins–
Primary Structure:
Amino acid sequence from N- to C-terminus
Ultimately determines all higher order structure and function
Driven and stabilized by covalent bonds
Secondary Structure:
Local, spatial interactions between functional groups of the protein backbone
Driven and stabilized by the hydrogen bond
Not usually a determinant of function
Tertiary Structure:
Three-dimensional folding of a polypeptide
Driven and stabilized largely by weak, hydrophobic interactions
Often dictates biological activity
Quaternary Structure:
Specific interactions between two or more proteins
Can be driven and stabilized by any combination of bond types
Figure illustrating the four hierarchal
levels of protein structure
Figure 3-6: Becker et. Al., World of the Cell 6th Ed.
Structure is a consequence of sequence.
Function is a consequence of structure.

8. Spider Dragline Silk–
Spiders have 7 different gland-spinneret complexes:
Each synthesizes a unique blend of structural polymer as a fiber with unique properties
Multiple fibers can be spun simultaneously
Dragline silk is used by spiders to build the frame and radii of their nets and as lifelines.
Dragline silk is produced by the largest gland (major ampullate) and is believed to have the most desirable properties for commercial use.
Potential applications include:
Biomedical sutures
Scaffolds for tissue engineering (bone & ligament)
Body armor
Photograph illustrating spider silk formation & stress-strain curves for
dragline and viscid spider silk
Top– Picture courtesy of Tiller et. Al., 1996.
Bottom– Figure courtesy of Gosline et. Al., 1999.

9. Macromolecular Structure of Silk Spidroin–
SEM of untreated and toluene treated spidroin fibers
Major ampullate dragline silk is comprised of two proteins joined together via 3 – 5 disulfide bonds near their C-termini:
Spidroin 1
Spidroin 2
The average diameter of major ampullate dragline silk spidroin  m.
Mucopolysaccharide is infused within, and on the surface of the silk fibers (removed by toluene treatment).
Figure courtesy of Rengasamy et. Al., 2005.

10. Primary Sequence of Spider Silk Spidroin–
Four motifs exist in the primary structure:
GPGXX (X often Q)
An or (GA)n
GGX
Spacer regions
Two residues predominate in the primary sequence:
42% Glycine
25% Alanine
Glu, Gln, Ser and Tyr are also prominent
Cys is concentrated near the C-terminus
Sequences of major ampullate spidroin
highlighting motif transitions
Figure courtesy of Gosline et. Al., 1999.

11. Secondary Structure Predictions from the Primary Sequence–
Double-quantum single-quantum correlation for static sample (DOQSY) NMR can measure the relative orientation of the peptide backbone carbonyl orientation when if 13C is present.
Feeding deuterated and 13C-L-alanine to spiders reveals that 40% of total alanine is involved in crystalline protein structure.
Chou-Fasman prediction of spidroin 2 structure indicates the -helix and turns should predominate.
Ala: P = 1.42, P = 0.83, Pturn = 0.66
Gly: P = 0.57, P = 0.75, Pturn = 1.56
Glu: P = 1.51, P = 0.37, Pturn = 0.74
Gln: P = 1.11, P = 1.10, Pturn = 0.98
Ser: P = 0.77, P = 0.75, Pturn = 1.43
Tyr: P = 0.69, P = 1.47, Pturn = 1.14
Cys: P = 0.70, P = 1.19, Pturn = 1.19
DOQSY Spectra and Ramachandran diagrams of silk spidroin fibers
Figure courtesy of van Beek et. Al., 2002.
Alanine torsion angles indicate  –135,  150
What does this data suggest?

12. Circular Dichroism Spectra Indicates -Sheet Structure–
CD Spectra and cooperative thermal transitions of spidroin segments against an -helical background
Circular dichroism measures the optical activity of proteins in the far UV-region.
Dissymmetry due to bias towards L-amino acids and the preferential twists of secondary structure can be distinguished.
-helices have a strong positive band at 192 nm and two negative bands at 208 and 222 nm.
CD spectra reveal no -helices and a cooperative and reversible disruption of protein 2 structure.
Fourier transform infrared spectroscopy (FTIR) confirms that -sheets are oriented parallel to the fiber axis.
Figure courtesy of Huemmerich et. Al., 2004.

13. Figure courtesy of Gosline et. Al., 1999.
X-Ray Crystallography Reveals A Composite, Hierarchal Block Co-Polymer–
Poly-Ala or (GA)n stretches form -sheets.
Glu and Tyr limit the size and spacing of -sheets by forcing loops to form and interact with the surrounding matrix.
-sheets stack on top of one another with crystal dimensions of  2nm X 5 nm X 7 nm.
-sheet crystals form intermolecular connections and are large and abundant enough to act as reinforcing filler particles to stiffen and strengthen the overall structure.
Major ampullate silk structure can be summarized as a crystal cross-linked, crystal-reinforced polymer network.
Summary figure of spidroin crystal structure in supercontracted vs. fibers
Figure courtesy of Gosline et. Al., 1999.

14. Physicochemical Analysis of Major Ampullate Spidroin–
Differential scanning calorimetry &
thermal mechanical analysis of spidroin fibers
Differential scanning calorimetry shows a broad endotherm with a peak at  90–95 C, consistent with the loss of water, and is stable up to 250 C.
Thermogravimetric analysis shows a two-step degradation profile above 150 C:
First step in the range of 200–501 C corresponds to the destruction of the amino acid side chains
Second step in the range of 501–896 C corresponds to destruction of the peptide bonds
Thermal mechanical analysis shows a change in the thermal expansion coefficient () from –6.59 X 10–4 to –8.2 X 10–3 at C (low glass transition temperature).
Figures courtesy of Rengasamy et. Al., 2005.

15. Physical Parameters of Major Ampullate Spidroin–
Stress () = the normalized force (F) such that:
 = F/A (A = initial cross-sectional area of the fiber)
Strain () = the normalized deformation such that:
 = L/L0 (L0 = initial fiber length and L = change in fiber length)
A stress-strain curve ( vs. ) gives:
Stiffness of the material (slope)
Strength of the material (max) as the maximum value of stress at the time the material fails
Extensibility of the material (max) as the maximum value of strain at the time the material fails
The integrated area under the stress-strain curve gives the energy required to break the material and is a quantification of toughness

16. Stress-Strain Curves in Different Solvents Reveals Unique Properties–
Silk shrinks by 40 – 50% and softens/weakens as a function of solvent: EtOH < MetOH < Water < Urea
The transition supercontraction is a function of solvent dielectric:
Big problem for engineering
Beneficial for the spider in environmental adaptation
Water and methanol act as plasticizers, and insinuates itself into the spidroin polymer to reduce inter-fiber interactions:
Decreases the elastic modulus
Decreases strength and toughness
Solvent absorbed during supercontraction is associated only with amorphous (non-crystalline) regions of the spidroin structure.
Stress-strain curves of major ampullate
spidroin in different solvents
Figures courtesy of Shao et. Al., 1999.

17. Dried Spidroin Fibers Do Not Recover Fully–
Stress-strain curves of major ampullate
spidroin in before and after submersion & drying in different solvents
Silk submerged in high dielectric solvents:
Exhibits a stress-strain profile more consistent with commercial rubber
Submerged silk that is dried only partially recovers:
Forms a semi-crystalline polymer
Stiffness decreases by 3 orders of magnitude
Mucopolysaccharide infusion and coating may partially protect spidroin from supercontraction.
Figures courtesy of Shao et. Al., 1999.

18. Figures courtesy of Shao et. Al., 1999.
Multiple Loading-Unloading Decreases Toughness and Extensibility Only Marginally After Drying–
Successive stress-strain curves of major ampullate spidroin after submersion & drying in water
Elastic recovery after submersion & drying is between 80 – 90% of maximum after stretching to 70% of breaking elongation.
Figures courtesy of Shao et. Al., 1999.

19. High-Strain-Rate Impact Reveals Hysteresis–
High-strain-rate analysis approximating common loads experienced by spidroin fibers
When dragline silk is first under strain it absorbs energy as the molecular chains reorient and slip against each other as H-bonds break.
After stretching, chains settle into a stable conformation.
Friction between chains and reformation of H-bonds induce a permanent set to prevent full recovery at relaxation.
A hysteresis value of 65%:
Allows 65% of transmitted kinetic energy to be absorbed and transformed into heat
Prevents prey from catapulting out of the web
Represents a balance between strength and extensibility yielding enormous toughness
Figures courtesy of Gosline et. Al., 1999.

20. Stress-Strain Comparisons With High-Performance Polymers–
Table courtesy of Gosline et. Al., 1999.
Major ampullate spidroin is amongst the stiffest and strongest biomaterials known.
Large extensibility (stretch), in spite of decreased strength, makes silk tougher than engineering materials.
Major ampullate spidroin has hard elastic properties that can outperform all synthetic fibers when energy absorption is important.
A Kevlar fiber of exactly the same breaking tension, but with an max one order of magnitude lower than spidroin would support a load less than 40% of a comparable silk fiber.
Major ampullate silk spidroin is 5-times stronger than steel by weight.

21. Rationalizing Spidroin Properties With Fiber Structure–
Proposed model for dragline silk fiber
GPGXX (GPGQQ)–
Likely a -turn spiral
Contributes to elasticity and connects crystalline sheets
P allows for retraction after stretching by providing torque
Serves as a focal point for retractive forces after stretching
(GA)n / An–
Crystalline -sheets that provide high tensile strength
Form zipper-like stacking of interdigitating sheets
GGX–
310 helix
Likely important for fiber alignment
Spacers–
Contributes to both elasticity and supercontraction
Serves as the matrix for embedding the crystalline regions of the polymer
May prevent premature fiber formation in the spider gland
Figure courtesy of van Beek et. Al., 2002.

22. Biology of the Major Ampullate Gland–
Silk proteins are stored in a liquid crystal form (elongated flexible rods) while in the gland.
Fibers are not formed until the protein passes trough the duct leading to the spinneret.
During thread assembly and spinning:
Water, sodium and chloride are removed
Lyotropic ions (K+ and PO43–) induce liquid crystal formation by increasing the surface tension of water and increasing hydrophobic interactions by changing structural water to bulk water
pH drops from 6.9 to 6.3
The mechanical stress of funneling through the gland and passing through the spinneret induced fiber alignment and assembly of the fiber by extensional flow
Fibers must be dehydrated to initiate -sheet formation and crystallization.
Micrograph of a single spider spinneret
highlighting internal anatomy
Image courtesy of:
biomimeticmaterials/files/spinningsystems.htm.

23. Considerations for Engineered Dragline Silk–
Expression of authentic spider silk in bacterial hosts is inefficient since some eukaryotic codons are not translated efficiently in bacteria.
Gene manipulation and amplification by PCR is difficult due to the repetitive nature of silk.
Drink your goat-milk silk!!!!
Dehydration and extensional flow must be reproduced in vitro to produce silk with the expected high strength, extensibility and toughness of native dragline silk.

24. Preliminary Attempts at Engineering Dragline Silk Has Been Successful–
Artificial spinning procedures of engineered dragline silk in hexafluoroisopropanol have produced films with a tensile strength on the order of 10 GPa and an elongation/extensibility 3-fold higher than native dragline silk.
Alteration of spinning conditions can markedly improve select characteristics of engineered silk:
Faster spinning produces stronger, more brittle fibers
Slower spinning produces weaker, more elastic fibers
The major hurdle for mass production and commercial application is producing engineered silk in mass quantity.

25. Questions, Comments, Screams of Fury and Pain???
Drink your goat milk!!!!
Questions, Comments, Screams of Fury and Pain???

26. References (Alphabetical)–
Allcock & Lampe. Contemporary Polymer Chemistry 2nd Ed. Prentice Hall, Inc., 1990.
Altman et. Al. Biomaterials 24: 401–416, 2003.
Becker et. Al. The World of the Cell 6th Ed. Pearson/Benjamin Cummings Press, 2005.
Chang et. Al. Polymer 46: 7909–7917, 2005.
Gosline et. Al. J. Exp. Biol. 202: 3295–3303, 1999.
Hinman et. Al. TIBTECH 18: 374–379, 2000.
Huemmerich et. Al. Biochemistry 43: 13604–13612, 2004.
Rengasamy et. Al. AUTEX Res. J. 5: 30–39, 2005.
Rising et. Al. Zoo. Sci. 22: 273–281, 2005.
Shao, Z. & Vollrath, F. Polymer 40: 1799–1806, 1999.
Tirrell, D. Science 271: 39 – 40, 1996.
van Beek et. Al. PNAS 99: 10266–10271, 2002.
Voet et. Al. Fundamentals of Biochemistry. John Wiley & Sons, Inc., 2001.