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)

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)

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.

Figure 2-17: Becker et. Al., World of the Cell 6 th Ed.

Scheme of biopolymer macromolecular assembly

Biopolymers Utilize a Variety of Functional Groups for Polymerization by Condensation–

Left– Figure 1-2: Voet et. Al., Fundamentals of Biochemistry. Right– Figure 3-6a: Voet et. Al., Fundamentals of Biochemistry.

Common functional groups employed for biopolymer formation

Nucleic acid structure highlightingchemically distinct ends

Candidate functional groups for condensation polymerization must either act as a

nucleophile or electrophile

Efficiency of Biopolymer Synthesis–• 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

Top– Figure 4-3: Voet et. Al., Fundamentals of Biochemistry. Right– Figure 26-28: Voet et. Al., Fundamentals of Biochemistry.

Scheme of amino acidpolymerization by condensation

Scheme of ribosomal (catalytic)protein synthesis

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.

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 3-6: Becker et. Al., World of the Cell 6 th Ed.

Figure illustrating the four hierarchallevels of protein structure

Structure is a consequence of sequence.

Function is a consequence of structure.

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.

Macromolecular Structure of Silk Spidroin–

• 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 2.53 + 0.4 m.

• Mucopolysaccharide is infused within, and on the surface of the silk fibers (removed by toluene treatment).

Figure courtesy of Rengasamy et. Al., 2005.

SEM of untreated and toluene treated spidroin fibers

Primary Sequence of Spider Silk Spidroin–

• 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

• Four motifs exist in the primary structure:– GPGXX (X often Q)

– An or (GA)n

– GGX– Spacer regions

Figure courtesy of Gosline et. Al., 1999.

Sequences of major ampullate spidroinhighlighting motif transitions

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

Figure courtesy of van Beek et. Al., 2002.

DOQSY Spectra and Ramachandran diagrams of silk spidroin fibers

Alanine torsion angles indicate –135, 150What does this data suggest?

Circular Dichroism Spectra Indicates -Sheet Structure–

• 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.

CD Spectra and cooperative thermal transitions of spidroin segments against

an -helical background

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.

Figure courtesy of Gosline et. Al., 1999.

Summary figure of spidroin crystal structure in supercontracted vs. fibers

Physicochemical Analysis of Major Ampullate Spidroin–

• 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 186.4 C (low glass transition temperature).

Figures courtesy of Rengasamy et. Al., 2005.

Differential scanning calorimetry & thermal mechanical analysis of spidroin fibers

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

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.

Figures courtesy of Shao et. Al., 1999.

Stress-strain curves of major ampullatespidroin in different solvents

Dried Spidroin Fibers Do Not Recover Fully–

• 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.

Stress-strain curves of major ampullatespidroin in before and after submersion &

drying in different solvents

Multiple Loading-Unloading Decreases Toughness and Extensibility Only Marginally After Drying–

• 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.

Successive stress-strain curves of major ampullate spidroin after submersion &

drying in water

High-Strain-Rate Impact Reveals Hysteresis–• 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.

High-strain-rate analysis approximating common loads experienced by spidroin fibers

Stress-Strain Comparisons With High-Performance Polymers–

• 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.

Table courtesy of Gosline et. Al., 1999.

Rationalizing Spidroin Properties With Fiber Structure–• 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

Proposed model for dragline silk fiber

Figure courtesy of van Beek et. Al., 2002.

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 PO4

3–) 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.

Image courtesy of: www.hubcap.clemson,edu/~ellisom/biomimeticmaterials/files/spinningsystems.htm.

Micrograph of a single spider spinneret highlighting internal anatomy

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.

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.

Drink your goat milk!!!!

Questions, Comments, Screams of Fury and Pain???

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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.• www.hubcap.clemson,edu/~ellisom/biomimeticmaterials/files/

spinningsystems.htm. • van Beek et. Al. PNAS 99: 10266–10271, 2002.• Voet et. Al. Fundamentals of Biochemistry. John Wiley & Sons,

Inc., 2001.