STRUCTURAL AND CONNECTIVE TISSUE PROTEINS

 Fibrous proteins – filamentous, elongated form; structural role, hold things together; major portions of skin, CT and fibers;
 AA sequence forms particular secondary structure conferring its mechanical properties
 Central Dogma of Proteins: Sequence  Structure  Function

AA and Properties α-keratin Fibroin Collagen Elastin

Gly – smallest, aliphatic, nonpolar 44.6 32.7 32.3
Ala – small 29.4 12.0 23.0
Ser – OH group 10.2 12.2
Glu + Gln 12.1
Cys – for disulfide crosslinks 11.2
Pro – rigid 22.1
Val 12.1

Keratin
 Mechanically durable, chemically unreactive
 In outer epidermis, hair, horns, nails and feathers
 α-keratins
o major proteins of hairs, fingernails and animal skin
o part of filament proteins for structural roles in nuclei, cytoplasm and surfaces
o Type I – acidic, Type II - basic
o > 300 residues, right-handed α-helical molecules in a left-handed coiled coil structure
o In hair: 2 coiled coils further twisted together into 4-molecule protofibril  8 protofibrils = circular / square arrangement
o In different tissues: hardened due to disulfide crosslinks (high cysteine content)
 β-keratins
o β sheet structure, found in birds, reptiles, feathers and scales
 Epidermolysis bullosa – keratin sequence defect  abnormal filament formation  loss of skin integrity  skin blistering

Fibroins
 used by silkworms and spiders, tough yet flexible fiber
 anti-parallel β sheet structure with polypeptide chains running parallel
 multiple repetitions of [G-A-G-A-G-S-G-A-A-G-(S-G-A-G-A-G)a]
 Almost every other residue is Gly with either Ala or Ser between  allows for fitting and stacking
 Strong, relatively inextensible (covalently bonded chains pre-stretched), flexible due to van der Waals forces between sheets

Collagen
 Major component of connective tissue, most abundant (25%) of all vertebrate protein with diverse functions and forms
 Liver 4%, Lung 10%, Aorta 12-14%, Cartilage 50%, Cornea 64%, Cortical bone 23%, Skin 74%
 Collagen + CaPO4 in bone, crystalline in eye, rope-like fibers in tendons, loosely woven fibers in skin
 Tropocollagen
o basic molecule of collagen, α-chain (3 left-handed polypeptide helices) coiled in a right-handed supercoil
o Complete turn every 3.0 residues, 0.31 nm pitch, 300nm long, 1.5 nm diameter
 Amino Acid Composition:
o 1/3 glycine (every 3rd position), 1/4 proline, many lysine
o Proline and lysine postranslationally modified into 4-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl) in the chain

 Pro + Pro hydroxylase + ascorbic acid + α-ketoglutarate  Hyp + succinate

 Lys + Lys hydroxylase + ascorbic acid + α-KG  Hyl
o Vit C deficiency (↓ ascorbic acid) = weak skin and blood vessels (scurvy)
o OH of Hyp participates in interchain hydrogen bonding, Hyl for polysaccharide attachment
 As a Glycoprotein
o Hyl residues for carb binding, usually with glucose-galactose in α(12) added by galactosyl transferase and glucosyl transferase
o Galactose bound to OH of Hyl via β glycosidic bond
o Unknown function
 Primary Structure
o Collagen I – most common type
o α1 and α2 – kinds of polypeptide helices, 2:1 ratio, forms type I helix
o wound in opposite directions to prevent unwinding of smaller strands

Type / Chain Type Molecular Formula Main Distribution Notes

 I (α1)2α2 Skin Most abundant, 90% of all collagen, most common fibrillar
II (α1)3 Cartilage
III (α1)3
IV (α1)3 Basal laminae Sheet-like meshwork
V (α1)2α2

o 5 different α1 chains (Type I to V), and 2 different α2 chains (Type I and V)

o Fibrillar collagen (Type I, II and III) – 10-300nm, cable-like, aggregate
o α1 and α2 chains – regularly repeating AA sequence (Gly-X-Y)n, Glycine every 3rd residue, followed by Pro and Hyp/Hyl
o Glycine – small size, since every 3rd residue of a strand makes unusually close contact with other 2 strands which only Gly can fit
o Proline – little conformational flexibility (pyrrolidine rings repel each other by steric hindrance)  rigidity
o intermolecular hydrogen bonds – amide hydrogen of Gly + carbonyl of Pro
 Covalent Cross-linking
o Tropocollagen molecules associate to form collagen microfibrils, covalent bonds for mechanical strength
1. ε-amino of Lys + lysine oxidase  allysine (aldehyde)
2. aldehyde of allysine + ε-amino of another Lys  Schiff base = cross-link OR
3. aldehyde of allysine + aldehyde of allysine  Aldol condensation = cross-link
 Stability
o Temperature can affect tropocollagen formation, can form gelatin (random coil)
o Melting temperature (Tm) – temperature at which half of the helical structure is lost
o Tm of tropocollagen – criterion for stability
o Different collagen species have different melting temperatures
o ↑ Hyp = ↑ Tm (due to imino acid content) = more stable
o Staiblity of helix of a single strand depends on locking of Pro and Hyp
 Biological Assembly
1. Synthesis & Entry of Fibroblast RER – ribosome translates procollagen (pro-α1 and pro-α2) that have extension peptides (extra 10 AA @ N-
termini and 300 in C-terminal)
2. Cleavage of Signal Peptide
3. Posttranslational Modification in SER – Pro and Lys converted into Hyp and Hyl
4. Initial Glycosylation – addition of sugar residues
5. Bundle formation in Golgi – procollagen aggregates in 3x stranded bundles
 Carboxyl of Extension peptides form disulfide bonds – helps in aligning of 3 pro-α chains into 3x helix, prevents premature
tropocollagen formation
6. Further Glycoslation
7. Transport vesicle
8. Secretion into ECM
9. Removal of Extension peptides – via procollagen peptidases  tropocollagen
10. Fiber aggregation on fibroblast surface – as microfibrils
11. Lateral association of collagen molecules and cross-linkages – maturation of collagen
 Staggered Array of Tropocollagen
o Tropocollagen fibers spontaneously associate  collagen
o Presence of dark bands at right angles to long axis due to quarter staggered array
o Cross-striations every 680 A, Length of 3000 A, gaps of 400 A between tropocollagen molecules (filled with hydroxyapatite in bone)
o Each tropocollagen molecule shifted by 1/4 of its length from next molecule  complete overlapping every 5 molecules
 Degradation
o Resistant to enzymatic attack, but collagenases cleave peptide bonds
o Clostridium histolyticum bacterium – gas gangrene, splits collagen in >200 sites, targets Glycine
o Tissue collagenases – required for rapid reorganization of tissue
 Defects
o Ehlers-Danlos Syndrome – genetic, defective removal of extension peptides (↓ procollagen peptidase), ↑ procollagen = stretchable skin,
hypermobile joints, short stature
o Dermatospraxis – recessive disease in cattle, no procollagen peptidase, disorganized collagen bundles, fragile dermis
o Osteogenesis Imperfecta (Brittle Bone disease) – deletion of part of all of COL1A1 & COL1A2, single AA change can cause disease,
skeletal deformities, easy fracturing
o Lathyrism – inhibition of lysine oxidase via nitriles / β-aminopropionitrile, spine deformation, bone deminleralization, joint dislocation,
aortic aneurysms, produced with chronic D-penicillamine (treatment of Wilson’s disease & Hg/Pb poisioning)
o Marfan’s Syndrome – genetic, lathyritic symptoms, inability to form lysine oxidase substrate

Elastins
 Yellow, fluorescent protein capable of stretching without tearing
 in ligaments, lungs, arterial walls, some in skin, tendon and loose CT, with collagen and polysaccharides
 830 AA, 64K-66K MW, irregular / random coil conformation (no 2° structure)
 Tropocollagen – basic building unit
 ↑ Gly, ↑ Pro, ↓ hydroxyproline, few polar AA, ↑ aliphatic / nonpolar
 Proelastin – precursor, 72kD, several regions of lysine separated by 2-3 Ala  for covalent cross-linking
 Elastin formation – covalent cross-linking of side-chains of adjacent polypeptides via:
o Lys + Lysine oxidase  allysine + ε-amino of Lys
o 3 allysine + ε-amino of Lys  desmosine – molecular know that ties 4 chains of elastin together, yellowish
 No fixed / regular conformations  net-like
 5x as stretchy as rubber band, too much stretching may deprive blood vessel stability  balance between strength (collagen) and elasticity (elastin)
 Cross-linking ↑ with age = ↓ elasticity of arterial walls  possible contribution to circulatory diseases
 William’s Syndrome – deletion of elastin gene, aortic valve stenosis

Fibronectin
 Large, fibril-forming adhesive glycoprotein, helps cells attach to extracellular matrix
 Dimer of 2 similar subunits (2500 AA) joined by a pair of disulfide bonds near carboxyl termini  folded into globular domains separated by a
flexible polypeptide chain
 Multifunctional due to various globular domains that bind to: collagen, heparin, receptors
 For cell adhesion and migration as it guides migration in developing embryos

Laminin
 Extracellular glycoprotein, connects epithelial cells to CT
 ~ 850kD, 3 very long polypeptide chains, crucifix formation, held together by disulfide bonds
 Functional domains bind with: collagen IV, heparan sulfate, laminin receptors on cells