1. BCH 443 Biochemistry of Specialized Tissues
2. Fibrous Proteins

2. Fibrous vs. Globular Proteins
Globular Fibrous
1. Compact protein structure Extended protein structure
2. Soluble in water (or in lipid Insoluble in water (or in lipid bilayers) bilayers)
3. Secondary structure is complex Secondary structure is simple
with a mixture of a-helix, b-sheet based on one type only
and loop structures
4. Quaternary structure is held Quaternary structure is usually together by noncovalent forces held together by covalent bridges
5. Functions in all aspects of Functions in structure of the body metabolism (enzymes, transport, or cell (tendons, bones, muscle, immune protection, hormones, etc). ligaments, hair, skin)

3. Fibrous Proteins
Fibrous proteins have high a-helix or b-sheet content. Most are structural proteins.
Examples include:
Collagen
Elastin
Keratin
Fibroin

4. Fibrous Proteins
Much or most of the polypeptide chain is parallel to a single axis
Fibrous proteins are often mechanically strong & highly cross-linked
Fibrous proteins are usually insoluble
Usually play a structural role

5. What makes collagen a strong tensile protein?

6. Questions?
1. How would you define the structure of a collagen molecule?
2. What are the dimensions of a collagen molecule?
3. What are the dimensions of a collagen fibril?
4. State the most important amino acids in collagen and explain their importance.
5. What is the periodicity of collagen? Why does it happen?

7. Collagen Background
The collagens are the most abundant proteins in the body.
They occur in connective tissues where tensile strength is needed.
Examples: skin, tendons, cartilage, bones.
Tensile strength results from the use of:
The triple helix secondary structure
The assembly of tropocollagen subunits into a fibre
Chemical cross linking to strengthen the fibre

8. Secondary structure - the triple helix
Collagen is formed from tropocollagen subunits. The triple helix in tropocollagen is highly extended and strong.
Features:
(1) Three separate polypeptide chains arranged as a left-handed helix (note that an a-helix is right-handed).
(2) 3.3 residues per turn
(3) Each chain forms hydrogen bonds with the other two: STRENGTH!

9. Collagen A Triple Helix
Principal component of connective tissue (tendons, cartilage, bones, teeth)
Basic unit is tropocollagen:
Three intertwined polypeptide chains (1000 amino acid residues each)
MW = 285,000
300 nm long, 1.4 nm diameter
Unique amino acid composition

10. Collagen Amino Acid Composition
Nearly one residue out of three is Gly
Proline content is unusually high
Many modified amino acids present:
4-hydroxyproline
3-hydroxyproline
5-hydroxylysine
Pro and HyPro together make 30% of res.

11. Collagen Amino Acid Sequence
AA sequence of C-terminal region of bovine type-I collagen

12. Hydroxylated residues found in collagen

13. Biosynthesis of hydroxyPro and hydroxyLys requires O2 and ascorbic acid (vitamin C). Vit. C deficiency leads to disorders in bone, skin and teeth.

14. The Collagen Triple Helix
The unusual amino acid composition of collagen is not favorable for a-helices OR b-sheets
But it is ideally suited for the collagen triple helix: three intertwined helical strands
Much more extended than a-helix, with a rise per residue of 2.9 Angstroms
3.3 residues per turn
Long stretches of Gly-Pro-Pro-HyPro

15. In collagen triple helix H-bonds form between separate chains
In collagen triple helix H-bonds form between separate chains. In a-helix H-bonds formed between residues of the same chain.

16. Collagen Fibers
Fibers are formed by staggered arrays of tropocollagens
Banding pattern in EMs with 68 nm repeat
Since tropocollagens are 300 nm long, there must be 40 nm gaps between adjacent tropocollagens (5 x 68 = 340 Angstroms)
40 nm gaps are called "hole regions" - they contain carbohydrate and are thought to be nucleation sites for bone formation

17. Electron micrographs of colagen fibers showing band pattern

18. Structure of collagen fibers
(a) and (b) the primary and secondary structure
(c) lower magnification emphasizes the triple-helix
(d) tropocollagen molecules align side by side to form collagen fiber

19. Biosynthesis and assembly of collagen
Synthesis on ribosome. Entry of chains into lumen of endoplasmic reticulum occurs with the first processing reaction removing signal peptide
Collagen precursor with N and C terminal extensions
Hydroxylation of selected protein and lysines

20. Biosynthesis and assembly of collagen (Con’t)
Addition of Asn-linked oligosaccharides to collagen
Initial glycosylation of hydroxylyine residues
Alignment of three polypeptide chains and formation of inter-chain disulfide bridges
Formation of triple helical procollagen
Transfer by endocytosis to transport vesicle
Exocytosis transfers triple helix to extracellular phase
Removal of N and C terminal propeptides by specific peptidase
Lateral association of collagen molecules coupled to covalent cross linking creates fibril

21. Structural Basis of Collagen Triple Helix
Every third residue faces the crowded center of the helix only Gly fits
Pro and HyPro suit the constraints of phi and psi
Interchain H-bonds involving HyPro stabilize helix
Fibrils are strengthened by intrachain lysine-lysine and interchain hydroxypyridinium cross links

22. Biosynthesis of Aldol Cross-links in Collagen

23. Lecture 9: Fibrous Proteins
Biosynthesis of cross links between Lys, His, and hydroxy-Lys residues in collagen.

24. Lecture 9: Fibrous Proteins
The Major Collagen Groups
In humans at least there are 19 different collagens. Within these 19 structural types four major classes are generally identified.

25. Classification of Collagens
Lecture 9: Fibrous Proteins
Classification of Collagens
Type
Chains
Tissue Found
Characteristics I
a1(I)2, a2(I)
Bone, skin, tendons
Low carbohydrate; <10%Hydroxylysines per chain II
a1(II)3
Cartilage, vitreous
10% carbohydrate; >20 hydroxylysines per chain
III
a1(III)3
Blood vessels, scar tissue, uterine wall

26. Classification Continued
Lecture 9: Fibrous Proteins
Classification Continued IV
[a1(IV)3
a2(IV)3]
Basement membrane
lens capsule
High carbohydrate,
>40 hydroxylysines per chain V
[a1(V)2a2(V)]
[a1(V)3]
[a1(V)a2(V)a3(V)]
Cell surface and exoskeleton
High carbohydrate, high glycine and hydroxylysine VI
Aortic intima, placenta, kidney
Low mol.weight, equal amounts of hydroxylysine and hydroxyproline

27. Lecture 9: Fibrous Proteins
Thermal Denaturation Curve
In normal collagens the transition midpoint temperature or Tm is related to the normal body temperature of the organism and for animal is above 40 oC as shown in blue line in the graph.. Tm
Triple helix stabilization is through HyPro and formation of H bonds with neighboring chains.

28. DISORDERS OF COLLAGEN DEPOSITION

29. Disorders of Collagen Deposition
insufficient collagen content
presence of chemically and/or morphologically abnormal collagen
excessive collagen content
insufficient collagen resorption
excessive collagen resorption

30. Disorders of Collagen Deposition
Genetic abnormalities of collagen
mutations that lead to aminoacid deletions or additions
deficient synthesis of a portion
disorders in post-translational modification (hydroxylation of lysine, hydroxylation of proline)
defects in enzymes essential for post-translational modification

31. Disorders of Collagen Deposition
Collagen is the building block; thus, its disorders lead to significant deterioration in the mechanical integrity of tissues
Several disorders
Ehlers-Danlos syndrome
Osteogenesis Imperfecta
Marfan syndrome

32. Three factors make it stretchy and elastic
ELASTIN
Three factors make it stretchy and elastic

33. Elastin
Elastin can stretch several times - then return to the original starting size
Elastin is found in large arteries (the aorta), ligaments, and the lung wall.
(1) The subunits of elastin are called tropoelastin – molecules 1, 2, 3 and 4.
The crosslinking of tropoelastin via lysine residues results in a stable starting network of elastin (i.e. when not stretched).
Either desmosine (4 Lys) links 4 molecules of tropoelastin, or lysinonorleucine (2 Lys) links 2 tropoelastin molecules.
It is clinically relevant in cardiovascular disease and lung emphysema

34. Desmosine
Desmosine is formed from 4 lysines, 3 of which are oxidised.

35. Elastin (2) Amino acid composition of elastin
33% Gly 10% Pro and Hyp 23% Ala 13% Val
Hence 79% of the residues come from 4 amino acids.
There are large hydrophobic peptides rich in Ala, Val, Ile and Leu.
As these sidechains do not interact with each other by hydrogen bonds, they enable the core of elastin to separate and stretch easily.
(3) Secondary structure of elastin
A different type of helix structure from those in the a-helix is present. This is able to stretch and relax like a coiled spring. So elastin is elastic!
This is constructed from a helix of repeated b-turns based on the sequence Val.Pro.Gly.Val, and is called the b-spiral.

36. Elastin Abundant in ligaments, lungs, artery walls, skin.
Provides tissues with ability to stretch in all directions without tearing.
Contains predominantly small hydrophobic residues: 1/3 Gly, 1/3 Ala + Val, many Pro but no hydroxyPro or hydroxyLys.
Lacks regular secondary structure.
Has unordered coil structure that is highly cross-linked into 3-dimensional network of fibers to provide rubber-like elasticity.

37. Elastin Cross-links formed from allyysine (aldehyde derivative of Lys)
Extracellular Lys oxidase specific for Lys-Ala-Ala-Lys and Lys-(Ala)3-Lys sequences
Lys + 3 allysine combine to from desmosine or isodesmosine cross-links responsible for yellow color of elastin
Also forms lysinorleucine cross-links from 2 allysine, as in collagen.
Cross-links responsible for elasticity & insolubility

38. Lysine amino
oxidase
Aldol condensations
Desmosine
cross-link
Biosynthesis of desmosine and isodesmosine cross-links unique to elastin

39. KERATIN a-Keratins are found in mammals
a-Keratins are found as a left-handed super helix
b-Keratins are found in birds and reptiles
b-Keratins are analogs to the silk fibroin structures produced by spiders and silkworms

40. Two reasons why this is a tough protective fibrous protein
a-KERATIN
Two reasons why this is a tough protective fibrous protein

41. a-Keratin
a-keratin is found in hair, nails, outer layer of skin. It forms almost the entire dry weight of these materials.
(1) The entire secondary structure is a dimer of two a-helices.
It is rich in amino acids that favours a-helix formation (Phe, Ile, Val, Met, Ala)
These hydrophobic side chains are on the a-helix surface-explaining its insolubility.
It is also rich in Cys residues.

42. Structure of dimer of two a-helices.

43. Proposed structure for a-keratin intermediate filaments
Two monomers (a) pair via a parallel coiled-coil to form 50- nm-long dimer (b)
These then associate to form 1st protofilament (c)
These then associate to form protofibril (d)
Regular spacing of 25 nm along the fibers is accounted for by overlap

44. Disulphide bridges and toughness in a-keratin
(2) Cys residues form disulphide bridges in a-keratin, and link the a-helices together.
The more disulphides, the stronger the a-keratin.
Cys
Disulphide bridges are also frequently used to stabilise the interior of a globular protein.
Cys

45. Quaternary structure a-keratin of
The association of long parallel a-helices also gives toughness to a-keratin.
The incorrect explanation of a-keratin structure states that THREE a-helices supercoil around each other to form a protofibril, and that the association of 2 and 9 protofibrils forms a hair microfibril.
Lippincott’s Fig 3.31 on page 45 is wrong!
UP-TO-DATE MODEL
Proto-filament of anti-parallel dimers
The up-to-date view is that TWO parallel a-helices supercoil around each other to form a dimer.
Then each dimer associates antiparallel with two other dimers to form the protofibril.
The association of four protofibrils forms a four-stranded rope. These successive overlaps explain why a-keratin is such a tough protein.
Clinical relevance in skin diseases: psioriasis – the overproduction of a-keratin

46. a-Keratin Structure

47. Fibroin Fibroins are the silk proteins. They also form the spider webs
Made with a -sheet structures (M6.12) with Gly on one face and Ala/Ser on the other
Fibroins contain repeats of [Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-Ala-Gly-Ala-Gly)8]
The -sheet structures stack on top of each other (M6.12b)
Bulky regions with valine and tyrosine interrupt the -sheet and allow the stretchiness

48. Structure of silk fibroin
(a)Three dimension view of the stacked b-sheets
(b) Interdigitation of Al or Ser and Gly side chain
The plane of the section is perpendicular to the folded sheets