III.

FIBRILLAR PROTEINS

Globular proteins have a spherical shape, variable molecular weights, high

water solubility and a variety of functional or dynamic roles.
Fibrous proteins usually have larger amounts of regular secondary
structure, rod shape, low solubility in water and mainly a structural role.
I. Intracellular fibrillar proteins

Cells contain three principal elements of the cytoskeleton: microtubules,

intermediate filaments and actin filaments (microfilaments) as well as
associated filament binding proteins: actin-binding proteins, MAP
(microtubule-associated proteins) and IFAP (intermediate filament associated
proteins). Intermediate filaments were named by their intermediate size
between microtubules (mini-tubes) and microfilaments. The cytoskeleton
maintains cellular morphology, is involved in intracellular transport, cell
motility, and cell division.
Microfilament
Actin - polymerization of globular subunits (G-actin) to long polymers (Factin) builds the microfilament that modulates cell shape and drives cell
locomotion.
Myosin a motor protein that works with actin to produce movement by
transducing chemical energy (ATP) to mechanical work. It is a well-developed
system in skeletal muscle cells. Myosin contains a head region (binds to
actin) and a coiled-coil tail.

Figure 24.29. Devlin Structural organization of skeletal muscle. Fibrillar molecules.

Microtubules
Globular alpha- and beta-tubulin monomers polymerize to build
microtubules, which are constructed as hollow tubes, and enable cells to
change shape in response to external and internal signals. Cells use
microtubules to reinforce other elements of the cytoskeleton, to construct cilia
and flagella, and to align and separate pairs of chromosomes during mitosis.

Plus (+) end is the fast- growing end; growth is achieved by the addition of
tubulin monomers.
Plus (+) ends of microtubules are oriented toward the cells periphery.
Minus (-) end is where actin depolymerizes (shortened).
Minus (-) ends of microtubules are oriented toward the cells periphery.

Drugs that interfere with microtubule formation:

Colchicine product of the meadow saffron plant - is used over 2000 years
to treat gout. Colchicine inhibits the action of the white blood cells that
mediate inflammation caused by the precipitation of uric acid in the joints by
disassembling microtubules.
Taxol extract of the Pacific yew tree - is used as an anti-cancer drug
because it blocks cell division by binding to beta-tubulin subunits in the
microtubules.
(Please, review the relationship of Alzheimers disease and microtubule
assembly.)
Microtubules support the movement of organelles and vesicles in the cell

The microtubules provide oriented tracks to support the movements of

transport vesicles outward toward the cell periphery or inward toward the
region around the nucleus. The redistribution of the Golgi apparatus and
much of the endoplasmic reticulum after mitosis is partly accountable to
movements of these organelles along the microtubules.
Molecular motors (ATP-dependent) are responsible for movement along
microtubules.
Kinesins move vesicles to the plus (+) end of microtubules anterograde
vesicular traffic. Similar to myosin, it has two heads that bind to the
microtubule and a tail that binds vesicles to be transported. Using ATP
energy, kinesin transports its cargo along the microtubules. Example: in
neurons neurotransmitters and membrane components are synthesized in
the cell body, where ribosomes are located, and moved to the end of the
axon by kinesins. Kinesin is also involved in fast axonal transport and
rapid movements of mitochondria.
Dyneins move vesicles to the minus (-) end of microtubules retrograde
vesicular traffic. For example endocytosed molecules are transported to
the interior of the cell.
Kinesin

Dynein

(-) (+)

(+) (-)

Intermediate filament
Keratin is an intermediate filament protein. Intermediate filament proteins
are exclusively structural; in many cells intermediate filaments are more
abundant than microfilaments or microtubules. Soft keratins - help define
internal body structures; hard keratins build skin, hair, claws.
Keratin forms coiled coils: a dimer of alpha-helices. The primary structure of
the keratin molecule contains seven-residue repeating units where the first
and fourth amino acids are apolar. These non-polar residues line up along
one side of the alpha-helix, then interact with similar apolar residues of
another helix twisting around each-other. These dimers then assemble to
multimers and the fibers are stabilized by disulfide bridges.
Skin keratin: epidermal cells synthesize keratin and when the innermost layer
of the skin cells die, their keratin content form a strong waterproof coating.
Keratin gene mutations:
Epidermolysis bullosa simplex (EBS) cells rupture when subject to normal
mechanical stress, resulting in visible separation of epidermal layers as
blistering.
Hair keratin: Multiple disulfide bonding between keratin molecules gives the
hair its strength. Hair helices however can be stretched by force. If force is not
extensive, original alpha-helical conformation can be regained. Hair can be
modified by reducing agents (breaking disulfide bonds) and reformed in a
different shape by oxidizing agents (permanent for curling or straightening
hair).

II. Extracellular fibrillar proteins

1. Collagen: is present in virtually all tissues and is the most abundant protein in
the human. Certain organs depend heavily on normal collagen structure to
function physiologically.
Abnormal collagen synthesis or structure causes dysfunction of
cardiovascular organs (aortic and arterial aneurysms and heart valve
malfunction),
bone (fragility and easy fracturing),
skin (poor healing and unusual distensibility),
joints (hypermobility and arthritis), and
eyes (dislocation of the lens).
Distribution of Collagen in Humans
Collagen is the most abundant protein in mammals; about 25 % of the
total proteins are a type of collagen.

Collagen by dry weight in tissues (FYI only):

o
o
o
o
o
o
o
o

4%
10%
12-24%
23%
50%
64%
74%
90%

liver
lung
aorta
cortical bone
cartilage
cornea
skin
demineralized bone

Amino Acid Composition of Collagen

The amino acid composition of the collagen is very unique: it contains 33%
Gly, 13% Pro, 9% hydroxyproline (OH-Pro) and also contains hydroxylysine
(OH-Lys).
OH-Pro and OH-Lys are modified amino acids, formed by posttranslational
enzymatic modifications. The enzymes that catalyze the hydroxylation of Pro
and Lys residues (Pro-hydroxylase and Lys-hydroxylase), require ascorbic
acid (vitamin C).
In vitamin C deficiency (scurvy), there is poor synthesis of collagen.

Figure 3.37, Devlin. Derived amino acids found in collagen

Primary structure of collagen

In collagens, Gly occurs at every third residue and Pro or OH- Pro (Hyp) at
every third in the same region.
Thus, the repeat is -Gly-Pro-Y- and -Gly-X-Hyp- where X and Y are any amino
acid.
What are the consequences of this unique sequence?
1. Gly is the smallest amino acid with a proton as a side chain will fit into
small places in the core of the structure.
2. Pro is a helix-breaker, thus regular alpha-helix cannot be formed.
3. Hydroxyl group can react further and form crosslinks.
Secondary and tertiary structure of collagen
One strand of collagen can form a so called polyproline type II helix.
This is a loose, left-handed helix, with three residues per turn. In contrast to the
alpha-helix, the plane of each peptide bond in the polyproline helix is
perpendicular to the axis of the helix.
The Gly at every third position, with its low bulkiness, allows formation of a
three-member superhelix since an apolar edge is generated into which Gly from
the other chains can insert. This is a right-handed triple helix.
The peptide carbonyl groups are pointed toward neighboring chains and form
strong interchain H-bonds with other collagen chains. The HO-Pro also allows
H-bonding.

Figure 3.38, Diagram of collagen demonstrating necessity for glycine in every third residue to
allow chains to be in close proximity.

Further structural arrangements of collagens:

The type I collagen molecule has a length of 300 nm and 1.5 nm in diameter.
Collagen fibrils can aggregate and form larger fibers (10-100 nm in diameter).
This special structure is needed for the function of collagens that either give the
tissue structural strength or form a meshwork with filtering capacity.

Figure 4.3. Lippincott Collagen structural organization

Formation of covalent cross-links in collagen

The N and C terminal segments of the triple helical region are called
telopeptides where covalent crosslinks between triple helices can form.
Some of the epsilon amino groups of Lys are enzymatically converted to
aldehyde groups to form allysine. The enzyme, lysyl amino oxidase (also called
lysyl oxidase) is cupper-dependent.
The aldehyde group spontaneously reacts with the non-modified Lys epsilon
amino groups or with another aldehyde group of another allysine to form
covalent bonds.
These link together superhelices to stabilize the collagen fiber.

Figure 4.9. Lippincott. Covalent cross-links formed in collagen through allysine intermediates.
Catalyzed by lysyl amino oxidase.

Types of collagens:
Collagen molecules belong to a family of more than 20 members, labeled with
roman numerals. The compositions of their helices (individual helices labeled
by and a number) are different and their tissue distribution and structure can
be different.
Depending on the structure of the collagen, there are two major categories of
collagens, fibril-forming and network-forming collagens. A third category is the
fibril-associated collagens (e.g. type IX).

Classification of Collagen Types (FYI only)

Type

Chain
Designations

[1(I)]2 2(I)

II

[ 1(II)]3

III

IV

VI

Tissue Found

Characteristics

Bone, skin, tendon,

scar tissue, heart
valve, intestinal,
and uterine wall
Cartilage, vitreous

Low carbohydrate; <10 hydroxylysines

per chain; two types of polypeptide
chains

10% carbohydrate; >20 hydroxylysines

per chain
[ 1(III)]3
Blood vessels,
Low carbohydrate; high hydroxyproline
newborn skin, scar and Gly; contains Cys
tissue, intestinal,
and uterine wall
[ 1(IV)]3
Basement
High 3-hydroxyproline; >40
[ 2(IV)]3
membrane, lens
hydroxylysines per chain; low Ala and
capsule
Arg; contains Cys; high carbohydrate
(15%)
[ 1(V)]2 2(V) Cell surfaces or
High carbohydrate, relatively high
[ 1(V)]3; 1(V) exocytoskeleton;
glycine, and hydroxylysine
2(V) 3(V)
widely distributed in
low amounts
1(VI) 2(VI) Aortic intima,
Relatively large globular domains in
3(VI)
placenta, kidney,
telopeptide region; high Cys and Tyr;
and skin in low
molecular weight relatively low
amounts
(~160,000); equimolar amounts of
hydroxylysine and hydroxyproline

Synthesis of collagen:
Collagens are coded by several different genes. Their formation includes several
posttranslational modifications.
Synthesis starts in the ER and hydroxylation of the Pro and Lys residues occur
parallel to the protein synthesis, before triple helix formation. Sugar residues can
be placed on the OH-Lys residues in the Golgi. Triple helix forms intracellularly
with globular propeptides on each end. The C-propeptide is heavily crosslinked
by disulfide bridges.
Procollagen is released by the cell. Outside the cell, procollagen peptidases
cleave off the propeptides from the amino and carboxyl terminals of the triple
helix, and the tropocollagen triple helices aggregate, crosslink and form fibers.

Biosynthesis of Type I collagen. Steps up through the formation of procollagen occur

intracellularly in fibroblasts; procollagen is then secreted from the cell and subsequent
steps occur extracellularly. (Pratt and Cornely: Essential Biochemisry, Wiley, 2003, p.
163)

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Collagen molecules aggregate in quarter-staggered parallel arrays and cross-link

to form fibrils. Specific banded appearance through microscope.
Degradation of collagens:
Collagens are rather stable; some has very long half-lives (e.g. in articular
cartilage). However, a specific class of matrix metalloproteinases, called
collagenases is able to catalyze the hydrolysis of collagens.
2. Elastin:

Elastin provides elasticity to tissue. It is fibrous and relatively insoluble in

water (lots of apolar amino acids, Ala, Val, etc).
Elastin is abundant in ligaments, lungs, arterial walls, and skin.

It lacks a regular secondary structure but contains an unordered coiled

structure in which amino acid residues are highly mobile.

Modified lysine residues, allysines are found in elastin. The conversion of Lys
to allysine is catalyzed by lysyle amino oxidase ((also called lysyl oxidase).
Three allysines and an unmodified lysine in these sequences, from different
regions of the polypeptide chains, react to form the heterocyclic structure of
desmosine and isodesmosine.
The desmosines covalently crosslink the chains in elastin.

The mobility of the coiled structure kept together with crosslinks gives the
molecule rubber-like qualities. E.g. lungs can change volume because the
coils uncoil during inhalation and recoil during exhalation.

Elastase, a serine proteinase that is specific for apolar amino acids, can
cleave elastin. When elastase inhibitor is decreased in concentration (alpha1-antitrypsin deficiency), or damaged by smoking, emphysema develops.

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Figure 3.40. Formation of desmosine covalent cross-link in elastin from lysine

and allysine.

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