Silk: A Fibrous Protein notes

Silk: A Fibrous Protein

Silk is produced by insects and spiders.
Its polypeptide chains are predominantly in the beta conformation, resembling a flat sheet.
Spider silk exhibits outstanding mechanical properties despite being spun at room temperature and normal pressure using water as a solvent.
Silk possesses unique properties that humans cannot fully replicate.
It's 10 times stronger than steel by mass and five times stronger than Kevlar, yet twice as elastic as nylon.
The simplicity of its structure contributes to these incredible properties.

Silk Structure and Properties

Spiders control the folding and crystallization of protein constituents to spin webs.
Silk has a beta composition where side chains of beta strands interdigitate, forming strong beta structures.
These beta structures provide tenacity and strength.
Amorphous regions separate beta structures, contributing to elasticity.
Stretching initially affects amorphous regions; further force engages the strong beta pleated sheets.

Silk Production and History

Silk was historically expensive, produced by domesticated silkworms (Bombyx mori) in Asia.
Silk threads spun from cocoons were a mainstay of world commerce.
World War II demands led to the development of man-made fibers from petrochemicals, superseding silk.
Today, nylon production far outweighs silk production.
Silk was crucial in the Middle Ages and remains important.

Fibrion Composition

Fibrin consists of layers of antiparallel sheets rich in alanine and glycine.
It features a repetitive motif of alanine-glycine, forming antiparallel beta-pleated sheets.
Small side chains of glycine and alanine interdigitate, enabling close packing of sheets.
This simple design accounts for the material's strength.
The structure is stabilized by extensive hydrogen bonding between peptide linkages and optimized by van der Waals interactions between sheets.
The structure is flexible due to numerous weak interactions rather than covalent bonds like disulfide bonds in alpha-keratin.

X-ray Diffraction of Silk Fibers

X-ray diffraction of silk fibers from Bombyx mori reveals a simplistic pattern.
X-ray crystallography can determine the three-dimensional structure of molecules.
The simple diffraction pattern indicates the simplicity of fibrous proteins like silk, keratin, and collagen.
Comparison with the complex diffraction pattern of globular proteins like myoglobin highlights this simplicity.

Spider Silk Production

Spiders have six to eight glands, each producing unique silk compositions suited for different needs (strong dragline, soft cocoons, sticky prey capture).
Silk within spider ducts is a liquid crystalline, disordered substance.
As silk is pulled from spinnerets through glands, dehydration and pH changes cause beta-pleated strands to align.

Globular Proteins

Most proteins are globular and include non-repetitive structures.
Globular proteins contain various secondary structure arrangements (helices, beta sheets).
A significant portion of protein structure may be irregular or unique.
Segments of polypeptide chains without similar dihedral angle values are sometimes referred to as simple coils.
Coils should not be confused with random coils, which refer to totally disordered, unfolded proteins.
Non-repetitive structures in native folded proteins are ordered but irregular.
Globular proteins are structurally more complex and compact than fibrous proteins.
They perform diverse functions: enzymes, transport, regulation, immune response, storage, etc.

X-ray Diffraction of Myoglobin

X-ray diffraction photograph of sperm whale myoglobin shows much more complexity compared to silk.
Each dot corresponds to intensity, which can be converted to interatomic distances.
This data is interpreted by computers to reveal three-dimensional structure.
Myoglobin is a classic example of a globular protein.
The intensity of diffraction maxima (darkness of spots) is a function of crystal electron density.

Compactness of Globular Proteins

Native globular proteins fold into unique three-dimensional shapes.
Human serum albumin (65.5 kDa, 585 residues) demonstrates compactness.
Extended beta conformation of 585 residues would be 200 angstroms long.
Compact helix conformation would be 900 angstroms long.
Native globular form of human serum albumin is only 100 x 60 angstroms.
Globular dimension is achieved through multiple side chain interactions and compact packing.
Myoglobin was the first protein to be crystallized (late 1800s) and have its structure determined by X-ray crystallography.

X-ray Crystallography Advancements

X-ray crystallography for large molecules became possible with computer advancements in the late 1950s and early 1960s.
Computers enabled the calculation of numerous variables related to atom distances and intensities.

Myoglobin Structure

Myoglobin is solely alpha-helical, with no beta-pleated sheets.
Alpha helices of different lengths are connected by loops.
Myoglobin contains a globin (amino acid portion) and a heme group (prosthetic group).
The heme group contains iron that binds oxygen.

Heme Group and Iron

Iron in the heme group has a coordination number of six.
Four bonds are to heme nitrogens; iron is in the ferrous ( $Fe^{2+}$ ) state.
Only the ferrous state can reversibly bind and transport oxygen; oxidation to ferric ( $Fe^{3+}$ ) state prevents this.
Nitrogens donate electron density to the iron to maintain the ferrous state.
The fifth bond connects to a histidine residue in myoglobin, and the sixth is free to bind ligands like oxygen, nitrous oxide, or carbon monoxide.

Amino Acid Distribution in Proteins

Distribution of amino acids differs between water-soluble and transmembrane proteins.
The folding main chain of myoglobin lacks symmetry.
The tertiary structure of a protein refers to the overall course of its polypeptide chain.
The interior of myoglobin consists almost entirely of nonpolar residues (leucine, valine, methionine, phenylalanine).
Charged/polar residues (aspartate, glutamate, lysine, arginine) are absent from the inside, except for two histidines essential for heme and oxygen binding.
The outside of myoglobin consists of both polar and nonpolar residues, with a predominance of polar/charged residues.

Protein Folding in Aqueous Environment

Protein folding in aqueous environments is driven by the tendency of hydrophobic residues to exclude water from the hydrophobic core.
The system is more thermodynamically stable when hydrophobic groups cluster together.
Polypeptide chains fold to bury hydrophobic side chains inside and expose polar side chains to the surface.
Many alpha helices and beta strands are amphipathic, with hydrophobic faces pointing inward and hydrophilic faces pointing outward.
Van der Waals interactions between tightly packed hydrocarbon side chains contribute to protein stability.

Transmembrane Proteins

Transmembrane proteins in hydrophobic membranes are exceptions to the typical rule.
Porins, found in bacterial outer membranes, transport water molecules.
To be lipid-soluble, porins have a reverse design: hydrophobic amino acids on the outside surface for contact with membrane lipids.
The inside features a collection of charged/polar amino acids forming a polar pore for water transport - "inside-out" design.

Protein Folding Patterns

Motifs: stable arrangements of several secondary structures, indicative of protein function and class.
Domains: parts of a polypeptide that are independently stable, distinct globular units - larger than motifs.
Domains may have similar or different functions and can function independently.

Examples of Domains

Polypeptide chains with >200 residues usually fold into two or more globular domains giving the protein a bi- or multi-global appearance.
Most domains consist of 100-200 amino acid residues.
Dehydrogenases have a specific domain for binding the coenzyme nicotinamide, known as the dinucleotide-binding domain or Rossmann fold.
Immunoglobulins have an immunoglobulin fold comprising of a pair of beta-pleated sheets stapled together by hydrophobic interactions and disulfide bonds.

Supersecondary Structures and Motifs

CD4 receptors on white blood cells have four similar domains of ~100 amino acids each.
Common supersecondary structures/motifs include:
- Beta-alpha-beta motif: an alpha helix connects two parallel strands of beta sheet.
- Beta hairpin motif: antiparallel strands connected by a tight reverse turn.
- Alpha-alpha motif: two successive antiparallel helices stacked together.
- Greek key motif: beta hairpin folded over to form a four-stranded antiparallel sheet.

Beta Barrels

Beta structures often form beta barrels, supersecondary structures with named topologies (e.g., Greek key motif, alpha/beta barrel motif).
Names are inspired by geometric motifs in Native American or Greek weaving and pottery.

Techniques to Determine 3D Structure

X-ray diffraction and Nuclear Magnetic Resonance(NMR).

X-Ray Diffraction

Requires protein crystallization, which can be challenging.
Proteins are not solids or crystals, and crystallizing them may result in different confirmations.
Provides the finest visualization of protein structure but results in an average structure (time-space average).
Excellent resolution thanks to the wavelength of X-rays being around the length of a covalent bond.
Involves a protein crystal, an X-ray source, and a detector.
The crystal is rotated to strike the beam from different directions.
The analysis results into an X-ray photograph with reflections (spots).
The intensity from each spot is then measured. They positions and intensities constitute the raw data of the analysis.
The intensity needs to be measured and an image constructed using it.
Recontructuing an image can be done with computers to obtain an electron density map.
Electron density reveals the distribution of electrons which is represented by parallel sections stack on top of each others to denote altitude.

Limitations of X-Ray diffraction

Resolution affects image quality.
Six angstroms resolution reveals the polypeptide chain course but fewer structural details.
Two point four and three-point angstrom resolution is easier to define details.
Two angstrom is the theoretical limit.
Since 170,000 proteins have had their structures determined by this method.
The atomic coordinates are stored in the Protein Data Bank and structures can be accessed to glean insight on protein function and ligand binding.

Nuclear Magnetic Resonance(NMR)

Structures are done in solution.
Unlike X-Ray diffraction, this is more suited to proteins and their dynamics in real conditions.
Requires greater concentrations of a protein.
Suitable if a protein is 15kDa.
Can be used to understand change and folding.
Exploits a quantum-mechanical property, namely "nuclear spin angular momentum".
Exploits that atomic nuclei are magnetic.
The environment of the sample can be revealed using One Dimensional NMR.

Two Dimensional NMR

Used to address interactions of different atoms.
COSY is used for inter atomic distances of atoms that are covalently bonded.
Nuclear Overhouses Spectroscopy is applied to atoms that are not on the protein sequence, but are close in space.
*One needs experimental methods to fully determine structure, and to account for uncertainty in specturm and different structures at any one time, so multiple can generated.
NMR is limited to proteins that at best are 40kDa.
It is valuable as a method to validate structures from crystallography.
NMR can validate that crystallised proteins were accurately determined.
Can be used to analyse protein that fails to crystallise, and for analysis protein dynamics.

Quaternary structures.

For larger proteins or proteins with subunit construction. Defects can be repaired by simply replacing a subunit. And in case of enzymes the the positions of its reacting groups are better fixed.
Subunits interact via noncovalent hydrogen bonds, electrostatic interaction and disulfide bonds.
*In this sense, multiple subunits are advantageous as they cooperate to function, bind to molecules and display phenomenon of elastarism.
Common examples include:
- Dimers with two identical subunits
  *Tetramer such as hemoglobin of 2beta and 2 alpha subunits

*One can use Bioinformatics to perform an experiment on a complex with native and denatured proteins.

The denatured separates the native and their elution volumes differ, which confirms the presence of subunits and to measure quantity.
Electrophoresis also performs this analysis but shows the quantities of Kilodaltons in the same manner.

Protein Stability

The native proteins are marginally more stable than denatured ones.
*Several factors dictate stability
Hydrophobic effects: Nonpolar residues aggregate to increase entropy of H2O, and has the greatest effect
Electrostatic interations: important to protein stability.
Hydrogen Bonds: contribute to protein stability and fine tune tertiary. and selects the unique structure fro the hydrophobic
*Ion pairs, of group groups with disparate charge can be formed to stabilise
*Disulfide bonds form extra cellular as cross linkages stabilize in these oxidising environment as the interacellular is is redox.
*Finally metal ions can contribute to stability.