BCH 443
Biochemistry of
Specialized Tissues
2. Fibrous Proteins
Lect. 6-1
Fibrous vs. Globular Proteins
Globular
Fibrous
1. Compact protein structure
Extended protein structure
2. Soluble in water (or in lipid
bilayers)
Insoluble in water (or in lipid
bilayers)
3. Secondary structure is complex
with a mixture of a-helix, b-sheet
and loop structures
Secondary structure is simple
based on one type only
4. Quaternary structure is held
together by noncovalent forces
Quaternary structure is usually
held together by covalent bridges
5. Functions in all aspects of
metabolism (enzymes, transport,
immune protection, hormones, etc).
Functions in structure of the body
or cell (tendons, bones, muscle,
ligaments, hair, skin)
Lect. 6-2
Fibrous Proteins
Fibrous proteins have high a-helix or b-sheet
content. Most are structural proteins.
Examples include:
• Collagen
• Elastin
• Keratin
• Fibroin
Lect. 6-3
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
Lect. 6-4
COLLAGEN
What makes collagen a strong
tensile protein?
Lect. 6-5
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?
Lect. 6-6
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:
(a) The triple helix secondary structure
(b) The assembly of tropocollagen subunits into a fibre
(c) Chemical cross linking to strengthen the fibre
Lect. 6-7
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!
Lect. 6-8
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
Lect. 6-9
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.
Lect. 6-10
Collagen Amino Acid Sequence
AA sequence of C-terminal region of bovine type-I collagen
Lect. 6-11
Hydroxylated residues found in collagen
Lect. 6-12
Biosynthesis of hydroxyPro and hydroxyLys requires
O2 and ascorbic acid (vitamin C). Vit. C deficiency
leads to disorders in bone, skin and teeth.
Lect. 6-13
The Collagen Triple Helix
• The unusual amino acid composition of
collagen is not favorable for a-helices OR bsheets
• 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
Lect. 6-14
In collagen triple helix Hbonds form between separate
chains. In a-helix H-bonds
formed between residues of
the same chain.
Lect. 6-15
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
Lect. 6-16
Electron micrographs of colagen fibers showing band pattern
Lect. 6-17
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
Lect. 6-18
Biosynthesis and assembly of collagen
11
OH
OH
OH
5
OH
OH
OH
OH
Collagen molecules covalently cross-linked to fibril
OH
6
OH
OH
OH
OH
S
OH
S
Tropocollagen with N and
C terminal peptides removed
10
OH
4
OH
S
S
S
S
OH
OH
OH
OH
OH
OH
3
OH
Tropocollagen
OH
OH
Extracellular region
9
Plasma
membrane
Exocytosis
2
N terminal
peptide
C terminal
peptide
7
1
S
S
Signal sequence
8
S
OH
Collagen
mRNA
OH
Endocytosis
OH
S
Transport vesicle
1. Synthesis on ribosome. Entry of chains into lumen of endoplasmic
reticulum occurs with the first processing reaction removing signal
peptide
2. Collagen precursor with N and C terminal extensions
3. Hydroxylation of selected protein and lysines
19
Biosynthesis and assembly of collagen (Con’t)
4. Addition of Asn-linked oligosaccharides to collagen
5. Initial glycosylation of hydroxylyine residues
6. Alignment of three polypeptide chains and formation of interchain disulfide bridges
7. Formation of triple helical procollagen
8. Transfer by endocytosis to transport vesicle
9. Exocytosis transfers triple helix to extracellular phase
10. Removal of N and C terminal propeptides by specific peptidase
11. Lateral association of collagen molecules coupled to covalent
cross linking creates fibril
Lect. 6-20
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 lysinelysine and interchain hydroxypyridinium
cross links
Lect. 6-21
Biosynthesis of Aldol Cross-links in Collagen
Lect. 6-22
Biosynthesis of cross links
between Lys, His, and hydroxyLys residues in collagen.
Lect. 6-23
The Major Collagen Groups
In humans at
least there are 19
different
collagens. Within
these 19
structural types
four major
classes are
generally
identified.
Lect. 6-24
Classification of Collagens
Type
Chains
Tissue Found
Characteristic
s
I
a1(I)2, a2(I)
Bone, skin,
tendons
Low
carbohydrate;
<10%Hydrox
ylysines per
chain
II
a1(II)3
Cartilage,
vitreous
10%
carbohydrate;
>20
hydroxylysine
s per chain
III
a1(III)3
Blood vessels,
scar tissue,
uterine wall
Lect. 6-25
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
Aortic intima,
placenta, kidney
Low mol.weight,
equal amounts of
hydroxylysine
and
hydroxyproline
VI
Lect. 6-26
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.
Lect. 6-27
DISORDERS OF COLLAGEN
DEPOSITION
Lect. 6-28
Disorders of Collagen Deposition
• Disorders of collagen deposition
– insufficient collagen content
– presence of chemically and/or morphologically
abnormal collagen
– excessive collagen content
– insufficient collagen resorption
– excessive collagen resorption
Lect. 6-29
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
Lect. 6-30
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
Lect. 6-31
ELASTIN
Three factors make it
stretchy and elastic
Lect. 6-32
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.
It is clinically relevant in
cardiovascular disease
and lung emphysema
(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.
Lect. 6-33
Desmosine
Desmosine is formed from 4 lysines, 3 of which are oxidised.
CO
CO
NH
a CH
a CH
Allysine
CH2
CH2
CH2
CH2
CH2
CH2
NH
NH
Allysine
a CH CH2
CH2
Allysine
CHO
CH2
CHO
CHO
CH2
CH2
CH2
NH
NH
a CH CH2
C
CH2
N+
CO
CH2
a CH
CO
CH2
CH2
Desmo sin e
CH2
NH
C
C
CH2
Lysine
C CH2
C
a CH
CO
NH3+
CO
NH
CH2
CH2
CH2
CH2
a CH
a CH
CO
NH
CO
Lect. 6-34
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.
Lect. 6-35
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 crosslinked into 3-dimensional network of fibers to
provide rubber-like elasticity.
Lect. 6-36
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
Lect. 6-37
(CH2)3
CH2
NH 2
NH 2
CH2
(CH2)3
(CH2)3
CH2
NH 2
Lysine amino
oxidase
O
H
NH 2
CH2
(CH2)3
(CH2)3
C H
(CH2)3
CH2
NH2
O
H O
C
C
(CH2)3
(CH2)3
Aldol condensations
Biosynthesis of
desmosine and
isodesmosine
cross-links
unique to elastin
CH2
(CH2)3
H 2C
CH2
N+
H 2C
(CH2)2
Desmosine
cross-link
CH2
CH2
Lect. 6-38
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
Lect. 6-39
a-KERATIN
Two reasons why this is a
tough protective fibrous
protein
Lect. 6-40
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.
Lect. 6-41
Structure of
dimer of two
a-helices.
Lect. 6-42
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
Lect. 6-43
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 akeratin.
Disulphide bridges
are also frequently
used to stabilise the
interior of a globular
protein.
CO
NH
a CH
CO
NH
Cys
aCH
CH2
CH2
SH
S
SH
S
CH2
CH2
Cys aCH
a CH
NH
CO
NH
CO
Lect. 6-44
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!
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.
UP-TODATE
MODEL
Protofilament
of antiparallel
dimers
Clinical relevance in skin diseases: psioriasis – the overproduction of a-keratin
Lect. 6-45
a-Keratin Structure
Lect. 6-46
Fibroin
• Fibroins are the silk proteins. They also form the
spider webs
• Made with a b-sheet structures (M6.12) with Gly
on one face and Ala/Ser on the other
• Fibroins contain repeats of [Gly-Ala-Gly-AlaGly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-Ala-Gly-AlaGly)8]
• The b-sheet structures stack on top of each other
(M6.12b)
• Bulky regions with valine and tyrosine interrupt
the b-sheet and allow the stretchiness
Lect. 6-47
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
Lect. 6-48