The structure and function of
fibrous proteins
Ka-Lok Ng
Asia University
Introduction
• Historically a division into globular and fibrous 纖維性
proteins – very different properties, different roles
• Today it is preferable to avoid this distinction and to treat
proteins as belonging to families that exhibit structural or
sequential homology
• Fibrous proteins – found to make up many of the fibres
found in the body
• Had a common role in conferring 賦予 strength and
rigidity
• Found in cells, connective tissues such as tendons (腱)
or ligaments (韌帶)
• These proteins tend to occur as rod-like structures
• Next chapter will deal with the membrane proteins
The amino acid composition and
organization of fibrous proteins
• Fibrous proteins - at least three different
structural designs
• (1) Coiled-coils of a helices – α keratins 角質素
• (2) extended β anti-parallel β sheets – such as
silk fibroin 蠶絲蛋白 a collection of proteins
made by spiders or silkworms
• (3) a triple helical arrangement of polypeptide
chains and shown by the collagen 膠原質 family
of proteins
The amino acid composition and
organization of fibrous proteins
• Amino acid composition of
fibrous proteins reveals
considerable differences in their
constituent amino acids to that
for globular proteins
• Collagen – high proline content,
> 20%, whilst silk fibroin < 1%
• a keratin – cysteine content is
11.2% but in collagen and silk
fibroin the level of cysteine ~ 0%
• In each case, the aa
composition influences the SS
formed by fibrous proteins
Keratins
• Found as hair, feathers, scales, nails
or hooves of animals
• Properties – mechanically strong,
unreactive
• At least two major groups of
keratins – α keratins are typically
found in mammals whilst β keratins
are found in birds and reptiles
β keratins are analogous to the silk
fibroin structures produced by spiders
and silkworms
α keratins are a subset of filamentous
proteins based on coiled-coils called
intermediate filament (IF)
• http://scop.berkeley.edu/search.cgi
(intermediate +filament, search PDB
using MSDlite Æ view)
• IF can be found as major components
of cytoskeleton structures
Keratins
• The most common arrangement for keratin is
a coiled-coil of two α helices
• Three helices stranded arrangements –
extracellular keratins domains
• Four stranded coiled-coil – found in insects
• Figure 4.1 – coiled-coil arrangement,
sometimes called a superhelix
• X-ray diffraction studies showed a periodicity
of 1.5 A (regular α helix) and 5.1 A (pitch)
repulsive
Keratins
A 500 interaction angle
Superhelix is left-handed
A 200 interaction angle
http://bmbiris.bmb.uga.edu/wampler/8010/lectures/motifs/sld018.htm
Keratins
• The coiled-coil is formed by each
helix interacting with the other and
by burying their hydrophobic
residues away from the solvent
interface
• The hydrophobic aa occur at regular
intervals throughout the chain
• Preference for charged side chains
at positions within helices that are in
contact with solvent Æ a repeating
unit of 7 residues, called the heptad
repeat Æ represented by a helical
wheel (Figure 4.2)
• The coiled-coil arrangement leads to
a slight decrease in the number of
residues/turn to 3.5
1, 4 position
Hydrophobic
Interaction,
usually Leu,
Ile and Ala
The Heptad Repeat of The Coiled-coil Structure
• Each strand of a coiled-coil protein may be
viewed as a repeated consensus substrings
of the form (a-b-c-d-e-f-g)n, where a, b, c,...,g
are the seven different structural positions on
the coil.
• The first and fourth position (a and d) are
generally apolar or hydrophobic amino acids.
Discontinuities in the heptad pattern, such as
stutters (disorder) are quite frequent.
• The mapping of the amino acids in the strand
to the seven positions is hence not always
continuous. When the two strands coil around
each other positions a and d are internalized,
stabilizing the structure, while positions b, c,
e, f, g are exposed on the surface of the
protein.
• Make a precise inter-digitating (指狀的)
surface Æ a slightly differ pitch 5.1 A
compared with 5.4 A in a regular α helix
http://cis.poly.edu/~jps/coilcoil.html
The Heptad Repeat of The Coiled-coil Structure
• The interleaving of side chain
protein – heptad repeat (1, 8, 15,
22 ..)
• Leucine zipper arrangement
• Coiled-coil arrangement not only
enhances the stability of a single
helix but also confers considerable
mechanical strength Æ analogous
to the interwinding of a rope
• High content of cysteine Æ
disulfide bridges that cross-link
neighbouring coiled-coils to build
up a filament Æ constituting hair or
nail
http://bmbiris.bmb.uga.edu/wampler/8010/lectures/motifs/sld014.htm
The 7, 11 and 18 Repeat of The Coiled-coil Structure
The 7, 11 and 18 Repeat of The Coiled-coil Structure
Other types of repeat found in coiled-coils
1,4 heptad repeat
1
1
1
Keratins - The classification of intermediate
filaments
星形神經膠質
間充質
Keratins
• Higher order of a keratin
structure
• Each coiled-coil aligns in a
head to tail arrangement Æ
staggered 錯開 row to form a
protofilament Æ
protofilament dimerizes to
form a protofibril Æ 4
protofibrils make a microfibril
Æ macrofibril
Keratins
Leucine zipper
DNA binding protein
GCN4
• Coiled-coil motif occurs in many
other proteins as a recognizable
motif
• It occurs in viral membrane-fusion
protein including the gp41 domain
found as part of the HIV/SIV
• Haemagglutinin component of the
influenza virus
• TF – leucine zipper protein GCN4
• Muscle proteins, tropomyosin
• Widely different folds of the coiledcoil (Figure 4.4)
c-jun proto-oncogene
dimerized with c-fos
Two views of GP41 core domain of the SIV
Keratins
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Mutation in gene coding for keratin lead to severe consequences on
individuals Æ the integrity (完全性) skin, cell adhesion, motility and
proliferation (增殖)
Keratins, the most abundant proteins in epithelial cells, are encoded
by two groups of genes designated type I and type II.
There are >20 type I and >15 type II keratin genes occurring in
clusters at separate loci
Type II keratin proteins – soft epithelia (K1 – K8), hard epithelia
(such as hair, nail and parts of the tongue epithelium) are
designated Hb1 – Hb8) Æ 8 + 8 = 16 types
Type I keratins – soft epithelia (K9 – K20) and hard epithelia (Ha1 –
Ha10) Æ 12 + 10 = 22 types
Hard and soft – refer to the sulfur content of keratins
A high cysteine refers to the sulfur content of keratins Æ hard
keratins
Keratins
• The distribution of some of the keratins – Table
4.3
• Type II keratins are the basic or neutral
courterparts to the acidic type I keratins. Each
type II keratin forms a heterodimer with a
specific acidic keratin, and the heterodimers are
organised into tetramers and then into chains
• Type I and type II keratin genes are in fact
regulated in a pairwise, epithelial tissue type-,
and differentiation-specific manner, giving rise
to "patterns" that have been and continue to be
useful to study epithelial growth and
differentiation.
• K10 – K 1 Æ suprabasal epidermal
keratinocyte上基底層角質化細胞
• K12 – K3 Æ cornea of eye 角膜
• K13 – K4 Æ epithelial layer, such as oral
cavity, tongue and the oesophagus食道
• K14 – K5 Æ basal layer keratinocytes 基底層
Keratins
• Disulfide bridges between cysteine Æ reduction
with reducing agents (mercaptans)
• Hair Æ reduction Æ from a curled state to a
straightened form
• Removal of the reducing agent Æ oxidation of
the thiol groups Æ formation of disulfide bridges
Æ reformed in a new curled conformation
• Reduction of disulfide bridges in hair Æ keratin
stretch to over twice their original length Æ β
keratin (found in feathers, silk-like sheets of
fibroin)
Fibroin
• Silk fibroin class made up of an extended array of β
strands assembled into a β sheet
• Insect and spider produce a variety of silks to assist in
the production of webs, cocoons 繭, and nests.
• Silk consists of a collection of antiparallel β strands leads
to a microcrystalline array of fibres in a highly ordered
structure.
• The silk filament is composed of a thread-like protein
called fibroin, which is bundled and arranged lengthwise
to form a continuous fiber. Fibroin is composed of two
subunits, Fibroin Heavy Chain and Fibroin Light Chain,
which are held together covalently (via disulfide bonds).
Fibroin
• Silk fibroin has a long stretches of repeating
composition. A six residue repeat of (Gly-SerGly-Ala-Gly-Ala)n is observed to occur
frequently – this motif lacks large aa
• These three aa appear to represent over 85% of
the total aa composition with ~45% Gly, 30% Ala,
and 15% Ser in silk fibroin
• The 6 aa motif is part of a larger repeating unit
that may be repeated up to 50 times
Fibroin
• The interaction between Gly surfaces
yields an inter-sheet spacing of 3.5 A
• The interaction of the Ser/Ala side chains
gives a spacing of 5.7 A
anti-// b strands
interaction of Ala
in an end on view
Ala/Ser interface
Gly interface
Fibroin
• Silk has many remarkable properties
• Weight-for-weight it is stronger than metal alloys such as
steel, it is more resilient 有彈力 than synthetic polymers
such as Dupont’s Kelvar, yet is finer than a human hair
• Many attempts to mimic the properties of silk –
biomimetic 仿生 chemistry
• Extremely strong – the fully extended conformation β
strands are any further extension would require the
breakage of strong covalent bonds
• Flexibility – a result of vdw interactions that exist
between the anti-// b strands
Spider silk proteins
Spider silk proteins
• http://biology.ucr.edu/people/faculty/
Hayashi.html
• Each individual spider produces as
many as six or seven distinct varieties
of silk from a battery of specialized
glands. The different silks serve
different purposes, ranging from web
construction and prey capture to
courtship and nest-building.
• Spider dragline silk have unmatched
characteristics of strength and
elasticity. It has a tensile strength
[200,000 psi (1 psi = 6.9 kPa), greater
than steel and of the same order of
magnitude as Dupont’s Kevlar. These
silk fibers also have an elasticity of up
to 35%.
• http://en.wikipedia.org/wiki/Spider_si
lk
Structure of spider silk. Inside a
typical fiber, one finds crystalline
regions separated by amorphous
linkages. The crystals are β-sheets
that have assembled together.
Collagen
• Collagen – a major component of skin, tendons 腱,
ligaments 韌帶, teeth and bones
• Collagen – a major component of connective tissue,
the most abundant tissue in vertebrates (also found
in C.elegan)
• at least 30 distinct types of collagen have been
identified from the respective genes with each
showing differences in aa sequences
• Collagens have the structure of a triple helix
• In humans at least 19 different collagens are
assembled Æ 4 major classes Æ Type I (2α1(I)
chains, one α2), Type II (3α1 chains)
• In a mature adult collagen fibres are extremely
robust and insoluble
• younger animals contained a greater proportion of
collagen with higher solubility (because the
extensive crosslinking of collagens is lacking)
Collagen
The structure and function of collagen
• Fundamental structural unit – tropocollagen
• 3 tropocollagen polypeptide chains (α chains, lefthanded helix) are supercoiled about a common axis
to form a right-handed superhelical structure
• ~1000 aa, ~300 nm in length, ~1.4 nm in diameter
• The length of collagen ~ 100 times of myoglobin, its
diameter is only half that of myoglobin
• Tropocollagen are unusual in their aa composition –
high proportions of glycine residues and proline
• Collagen has a repetitive primary sequence in which
every third residue is glycine
• -Gly-Xaa-Yaa-Gly-Xaa-Yaa-Gly-Xaa-Yaa• Xaa and Yaa are often found to be proline or lysine
• Many of the proline and lysine are hydroxylated via
PTM to yield HyP or Hyl Æ Gly-Pro-Hyp occurs
frequently in collagen
Collagen
• Each polypeptide chain intertwines with
the remaining two chains to form a triple
helix Æ Figure 4.6
• Each chain has the sequence (Gly-X-Y)n
and forms a LH superhelix with the
other two chains Æ supercoiled in a RH
manner about a common axis ≠ α-helix
structure
• Translation distance per residue for each
chain in the triple helix is 0.286 nm
whilst the number of residues per turn is
3.3 (3.6 in regular α-helix, p.43) Î
0.286 x 3.3 ~ 0.95 nm for the helix pitch
Collagen
• As a result of the presence of both glycine
and proline in high frequency in the collagen
sequences the triple helix is forced to adopt
a different strategy in packing polypeptide
chains.
• The close packing of chains stabilizes the
triple helix through vdw interactions +
extensive H-bonding between polypeptide
chains (NH group of one Gly --- C=O group
of residue X on adjacent chains) – direction
of H-bonding are transverse the axis of the
helix
1
2
3
Y – Gly – X
Gly – X – Y occur at ~ the same level
X – Y – Gly
Proline and HyP are shown in yellow
Collagen
Melting temperature of the collagen triple helix
- The temperature at which half the helical structure has been lost
- A sharp transition at a certain temperature reflecting the loss of ordered structure
Î a progressive loss of function
- (Gly-Pro-Pro)n Tm ~ 24℃
- (Gly-Pro-HyP)n Tm ~ 60 ℃
Sharp transition
Collagen
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Further strength arises from the association of tropocollagen molecules together as
part of a collagen fibre
Each tropocollagen molecule packs together with neighbouring molecules to produce
a characteristic banded appearance of fibres in electron micrographs
Intramolecular and intermolecular cross-links – result of covalent bond formation
Collagen
Collagen biosynthesis
• Collagen (synthesized at the ribosome) Æ PTM
Æ mature collagen which is very different from
the initial collagen
• Any process interfering with modification of
collagen tends to result in severe forms of
disease
• The biosynthesis of collagen is divided into
discrete reactions that differ not only in the
nature of the modification but their cellular
location
Collagen
Collagen
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initial formation of preprocollagen, the initial translation product
formed at the ribosome. The collagen precursor contains a signal
sequence that directs the protein to the endoplasmic reticulum
membrane
the polypeptide is hydroxylated resulting in the formation of
hydroxyproline (Hyp) and hydroxylysine (Hyl)
glycosylation of the collagen precursor and the attachment of sugars,
chiefly glucose and galactose, occurs via the hydroxyl group of Hyl
Assembly of three α-chains to form procollagen, and it is transported
to the Golgi system prior to secretion from the cell
procollagen peptidases remove the disulfide-rich N and C terminal
extensions leaving the triple helical collagen in the extracellular matrix
(Figure 4.11)
A better graphical description
http://staff.um.edu.mt/acus1/Extracellular.htm
Collagen
• the triple helical collagen in the extracellular matrix
can then associate with other collagen molecules to
form staggered 錯開, parallel arrays (Figure 4.12)
Disease states associated with collagen defects
• Involvement of collagen – tendons, ligaments, skin, blood vessels
• Mutation in collagen genes often result in severe disorders
affecting many organ systems
• Defects in the enzymes responsible for the assembly and
maturation of collagen creating a further group of disease states.
• Defects in collagen genes lead to osteogenesis (the formation or
growth of bone) imperfecta, hereditary osteoporosis 骨質疏鬆症,
and familial aortic aneurysm 家族性大動脈瘤
• the most common mutation – substitution of Gly by a different aa
Æ destroying the characteristic repeating sequence of Gly – X –
Y Æ consequence of these mutation is the incorrect folding of
collagen Æ serious diseases Æ Osteogenesis imperfecta and
Ehlers – Danlos syndrome (EDS)先天結締組織異常
Disease states associated with collagen defects
• Osteogenesis imperfecta is a
genetic disorder characterized by
bones that break comparatively
easily often without obvious
cause – also called brittle bone
disease. At least four different types
of osteogenesis imperfecta are
recognized (~ 1 in 20000, Table
4.5 – ranging in severity from lethal
to mild)
• Osteogenesis imperfecta is caused
by a mutation in one allele of either
the α1 or α2 chains of the major
collagen in bone, type I collagen.
• Type I collagen contains two α1
chains + α2 chain
Disease states associated with collagen defects
Ehlers – Danlos syndrome
• Caused by mutations in a single collagen gene
• Variable phenotype – some relatively benign 良性 whilst
others are life threatening
• Joint hypermobility, skin extensibility, vascular fragility
• Mutations can lead to changes in the levels of collagen
molecules, changes in the cross-linking of fibres, a decreased
hydroxylysine content and a failure to process collagen
correctly by removal of the N-terminal regions
• The common effect of all mutations is to create a structural
weakness in connective tissue as a result of a molecular defect
in collagen
http://staff.um.edu.mt/acus1/Extracellular.htm
Related disorders characterized at a molecular level
Marfan syndrome馬凡氏症候群
• http://www.tfrd.org.tw/rare/typeCont.php?sno=1101&kind
_id=11
• is an inherited disorder of connective tissue affecting
multiple organ systems including the skeleton, lungs,
eyes, heart and blood vessels. The defect is now known
to reside in a related protein that forms part of the
microfibrils making up the extracellular matrix that
includes collagen.
• Marfan syndrome is caused by a molecular defect in the
gene coding for fibrillin, an extracellular protein found in
connective tissue, where it is an integral component of
extended fibrils.
Related disorders characterized at a molecular level
• Humans have two highly homologous fibrillins, fibrillin-1 (Figure 4.13) and
fibrillin-2, mapping to chromosomes 15 and 5 respectively.
• A characteristic feature of both fibrillins is their mosaic (拼圖, 鑲嵌) composition
where numerous small modules combine to produce the complete, very large,
protein of 350 kDa.
• The majority of fibrillin consists of epidermal growth factor-like subunits (47
EGF-like modules) of which 43 have a consensus sequence for calcium binding.
• Each of these domains is characterized by 6 cysteine aa three S-S bridges and
a calcium binding sequence of D/N – x – D/N – E/Q – xm – D/N* - xn – Y/F
(where m and n are variable and * indicates possible PTM by hydorxylation).
• Other modules : TB domain, hybrid domain
Related disorders characterized at a molecular level
• The EGF domain occurs in many other proteins
including blood coagulation proteins such as
factors VII, IX, X, and the low density lipoprotein
receptors.
• Structure of EGF like domain and a pair of
calcium-binding domains confirmed a rigid rodlike arrangement stabilized by calcium binding
and hydrophobic interactions (Figure 4.14).
• Mutations known to result in Marfan’s syndrome
lead to decreased calcium binding to fibrillin Æ
play an important physiological role
• A single aa substitution Æ disrupts the structural
organization of individual EGF-like motifs
• Victims – Flo Hyman, a famous American
Olympian volleyball player, Abraham Lincoln
(speculated on the basis of their physical
appearance), president of USA, and the virtuoso
鑒賞家 violinist Niccolo Paganini
Summary
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8.
Fibrous proteins represent a contrast to the normal topology of
globular domains.
Fibrous proteins lack true tertiary structure showing elongated
structures and interactions confined to those between local
residues.
The amino acid composition of fibrous proteins differs
considerably from globular proteins but also varies widely within
this group.
Variation in composition reflects the different roles performed by
each group of fibrous proteins.
Three prominent groups of fibrous proteins are collagens, silk
fibroin and keratins - all occupy pivotal roles within cells.
Collagen is very abundant in vertebrates and invertebrates
A triple helix provides a platform for a wide range of structural
roles in the extracellular matrix delivering strength and rigidity to a
wide range of tissues.
The triple helix is a repetitive structure containing the motif (GlyXaa-Yaa) in high frequency with Xaa and Yaa often found as
proline and lysine residues.
Summary
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Repeating sequences of amino acids are a feature of many fibrous proteins and
help to establish the topology of each protein.
In collagen the presence of glycine at every third residue is critical because its
small side chain allows it to fit precisely into a region that forms from the close
contact of three polypeptide chains.
Although based around a helical design the helix differs considerably in
dimensions to the typical α helix.
The triple helix of collagen undergoes PTM to increase strength and rigidity.
Keratins make up hair and nails and contain polypeptide chains arranged in
helical conformations. The helices interact by supercoiling to form “coiled-coils”.
Specific non-polar interactions between residues in different helices confer stability
with the basis of this interaction being a heptad of repeating residues along the
primary sequence.
A heptad repeat possesses leucine or other residues with hydrophobic side chains
arranged periodically to favour inter-helix interactions.
In view of their widespread distribution in all animal cells mutations in fibrous
proteins such as keratins or collagens leads to serious medical conditions.
Many disease states arise from inherited disorders that lead to impaired structural
integrity in these groups of proteins.