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STRUCTURAL PROTEINS

Proteins are polymers in which the 20 natural amino acids are linked by amide bonds.
In addition to the 20 natural amino acids, there are amino acids that are not directly
synthesized from ribosomes, such as L-3,4-dihydroxyphenylalanine (DOPA),
hydroxyproline (Hyp), dityrosine, and selenomethionine, and these compounds are
synthesized via posttranslational modifications. These nonribosomal peptides and
amino acids often play an important role in structural and functional proteins. In
many cases, structural proteins have a characteristic amino acid sequence that repeats
to form a higher-order structure by intermolecular and/or intramolecular hydrogen
bonding. One example is the disulfide bond formed between two cysteine residues,
which can induce dimerization and hierarchical structures in proteins. However, in
general, the amino acid sequence and the structure being formed depend on the type
of structural protein, and it is difficult to identify a structural protein based on the
sequence or structure alone. In particular, when nonnatural sequences created by gene
recombination techniques and chemical synthesis are considered, defining a
structural protein is even more difficult and complicated. A structural protein is a
protein that possesses a characteristic amino acid sequence or motif that repeats and
forms a skeleton or contributes to the mechanical properties of a living organism,
cell, or material. Structural proteins can be globular or fibrillar proteins. In most
cases, such motifs are expected to form higher-order structures by intermolecular or
intramolecular interactions, leading to the expression of physical characteristics.
However, considering protein-based amorphous materials, the requirement of a
hierarchical structure should not be included in this definition.

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Examples of structural proteins in nature. Their roles/functions in nature and typical
repeat sequences are listed

1. Collagen

Collagen is one of the most abundant proteins in mammals and is a major structural
component of extracellular matrices. It contains many periodically repeating 3-amino-
acid sequences containing Gly. The Gly-containing repeating sequences include ~1400
amino acid residues. The most common tripeptide unit of collagen is Gly-Pro-Hyp, and
these three residues form one helical turn. A typical collagen molecule is a long, rigid
structure in which three polypeptides (referred to as α chains) are wound around one
another in a rope-like triple helix. Although these molecules are found throughout
the body, their types and organization are dictated by the structural role collagen
plays in a particular organ. In some tissues, collagen may be dispersed as a gel that

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gives support to the structure, as in the extracellular matrix or the vitreous humor of
the eye. In other tissues, collagen may be bundled in tight, parallel fibers that provide
great strength, as in tendons. In the cornea of the eye, collagen is stacked so as to
transmit light with a minimum of scattering. Collagen of bone occurs as fibers
arranged at an angle to each other so as to resist mechanical shear from any direction.

As a structural protein, collagen has excellent biocompatibility and cell adhesion and
can promote cell proliferation and differentiation.

Types of collagen

The collagen superfamily of proteins includes more than 25 collagen types as well
as additional proteins that have collagen-like domains. The three polypeptide α
chains are held together by interchain hydrogen bonds. Variations in the amino acid
sequence of the α chains result in structural components that are about the same size
(approximately 1,000 amino acids long) but with slightly different properties. These
α chains are combined to form the various types of collagen found in the tissues. For
example, the most common collagen, type I, contains two chains called α1 and one
chain called α2 (α12α2), whereas type II collagen contains three α1 chains (α13).
The collagens can be organized into three groups, based on their location and
functions in the body

• Fibril-forming collagens
• Network-forming collagens
• Fibril-associated collagens:

Structure of collagen

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1. Amino acid sequence: Collagen is rich in proline and glycine, both of which are
important in the formation of the triple-stranded helix. Proline facilitates the
formation of the helical conformation of each α chain because its ring structure
causes “kinks” in the peptide chain. [Note: The presence of proline dictates that the
helical conformation of the α chain cannot be an α helix. Glycine, the smallest amino
acid, is found in every third position of the polypeptide chain. It fits into the restricted
spaces where the three chains of the helix come together. The glycine residues are
part of a repeating sequence, –Gly–X–Y–, where X is frequently proline, and Y is
often hydroxyproline (but can be hydroxylysine). Thus, most of the α chain can be
regarded as a polytripeptide whose sequence can be represented as (–Gly–Pro–Hyp–
).

2. Triple-helical structure: Unlike most globular proteins that are folded into
compact structures, collagen, a fibrous protein, has an elongated, triple-helical
structure that is stabilized by interchain hydrogen bonds. Electron micrograph of a
polygonal network formed by association of collagen type IV monomers.

3. Hydroxyproline and hydroxylysine: Collagen contains hydroxyproline and

hydroxylysine, which are not present in most other proteins. These residues result
from the hydroxylation of some of the proline and lysine residues after their
incorporation into polypeptide chains. The hydroxylation is, thus, an example of
posttranslational modification. [Note: Generation of hydroxyproline maximizes
formation of interchain hydrogen bonds that stabilize the triple-helical structure.]

4. Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen

may be enzymatically glycosylated. Most commonly, glucose and galactose are
sequentially attached to the polypeptide chain prior to triple-helix formation. Figure

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4.5 Amino acid sequence of a portion of the α1 chain of collagen. [Note: Hyp is
hydroxyproline, and Hyl is hydroxylysine.]

BIOSYNTHESIS OF COLLAGEN

The polypeptide precursors of the collagen molecule are synthesized in fibroblasts

(or in the related osteoblasts of bone and chondroblasts of cartilage). They are
enzymically modified and form the triple helix, which gets secreted into the
extracellular matrix. After additional enzymic modification, the mature extracellular
collagen monomers aggregate and become cross-linked to form collagen fibers.

1. Formation of pro-α chains: Collagen is one of many proteins that normally

function outside of cells. Like most proteins produced for export, the newly
synthesized polypeptide precursors of α chains (prepro-α chains) contain a special
amino acid sequence at their N-terminal ends. This sequence acts as a signal that, in
the absence of additional signals, targets the polypeptide being synthesized for
secretion from the cell. The signal sequence facilitates the binding of ribosomes to
the rough endoplasmic reticulum (RER), and directs the passage of the prepro-α
chain into the lumen of the RER. The signal sequence is rapidly cleaved in the RER
to yield a precursor of collagen called a pro-α chain

2. Hydroxylation: The pro-α chains are processed by a number of enzymic steps

within the lumen of the RER while the polypeptides are still being synthesized.
Proline and lysine residues found in the Y-position of the –Gly–X–Y– sequence can
be hydroxylated to form hydroxyproline and hydroxylysine residues. These

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hydroxylation reactions require molecular oxygen, Fe2+ , and the reducing agent
vitamin C (ascorbic acid), without which the hydroxylating enzymes, prolyl
hydroxylase and lysyl hydroxylase, are unable to function. In the case of ascorbic
acid deficiency (and, therefore, a lack of proline and lysine hydroxylation),
interchain H-bond formation is impaired, as is formation of a stable triple helix.
Additionally, collagen fibrils cannot be cross-linked (see below), greatly decreasing
the tensile strength of the assembled fiber. The resulting deficiency disease is known
as scurvy. Patients with ascorbic acid deficiency also often show bruises on the limbs
as a result of subcutaneous extravasation (leakage) of blood due to capillary fragility.

3. Glycosylation: Some hydroxylysine residues are modified by glycosylation with

glucose or glucosyl-galactose.

4. Assembly and secretion: After hydroxylation and glycosylation, three pro-α

chains form procollagen, a precursor of collagen that has a central region of triple
helix flanked by the nonhelical amino- and carboxyl-terminal extensions called
propeptides. The formation of procollagen begins with formation of interchain
disulfide bonds between the C-terminal extensions of the pro-α chains. This brings
the three α chains into an alignment favorable for helix formation. The procollagen
molecules move through the Golgi apparatus, where they are packaged in secretory
vesicles. The vesicles fuse with the cell membrane, causing the release of
procollagen molecules into the extracellular space.

5. Extracellular cleavage of procollagen molecules: After their release, the

procollagen molecules are cleaved by N- and C-procollagen peptidases, which
remove the terminal propeptides, releasing triple-helical tropocollagen molecules.

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6. Formation of collagen fibrils: Tropocollagen molecules spontaneously associate
to form collagen fibrils. They form an ordered, overlapping, parallel array, with
adjacent collagen molecules arranged in a staggered pattern, each overlapping its
neighbor by a length approximately three-quarters of a molecule.

7. Cross-link formation: The fibrillar array of collagen molecules serves as a

substrate for lysyl oxidase. This Cu2+-containing extracellular enzyme oxidatively
deaminates some of the lysine and hydroxylysine residues in collagen. The reactive
aldehydes that result (allysine and hydroxyallysine) can condense with lysine or
hydroxylysine residues in neighboring collagen molecules to form covalent cross-
links and, thus, mature collagen fibers

Degradation of collagen

Normal collagens are highly stable molecules, having half-lives as long as several
years. However, connective tissue is dynamic and is constantly being remodeled,
often in response to growth or injury of the tissue. Breakdown of collagen fibers is
dependent on the proteolytic action of collagenases, which are part of a large family
of matrix metalloproteinases. For type I collagen, the cleavage site is specific,
generating three-quarter and one-quarter length fragments. These fragments are
further degraded by other matrix proteinases.

Collagen diseases: Collagenopathies

Defects in any one of the many steps in collagen fiber synthesis can result in a
genetic disease involving an inability of collagen to form fibers properly and,
therefore, an inability to provide tissues with the needed tensile strength normally

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provided by collagen. More than 1,000 mutations have been identified in 23 genes
coding for 13 of the collagen types. The following are examples of diseases that are
the result of defective collagen synthesis. Figure 4.10 Stretchy skin of classic Ehlers-
Danlos syndrome.

1. Ehlers-Danlos syndrome: Ehlers-Danlos syndrome (EDS) is a heterogeneous

group of connective tissue disorders that result from inheritable defects in the
metabolism of fibrillar collagen molecules. EDS can be caused by a deficiency of
collagen-processing enzymes (for example, lysyl hydroxylase or N-procollagen
peptidase) or from mutations in the amino acid sequences of collagen types I, III, or
V. The classic form of EDS, caused by defects in type V collagen, is characterized
by skin extensibility and fragility and joint hypermobility. The vascular form, due to
defects in type III collagen, is the most serious form of EDS because it is associated
with potentially lethal arterial rupture. [Note: The classic and vascular forms show
autosomal dominant inheritance.] Collagen containing mutant chains may have
altered structure, secretion, or distribution. It frequently is degraded. [Note:
Incorporation of just one mutant chain may result in degradation of the triple helix.
This is known as a dominant-negative effect.]..

2. Osteogenesis imperfecta: This syndrome, known as brittle bone disease, is a

genetic disorder of bone fragility characterized by bones that fracture easily, with
minor or no trauma. Over 80% of cases of osteogenesis imperfecta (OI) are caused
by dominant mutations to the genes that code for the α1 or α2 chains in type I
collagen. The most common mutations cause the replacement of glycine (in –Gly–
X–Y–) by amino acids with bulky side chains. The resultant structurally abnormal α
chains prevent the formation of the required triple-helical conformation. Phenotypic
severity ranges from mild to lethal. Type I OI, the most common form, is

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characterized by mild bone fragility, hearing loss, and blue sclerae. Type II, the most
severe form, is typically lethal in the perinatal period as a result of pulmonary
complications. In utero fractures are seen. Type III is also a severe form. It is
characterized by multiple fractures at birth, short stature, spinal curvature leading to
a “humped-back” (kyphotic) appearance, and blue sclerae. Dentinogenesis
imperfecta, a disorder of tooth development, may be seen

ELASTIN

Elastin in contrast to collagen, which forms fibers that are tough and have high
tensile strength, elastin is a connective tissue protein with rubber-like properties.
Elastic fibers composed of elastin and glycoprotein microfibrils are found in the
lungs, the walls of large arteries, and elastic ligaments. They can be stretched to
several times their normal length but recoil to their original shape when the
stretching force is relaxed.

Consisting mainly of glycine, valine, alanine, and proline residues and possessing a
molecular weight of ~66 kDa, elastin is present in connective, vascular, and load-
bearing tissues and has very elastic mechanical properties. In rat skin, elastin plays a
mechanical role at small stress with small deformations. The elastic modulus, strength,
extensibility, toughness, and resilience are 0.0011 GPa, 0.002 GPa, 150%, 1.6
MJm−3 and 90%, respectively. Therefore, elastin is considered an important structural
protein and material for scaffolds that require elastic physical properties in cell culture.
However, elastin is poorly soluble in aqueous solutions, making it difficult to process,
and this is a common problem among structural proteins. In addition, since elastin is
difficult to obtain, synthesize, and produce in large quantities, reports on biomaterials
composed of elastin alone are very limited compared with reports on scaffolds

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composed of other structural proteins.. The elasticity of elastin makes it an effective
additive for improving collagen-based tissue engineering vessels, showing that elastin
hybrid composites offer unique mechanical properties such as significant elasticity,
indicating that elastin is a useful structural protein for preparing elastic structural
materials.

STRUCTURE OF ELASTIN

Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin,

which is a linear polypeptide composed of about 700 amino acids that are primarily
small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich
in proline and lysine but contains scant hydroxyproline and hydroxylysine.
Tropoelastin is secreted by the cell into the extracellular space. There, it interacts
with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold
onto which tropoelastin is deposited. Some of the lysyl side chains of the
tropoelastin polypeptides are oxidatively deaminated by lysyl oxidase, forming
allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain
from the same or neighboring polypeptides form a desmosine cross-link (Figure
4.12). This produces elastin, an extensively interconnected, rubbery network that
can stretch and bend in any direction when stressed, giving connective tissue
elasticity (Figure 4.13). Mutations in the fibrillin-1 protein are responsible for
Marfan syndrome, a connective tissue disorder characterized by impaired structural
integrity in the skeleton, the eye, and the cardiovascular system. With this disease,
abnormal fibrillin protein is incorporated into microfibrils along with normal
fibrillin, inhibiting the formation of functional microfibrils. [Note: Patients with
Marfan syndrome, OI, or EDS may have blue sclerae due to tissue thinning that
allows underlying pigment to show through.]

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B. Role of α1-antitrypsin in elastin degradation

1. α1-Antitrypsin: Blood and other body fluids contain a protein, α1-antitrypsin

(AAT or A1AT), which inhibits a number of proteolytic enzymes (called proteases
or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was
originally named α1-antitrypsin because it inhibits the activity of trypsin, a
proteolytic enzyme synthesized as trypsinogen by the pancreas (see p. 248).] AAT
has the important physiologic role of inhibiting neutrophil elastase, a powerful
protease that is released into the extracellular space and degrades elastin of alveolar
walls as well as other structural proteins in a variety of tissues (Figure 4.14). Most
of the AAT found in plasma is synthesized and secreted by the liver. AAT comprises
more than 90% of the α1-globulin fraction of normal plasma. Extrahepatic synthesis
occurs in monocytes and alveolar macrophages, and may be important in the
prevention of local tissue injury by elastase.

2. Role of α1-antitrypsin in the lungs: In the normal lung, the alveoli are
chronically exposed to low levels of neutrophil elastase released from activated and
degenerating neutrophils. The proteolytic activity of elastase can destroy the elastin
in alveolar walls if unopposed by the action of AAT, the most important inhibitor
of neutrophil elastase. Because lung tissue cannot regenerate, the destruction of the
connective tissue of alveolar walls results in emphysema.

3. Emphysema resulting from α1-antitrypsin deficiency: In the United States,

approximately 2%–5% of patients with emphysema are predisposed to the disease
by inherited defects in AAT. A number of different mutations in the gene for AAT
are known to cause a deficiency of the protein, but one single purine base mutation
(GAG to AAG, resulting in the substitution of lysine for glutamic acid at position

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342 of the protein) is clinically the most widespread. The mutation causes the
normally monomeric AAT to polymerize within the endoplasmic reticulum of
hepatocytes, resulting in decreased secretion of AAT by the liver. Consequently,
blood levels of AAT are reduced, decreasing the amount that gets to the alveoli. The
polymer that accumulates in the liver may result in cirrhosis (scarring of the liver).
In the United States, the AAT mutation is most common in Caucasians of Northern
European ancestry. An individual must inherit two abnormal AAT alleles to be at
risk for the development of emphysema. In a heterozygote, with one normal and one
defective gene, the levels of AAT are sufficient to protect the alveoli from damage.
[Note: Methionine 358 in AAT is required for the binding of the inhibitor to its
target proteases. Smoking causes the oxidation and subsequent inactivation of the
methionine, thereby rendering the inhibitor powerless to neutralize elastase.
Smokers with AAT deficiency, therefore, have a considerably elevated rate of lung
destruction and a poorer survival rate than nonsmokers with the deficiency.] The
deficiency of elastase inhibitor can be treated by weekly augmentation therapy, that
is, intravenous administration of AAT. The AAT diffuses from the blood into the
lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial
cells.

RESILIN

Resilin, a structural protein, is an entropy elastic material (rubber) found in biological

structures that require energy storage and long-range elasticity, and it exhibits 300–
400% elongation at break, low solubility, and thermal stability up to ~140 °C.
Resilience (the ratio of the energy elastically returned to the added energy) of resilin is
~92% due to covalent crosslinking between tyrosine residues. The mechanical
properties of elastin are similar to those of resilin, but resilin shows higher resilience

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and lower modulus. Resilin can be found in various biological joints, such as the vein
joints of dragonfly wings; however, natural resilin is not widely available for biological
and physical characterization. Therefore, by partially cloning and expressing
the Drosophila gene that has been identified to encode resilin, a recombinant protein
for resilin was successfully designed and obtained, and a rubber-like material could be
prepared by rapid photochemical crosslinking . This crosslinking was achieved by
peroxidase, which catalyzes dityrosine formation. The recombinant resilin proteins
were expressed and evaluated as biomaterials. Due to the attractive physical
characteristics of resilin, combinations of resilin with other proteins have also been
investigated. Elastomers composed of biomolecules, including resilin, are often
photochemically or chemically cross-linked. These biomaterials behave as rubber-like
materials and exhibit high elasticity with small strains, and they are expected to be
applicable as new muscle-mimetic biomaterials. On the other hand, examples of
research on resilin are limited compared to the number of studies on elastin, as many
researchers have studied the use of the latter as a biologically derived elastomer.

KERATIN

Keratin is the main structural protein that forms the hair, wool, feathers, nails, and horns
of many types of animals. This protein has a high content of cysteine (7–20% of the
total amino acid residues), which is known to form intramolecular and intermolecular
disulfide bonds. It has been reported that α-keratin, which has a helical structure, can
be reduced to form β-keratin upon stretching treatment, and this structural change
affects the mechanical, thermal, and chemical properties of the material. The disulfide
bonds in a tissue containing keratin, such as wool, can be disrupted by reductive
treatment, affording highly soluble keratin. Keratin can be extracted with an aqueous

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solution containing urea, mercaptanol, and sodium dodecyl sulfate as a surfactant, and
then, insoluble films and biodegradable sponge scaffolds can be prepared.

REFLECTIN

Reflectin has been found in certain cephalopods (especially squid),

including Euprymna scolopes and Doryteuthis opalescens. Squid can change their body
color to camouflage themselves, that is, mimic their surroundings, through the function
of reflectin, but research on reflectin protein is very limited, as is our understanding of
the molecular mechanism. Reflectin is speculated to have random coil and beta barrels
as the main motifs, but the structure has not been fully elucidated. Protein families that
exist in cephalopod pigment cells (rainbow cells, a type of pigment cell that changes
the wavelength of reflected light) and white pigment vesicles and contribute to
camouflage function have been recently reported. The amino acid sequence of reflectin
is rich in aromatic and sulfur-containing amino acids, and these motifs are used to
refract incident light in certain environments in certain cephalopods. However, not all
the molecular mechanisms have been fully clarified. Aliivibrio fischeri, a marine
luminescent bacterium, possesses the reflectin gene of cephalopods due to horizontal
gene transmission. Recently, several material scientists have been interested in reflectin
as a new functional biopolymer and have reported novel optical biomaterials containing
recombinant reflectin.

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