Proteins

Learning Objectives
• At the end of this lecture, you will be
able to
– Define proteins
– Describe the biomedical importance of
proteins
– Know composition of proteins and that
basic monomeric unit of proteins is amino
acid
 Biomedical importance
• Proteins are the most abundant
biological macromolecules occurring
in all cells and all parts of cells
• Proteins also occur in great variety;
thousands of different kinds may be
found in a single cell
• Proteins are the molecular instruments
through which genetic information is
expressed
• Biochemical catalysts known as enzymes
are proteins
• Immunoglobulins that serve as the first
line of defense against bacteria and
viruses and other foreign agents are
proteins
• Several hormones are proteins
• Structural proteins provide
mechanical support
• Contractile proteins help in the
movement of muscle fibre and
microvilli
• Some proteins present in the cell
membrane, cytoplasm and nucleus act as
receptors
• Certain other proteins in the cell
membrane act as channels and
transporters
• The transport proteins carry out the
function of transporting specific
substances across the membrane or in
the body fluids
• Storage proteins bind with specific
substances and store them

• Under certain conditions, proteins

can be catabolized to provide energy

• Proteins help in the maintenance of

water and electrolyte balance in the
body by exerting osmotic pressure
• Hence proteins are the most versatile
macromolecules in living systems and
serve crucial functions in essentially all
biological processes
• They function as catalysts, they transport
and store other molecules such as
oxygen, they provide mechanical support
and immune protection, they generate
movement, and they control growth and
differentiation
 Several key properties enable proteins to
participate in such a wide range of functions
1. Proteins are linear polymers built of
monomer units called amino acids
• The function of a protein is directly
dependent on its three dimensional structure
• Remarkably, proteins spontaneously fold up
into three-dimensional structures that are
determined by the sequence of amino acids
in the protein polymer
2. Proteins contain a wide range of
functional groups
• These functional groups include alcohols,
thiols, thioethers, carboxylic acids,
carboxamides, and a variety of basic
groups
• When combined in various sequences, this
array of functional groups accounts for the
broad spectrum of protein function
3. Proteins can interact with one another and
with other biological macromolecules to form
complex assemblies
• The proteins within these assemblies can act
synergistically to generate capabilities not
afforded by the individual component proteins
• These assemblies include macromolecular
machines that carry out the accurate
replication of DNA, the transmission of signals
within cells, and many other essential
processes
4. Some proteins are quite rigid, whereas others
display limited flexibility
• Rigid units can function as structural elements in
the cytoskeleton (the internal scaffolding within
cells) or in connective tissue
• Parts of proteins with limited flexibility may act
as hinges, springs, and levers that are crucial to
protein function, to the assembly of proteins
with one another and with other molecules into
complex units, and to the transmission of
information within and between cells
 Composition of proteins
• All proteins, whether from the
most ancient lines of bacteria or
from the most complex forms of
life, are constructed from the
same set of 20 amino acids,
covalently linked in characteristic
linear sequence
• Each of these amino acids has a
side chain with distinctive
chemical properties
• This group of 20 precursor
molecules maybe regarded as the
alphabet in which the language
of protein structure is written
• Proteins are found in a wide range of
sizes, from relatively small peptides with
just a few amino acid residues to huge
polymers with molecular weights in the
millions
• Cells can produce proteins with strikingly
different properties and activities by
joining the same 20 amino acids in many
different combinations and sequence
• From these building blocks different organisms can
make such widely diverse products as
– enzymes,
– hormones,
– antibodies,
– transporters,
– muscle fibers,
– the lens protein of the eye,
– feathers,
– spider webs,
– rhinoceros horn,
– milk proteins,
– antibiotics,
– mushroom poisons
• Proteins are polymers of amino acids,
with each amino acid residue joined
to its neighbor by a specific type of
covalent bond termed the peptide
bond
• The term "residue" reflects the loss of
the elements of water when one
amino acid is joined to another
• Twenty different amino acids are
commonly found in proteins
• All of the 20 common amino acids
are α amino acids
• They have a carboxyl group and an
amino group bonded to the same
carbon atom (the α carbon)
• They differ from each other in their
side chains, or R groups, which vary in
–structure,
–size,
–electric charge,
and which influence the solubility of
the amino acids in water
 Standard amino acids

• The 20 amino acids that constitute

the monomer units of proteins are
the standard amino acids as the
genetic code specifies only these 20
L-α-amino acids
• In addition to these 20 amino acids
there are many less common ones
i.e., the non standard amino acids
• (for which the genetic code is not specified)

• Some proteins contain additional

amino acids that arise by
modification of an amino acid already
present in a peptide i.e., after the
protein has been synthesized
• Examples include
–conversion of peptidyl proline and lysine
to 4-hydroxyproline and 5-hydroxylysine

–the conversion of peptidyl glutamate to -

carboxyglutamate

–the methylation, formylation, acetylation,

prenylation, and phosphorylation of
certain aminoacyl residues
• Among these uncommon amino acids

–4-hydroxyproline, a derivative of
proline
–5-hydroxylysine, derived from lysine
• The former is found in plant cell wall
proteins, and both are found in collagen
–G-N-Methyllysine is a constituent of
myosin, a contractile protein
• Another important uncommon
amino acid is
γ -carboxyglutamate, found in
the blood clotting protein
prothrombin and in certain
other proteins that bind Ca as
2+

part of their biological function

• Some 300 additional amino acids have
been found in cells but not as
constituents of proteins
• Examples include
–Ornithine and citrulline (intermediates in
urea cycle)
–Taurine (found in bile acids)

–δ-Aminolevulinic acid (intermediate in

haem synthesis)
 D- AMINO ACIDS
• D-amino acids are also non standard amino
acids that occur naturally and include
–free D- serine, and D-aspartate in brain tissue

–D-alanine & D-glutamate in the cell walls of

gram positive bacteria
• D-amino acids are also found in some
antibiotics
 Selenocysteine, the 21st L-α-Amino Acid?
• Selenocysteine is an L-α-amino acid found in a
handful of proteins, including certain peroxidases
and reductases where it participates in the
catalysis of electron transfer reactions
• Its synthesis is not a post translational
modification, however, but a modification to a
serine that occurs while the serine is bound to a
unique RNA
• The hydroxyl group of serine is replaced by a
selenium atom
• The selenocysteine is then inserted into a
protein as it is being synthesized
• Since selenocysteine is inserted into
polypeptides during translation, it is
commonly referred to as the "21st amino
acid"
• However, unlike the other 20 genetically
encoded amino acids, selenocysteine is not
specified by a simple three-letter codon
 Chiral centres and isomerism
• For all the common
amino acids except
glycine (the R group
is another hydrogen
atom), the α carbon
is bonded to four
different groups:
–a carboxyl group,

–an amino group,

–an R group,

–a hydrogen atom
• The α-carbon atom is thus a chiral
center and thus amino acids have
two possible stereoisomers

• All molecules with a chiral center

are also optically active-that is, they
rotate plane polarized light
• Special nomenclature has been
developed to specify the absolute
configuration of the four substituents
of asymmetric carbon atom
• The absolute configurations of simple
sugars and amino acids are specified
by the D, L system, based on the
absolute configuration of the three-
carbon sugar glyceraldehyde
• Thus the amino group of
L-alanine occupies the
same position about the
chiral carbon as does the
hydroxyl group of L-
glyceraldehyde
• Since they are non-
superimposable mirror
images of each other, the
two forms represent a
class of stereoisomers
called enantiomers
Two conventions are used to identify the
carbons in an amino acid
• The additional carbons in an R group are
commonly designated β, γ, δ and so forth,
proceeding out from the α carbon
• For most other organic molecules, carbon
atoms are simply numbered from one end,
giving highest priority to the carbon with the
substituent containing the atom of highest
atomic number
• Within this latter
convention, the carboxyl
carbon of an amino acid
would be C-1 and the α
carbon would be C-2
• In some cases, such as
amino acids with
heterocyclic R groups (such
as histidine), the Greek
lettering system is
ambiguous and the
numbering convention is
therefore used
• For branched amino
acid side chains,
equivalent carbons
are given numbers
after the Greek
letters

• Leucine thus has δ1

and δ2 carbons
Acidic and basic properties of amino acids
• The form of an amino acid that has both a positive
and a negative charge is called a zwitterion
• Amino acids in solution at neutral pH exist
predominantly as dipolar ions (zwitterions)
• In this form, the amino group is
protonated (–NH3+) and the carboxyl
group is deprotonated(–COO-)
• A zwitterion can act either as an acid
(proton donor) or as a base (proton
acceptor)
• Substances having this dual nature are
amphoteric and are often called
ampholytes
• Acting as acid (proton donor)

H H

R C COO- R C COO- + H+

NH3+ NH2

Zwitterion
• Acting as base (proton acceptor)

H H

R C COO- + H+ R C COOH

NH3+ NH3+

Zwitterion
• The ionization state of an amino
acid varies with pH
• At physiological pH, the carboxyl
group is dissociated , forming the
negatively charged carboxylate ion
(-COO ) and the amino group is
-

protonated (-NH3+ )
• A simple monoamino monocarboxylic -
amino acid, such as alanine, is a diprotic
acid when fully protonated—it has two
groups, the-COOH group and the - NH3+
group, that can yield protons
• Each acid has a characteristic tendency to
lose its proton in an aqueous solution
• The stronger the acid, the greater its
tendency to lose its proton
• The tendency of any acid (HA) to lose
a proton and form its conjugate base
(A-) is defined by the equilibrium
constant (Keq) for the reversible
reaction

HA A - + H+
• Equilibrium constants for ionization
reactions are usually called
ionization constants or acid
dissociation constants, often
designated pKa

• The stronger the tendency to

dissociate a proton, the stronger is
the acid and the lower its pKa
• pKa is the measure of the tendency of a group
to give up (donate) a proton with the tendency
decreasing ten-fold as the pKa increases by one
unit
• Each titratable group has a pKa that is
numerically equal to the pH at which exactly
one half of the protons have been removed
from that group
• The pKa for the most acidic group (COOH) is
pK1, whereas the pKa for the next most acidic
group (NH3+) is pK2
• In acid solution (e.g., pH 1), the amino
group is protonated (–NH3+) and the
carboxyl group is not dissociated(–COOH)
• As the pH is raised, the carboxylic acid is
the first group to give up a proton, as its
pKa is near 2
• The dipolar form persists until the pH
approaches 9, when the protonated
amino group loses a proton
Titration of an amino acid
Dissociation of the carboxyl
group:
• The titration curve of an
amino acid can be analyzed
• Consider alanine, for example,
which contains both an α-
carboxyl and an α-amino
group
• At a low (acidic) pH, both of
these groups are protonated
• As the pH of the
solution is raised, the
-COOH group of form I
can dissociate by
donating a proton to
the medium
• The release of a
proton results in the
formation of the
carboxylate group –
COO-
• This structure is shown
as form II, which is the
dipolar form of the
molecule

• This form, also called a

zwitterion, is the
isoelectric form of
alanine-that is, it has
an overall charge of
zero
• The dissociation constant of the carboxyl
group of an amino acid is called pK1 rather
than pKa because the molecule contains a
second titratable group
Dissociation of the amino group:
• The second titratable group of alanine is the
amino group
• This is a much weaker acid than the -COOH
group and, therefore, has a much bigger
dissociation constant, pK2
• Release of a
proton from the
protonated
amino group of
form II results in
the fully
deprotonated
form of alanine,
form III
• A simple amino acid e.g. glycine
is a diprotic acid when fully
protonated

• This means that it has two

groups, the COOH and the NH3 +

group that can yield protons

• Figure shows the titration curve of
the diprotic form of glycine
• The two ionizable groups of glycine,
the carboxyl group and the amino
group, are titrated with a strong base
• The plot has two distinct stages,
corresponding to deprotonation of
two different groups on glycine
• At very low pH, the predominant ionic species of
glycine is the fully protonated form,
+
H3N-CH2-COOH
• At the midpoint of any titration, a point is
reached where the pH is equal to the pKa of the
protonated group being titrated
• At the midpoint in the first stage of the titration,
in which the -COOH group of glycine loses its
proton, equimolar concentrations of the
protonated (+H3N-CH2-COOH) and the
deprotonated (+H3N-CH2-COO-) species are
• As the titration proceeds, another
important point is reached at pH 5.97
• This is a point, at which removal of the
first proton is essentially complete and
removal of the second has just begun
• At this pH glycine is present largely as
the dipolar ion (zwitterion)
+
H3N-CH2-COO-
• The second stage of the titration
corresponds to the removal of a proton
from the –NH3+ group of glycine
• The pH at the midpoint of this stage is
9.60, equal to the pKa for the –NH3 group
• The titration is essentially complete at a
pH of about 12, at which point the
predominant form of glycine is
H2N-CH2-COO-
• From the titration curves of glycine
and alanine we can derive several
important pieces of information
• First, it gives a quantitative
measure of the pKa, of each of the
two ionizing groups:
–2.34 for the -COOH group
– 9.60 for the –NH3+ group
• The second piece of information is
that this amino acid has two regions
of buffering power
• One of these is the relatively flat
portion of the curve, extending for
approximately 1 pH unit on either
side of the first pKa, of 2.6, indicating
that glycine is a good buffer near this
pH
• The other buffering zone is
centered around pH 9.60

• Note that glycine is not a

good buffer at the pH of
intracellular fluid or blood,
about 7.4
• Another important piece of information
derived from the titration curve of an amino
acid is the relationship between its net
charge and the pH of the solution
• At pH 5.97, glycine is present predominantly
as its dipolar form, fully ionized but with no
net electic charge
• The characteristic pH at which the net
electric charge is zero is called the isoeleetric
point or isoelectric pH, designated pI
• For glycine, which has no
ionizable group in its side
chain, the pI is simply the
arithmetic mean of the two
pKa values
• pI = ½ (pK1+pK2)
= ½ (2.34+9.60) = 5.97
• The farther the pH of a glycine solution is
from its pI, the greater the net electric
charge of the population of glycine
molecules
• At any pH below its pI, glycine has a net
positive charge and will move toward the
negative electrode (cathode)
• At any pH above its pI, glycine will have a
net negative charge and thus will move
towards the positive electrode (anode)
• All amino acids with a single α-amino group, a
single α-carboxyl group, and an R group that
does not ionize have titration curves
resembling that of glycine
• These amino acids have very similar, although
not identical, pKa values:
– pKa of the –COOH group in the range of 1.8 to 2.4
– pKa of the -NH3+ group in the range of 8.8 to 1 1

• The differences in these pKa values reflect the

effects of the R groups
• Alanine has only two dissociable
hydrogens (one from α carboxyl & one
from α amino group)
• Its pI can be calculated as follows:
• pI = pK1 + pK2 ÷ 2
pK1 = 2.3 (for carboxyl group)
pK2 = 9.1 (for amino group)
• pI = 2.3 + 9.1 ÷ 2 = 5.7
• Amino acids with an ionizable R
group have more complex titration
curves, with three stages
corresponding to the three possible
ionization steps and thus they have
three pKa values
• The additional stage for the titration
of the ionizable R group merges to
some extent with the other two
• Each of the acidic and basic amino
acids contains an ionizable group in
its side chain
• Thus, both free amino acids and
some amino acids combined in
peptide linkages can act as buffers
• A buffer is a solution that resists
change in pH following the addition
of an acid or base
• The isoelectric points reflect the
nature of the ionizing R groups
present
• For example, glutamate has a pI of
3.22, considerably lower than that of
glycine
• This is due to the presence of two
carboxyl groups, with an average of
their pKa values of 3.22
• Histidine has got 3 dissociable
hydrogens
–one from carboxyl, pK1=1.8,

–one from imidazole group,

pK2=6.0
–one from amino group,
pK3=9.2
• The pI of Histidine, with two groups
that are positively charged when
protonated, is 7.59 (the average of the
pKa values of the amino and imidazole
groups)
• Only histidine has an R group (pKa =
6.0) providing significant buffering
power near the neutral pH usually
found in the intracellular and
extracellular fluids of most animals
 IMPORTANCE OF pI
• At physiologic pH, all amino acids
have both a negatively charged group
(-COO ) & a positively charged group
-

(-NH3+)
• At physiologic pH, all amino acids are
dipolar ions (zwitterions)
• Amino aids can act both as an acid or
a base, therefore they are
‘Ampholytes’
• The buffering capacity of amino
acids & proteins is maximum
nearest to their pI values
• The net charge on an amino acid
or a protein molecule will be
negative at pH above the pI &
positive at pH below the
isoelectric point
Abbreviations and symbols for the
commonly occurring amino acids
•Each amino acid name has an
associated three-letter abbreviation
and a one-letter symbol
•The one-letter codes are determined
by the following rules:
 1. Unique first letter:

• If only one amino acid begins with

a particular letter, then that letter
is used as its symbol, for example,

I = isoleucine
 2. Most commonly
occurring amino acids
have priority:
• If more than one amino
acid begins with a
particular letter, the
most common of these
amino acids receives
this letter as its symbol
• For example, glycine is
more common than
glutamate, so G =
glycine
Similar sounding
names:
• Some one-letter
symbols sound like
the amino acid they
represent
• For example, F =
phenylalanine, or
W= tryptophan
("twyptophan“)
Letter close to initial letter:
• For the remaining amino
acids, a one-letter symbol is
assigned that is as close in
the alphabet as possible to
the initial of the amino acid
• Further, B is assigned to
Asx, signifying either
aspartic acid or asparagine,
Z is assigned to Glx
signifying either glutamic
acid or glutamine and X is
assigned to an unidentified
amino acid
Classification of amino acids
• Amino acids can be classified on the basis of
four different criteria:
– Side chains
• Polarity of the side chain
• Functional groups
– Nutritional importance
– Metabolic fate
• However, some amino acid side chains fit into
a number of different classifications and are
therefore grouped differently in different
textbooks
Polarity of Bonds and Partial Charges
• Polar bonds are covalent bonds in which the
electron cloud is denser around one atom (the
atom with the greater electronegativity) than
the other
• Oxygen is more electronegative than carbon,
and a carbon–oxygen bond is therefore polar,
with the oxygen atom carrying a partial
negative charge and the carbon atom carrying
a partial positive charge
• In nonpolar carbon–carbon bonds and
carbon–hydrogen bonds, the two
electrons in the covalent bond are shared
almost equally
• Nitrogen, also carries a partial negative
charge relative to carbon, and the
carbon–nitrogen bond is polarized
• Sulfur can carry a slight partial negative
charge
SOLUBILITY
• Water is a dipolar molecule in
which the oxygen atom
carries a partial negative
charge and the hydrogen
atoms carry partial positive
charges
• For molecules to be soluble in
water, they must contain
charged or polar groups that
can associate with the partial
positive and negative charges
of water
• Thus, the solubility of organic
molecules in water is determined
by both
–the proportion of polar to nonpolar
groups attached to the carbon–
hydrogen skeleton

–to their relative positions in the

molecule
• Polar groups or molecules are called hydrophilic
(water-loving), and nonpolar groups or
molecules are hydrophobic (water-fearing)
• The water molecules interacting with a polar or
ionic compound form a hydration shell around
the compound
• Compounds that have large nonpolar regions are
relatively water insoluble
• They tend to cluster together in an aqueous
environment and form weak associations through
van der Waals forces, often termed hydrophobic
bonds
• Hydrophobic compounds are essentially pushed
together as the water molecules maximize the
number of energetically favorable hydrogen bonds
they can form with each other in the water lattice

• Thus, lipids form droplets or separate layers in an

aqueous environment
A. Nonpolar, Aliphatic R Groups

• These are hydrophobic

• The amino acids in this group

have non polar side chains
• They do not participate in
hydrogen bonds

• They are ‘oily’ or lipid like and

promote hydrophobic interactions

• These side chains cluster together

in the interior of the protein
• This class
includes
–Glycine
–Alanine
–Proline
–Valine
–Leucine
–Isoleucine
–Methionine
• Electrons are shared equally
between the carbon and hydrogen
atoms in these side chains, so that
they cannot hydrogen bond with
water

• Within proteins, these amino acid

side chains will cluster together to
form hydrophobic cores
• Glycine is the simplest amino
acid, and it really does not fit
well into any classification
because its side chain is only a
hydrogen atom
• Because of this, it causes the
least amount of steric hindrance
in a protein
• Therefore glycine is often found
in bends or in the tightly packed
chains of fibrous proteins
• Alanine and the branched
chain amino acids (valine,
leucine, and isoleucine)
have bulky, nonpolar,
aliphatic(open chain
hydrocarbon) side chains
• Methionine, one of the two
sulfur-containing amino
acids, has a nonpolar
thioether group in its side
chain
• The role of proline in amino
acid structure differ from those
of the nonpolar amino acids
• The amino acid proline contains
a ring involving its α–carbon
and its α-amino group, which
are part of the peptide
backbone
• It is an imino acid
• This rigid ring causes a kink in
the peptide backbone that
prevents it from forming its
usual configuration
B. Aromatic Amino Acids
• The aromatic amino acids have been
grouped together because they all
contain ring structures with similar
properties, but their polarity differs a
great deal
• The aromatic ring is a six-membered
carbon–hydrogen ring with three
conjugated double bonds (the benzene
ring or phenyl group)
• The substituents on this ring
determine whether the amino acid
side chain engages in polar or
hydrophobic interactions
• This group includes
–Phenylalanine

–Tyrosine
• In the amino acid phenylalanine, the ring
contains no substituents, and the electrons
are shared equally between the carbons in
the ring, resulting in a very nonpolar
hydrophobic structure in which the rings can
stack on each other

• In tyrosine, a hydroxyl group on the phenyl

ring engages in hydrogen bonds, and the
side chain is therefore more polar and more
hydrophilic
• The more complex ring structure in
tryptophan is an indole ring with a
nitrogen that can engage in hydrogen
bonds
• Tryptophan is therefore also more polar
than phenylalanine
• Tyrosine and tryptophan are significantly
more polar than phenylalanine, because
of the tyrosine hydroxyl group and the
nitrogen of the tryptophan indole ring
C. Aliphatic, Polar, Uncharged R Groups
• The R groups of these amino acids are
more soluble in water, or more
hydrophilic, than those of the
nonpolar amino acids, because they
contain functional groups that form
hydrogen bonds with water
• These Amino acids have zero net
charge at neutral pH
• This class includes

– Serine & threonine

which contain a polar
hydroxyl group

– Similarly side chains of

asparagine &
glutamine contain a
carbonyl group &
amide group, both of
which can participate in
hydrogen bonding
• These side chains are sites of attachment
for other compounds
• The polar hydroxyl groups of serine &
threonine serve as a site of attachment
for phosphate groups
• The amide group of asparagine & the
hydroxyl group of serine & threonine
serve as a site of attachment for
oligosaccharide chains in glycoproteins
• Amino acids with side chains that
contain an amide group (asparagine
and glutamine) or a hydroxyl group
(serine and threonine) can be
classified as aliphatic, polar,
uncharged amino acids
• Asparagine and glutamine are amides
of the amino acids aspartate and
glutamate
• As a consequence of their
hydrophilicity, these amino acids are
frequently found on the surface of
water-soluble globular proteins

• Cysteine, which is sometimes

included in this class of amino acids,
has been separated into the class of
sulfur-containing amino acids
D. Sulfur-Containing Amino Acids
• Both cysteine and methionine contain sulfur
• The side chain of cysteine contains a sulfhydryl
group that has a pKa of approximately 8.4 for
dissociation of its hydrogen, so cysteine is
predominantly undissociated and uncharged at the
physiologic pH of 7.4
• The polarity of cysteine is contributed by its
sulfhydryl group, which is a weak acid and can make
weak hydrogen bonds with oxygen or nitrogen
• Cysteine is readily oxidized to form a
covalently linked dimeric amino acid called
cystine, in which two cysteine molecules or
residues are joined by a disulfide bond
• The disulfide-linked residues are strongly
hydrophobic (nonpolar)
• Disulfide bonds play a special role in the
structures of many proteins by forming
covalent links between parts of a polypeptide
molecule or between two different polypeptide
chains
• Methionine, although it
contains a sulfur group,
is a nonpolar amino acid
with a large bulky side
chain that is hydrophobic

• It does not contain a

sulfhydryl group and
cannot form disulfide
bonds
• Its important and
central role in
metabolism is related
to its ability to
transfer the methyl
group attached to
the sulfur atom to
other compounds
E. The Acidic and Basic Amino Acids
• The most hydrophilic R groups are those that
are either positively or negatively charged
• The amino acids aspartate and glutamate
have carboxylic acid groups that carry a
negative charge at physiologic pH
• The basic amino acids histidine, lysine, and
arginine have side chains containing nitrogen
that can be protonated and positively charged
at physiologic and lower pH values
The amino acids in which the R groups have significant
positive charge at pH 7.0 are:
– Lysine, which has a second primary amino group at the ε
position on its aliphatic chain
– Arginine, which has a positively charged
guanidinium group
– Histidine, which has an aromatic imidazole group
• The side chains of the two basic amino acids,
arginine and lysine, have pKa values above 10,
so that the positively charged form always
predominates at physiologic pH
• The side chain of histidine (pKa ~ 6.0)
dissociates near physiologic pH, so only a
portion of the histidine side chains carry a
positive charge
• Histidine is therefore weakly basic & the free
amino acid is mainly uncharged at physiologic
pH
• The amino acid side chains might have
very different pKas than those of the free
amino acids if they are involved in
hydrogen or ionic bonds with other
amino acid side chains
• Therefore, when histidine is incorporated
into a protein, its side chain can be either
positively charged or neutral depending
on the ionic environment provided by the
polypeptide chains of the protein
Negatively Charged (Acidic) R Groups

• At neutral pH, the side chains of

these amino acids are fully ionized,
containing a negatively charged
carboxylate group (--COO )-
• Therefore these amino
acids are negatively
charged at phyisologic
pH
• The two amino acids
having R groups with a
net negative charge at
pH 7.0 are aspartate
and glutamate, each
of which has a second
carboxyl group
• Humans have no dietary requirement for protein,
per se, but, the protein in food does provide
essential amino acids
• Ten of the twenty amino acids needed for the
synthesis of body proteins are essential-that is,
they cannot be synthesized in humans at an
adequate rate
• Of these ten, eight are essential at all times,
whereas two (arginine and histidine) are required
only during periods of rapid tissue growth,
characteristic of childhood or recovery from illness
• Amino acids can be classified as glucogenic or
ketogenic based on what intermediates are
produced during their catabolism
A. Glucogenic amino acids
• Amino acids whose catabolism yields
pyruvate are termed glucogenic or glycogenic
• These intermediates are substrates for
gluconeogenesis and, therefore, can give rise
to the net formation of glucose or glycogen in
the liver and glycogen in the muscle
B. Ketogenic amino acids
• Amino acids whose catabolism yields
either acetoacetate or its precursor,
(acetyl CoA or acetoacetyl CoA) are
termed ketogenic
• Acetoacetate is one of the ketone
bodies which also include 3-
hydroxybutyrate and acetone
• Leucine and lysine are the only
exclusively ketogenic amino acids
found in proteins
• Their carbon skeletons are not
substrates for gluconeogenesis and,
therefore, cannot give rise to the net
formation of glucose or glycogen in
the liver, or glycogen in the muscles
• We now turn to polymers of
amino acids, the peptides and
proteins

• Biologically occurring
polypeptides range in size from
small to very large, consisting of
two or three to thousands of
linked amino acid residues
• Two amino acid
molecules can
be covalently
joined through
an amide
linkage, termed
a peptide bond
• This linkage is formed by removal of
the elements of water from the α-
carboxyl group of one amino acid and
the α-amino group of another
• Two configurations are possible for a
planar peptide bond

• In the trans configuration, the two -

carbon atoms are on opposite sides
of the peptide bond

• In the cis con-, these groups are on

the same side of the peptide bond
• Almost all peptide bonds
in proteins are trans
• This preference for trans
over cis can be
explained by the fact
that steric clashes
between groups
attached to the -carbon
atoms hinder formation
of the cis form but do
not occur in the trans
configuration
• The peptide
C-N bonds,
have a
partial
double-bond
character,
and thus
cannot
rotate freely
• Three amino acids can be joined by two
peptide bonds to form a tripeptide;
similarly, four amino acids can be linked
to form tetrapeptides, five to form
pentapeptides, and so forth
• When a few amino acids are joined in this
fashion, the structure is called an
oligopeptide
• When many amino acids are joined, the
product is called a polypeptide
• Proteins may have thousands of
amino acid residues
• Although the terms "protein" and
"polypeptide" are sometimes used
interchangeably, molecules referred
to as polypeptides generally have
molecular weights below 10,000, and
those called proteins have higher
molecular weights
• The figure below shows the structure of a
pentapeptide
• An amino acid unit in a peptide is often called
a residue (the part left over after losing a
hydrogen atom from its amino group and the
hydroxyl moiety from its carboxyl group)
• In a peptide, the amino acid residue at the
end with a free α-amino group is the amino-
terminal (or N-terminal) residue
• The residue at the other end, which has a free
carboxyl group, is the carboxyl-terminal (C-
terminal) residue
• When an amino acid sequence of a
peptide, polypeptide, or protein is
displayed the aminoterminal end is
placed on the left, the carboxyl-
terminal end on the right

• The sequence is read left to right,

beginning with the amino-terminal
end
• Peptides contain only one free α-
amino group and one free α-carboxyl
group, at opposite ends of the chain
• These groups ionize as they do in free
amino acids, although the ionization
constants are different because an
oppositely charged group is no longer
linked to the α carbon
• The α-amino and α-carboxyl
groups of all non terminal
amino acids are covalently
joined in the peptide bonds,
which do not ionize and thus
do not contribute to the total
acid-base behavior of
peptides
• However, the R groups of some amino
acids can ionize, and in a peptide
these contribute to the over all acid-
base properties of the molecule
• Like free amino acids, peptides have
characteristic titration curves and a
characteristic isoelectric pH (pI) at
which they do not move in an electric
field
• The pKa value for an ionizable R
group can change somewhat when
an amino acid becomes a residue in
a peptide
• The Ioss of charge in the α-carboxyl
and α-amino groups, the
interactions with other peptide R
groups, and other environmental
factors can affect the pKa
• No generalizations can be made about
the molecular weights of biologically
active peptides and proteins in relation to
their functions
• Even the smallest peptides can have
biologically important effects, e.g. the
commercially synthesized dipeptide l-
aspartyl-l-phenylalanine methyl ester, the
artificial sweetener better known as
aspartame or NutraSweet
• Many small peptides exert their effects at very low
concentrations.
• For example,a number of vertebrate hormones are
small peptides.
– oxytocin (nine amino acid residues), which is secreted by
the posterior pituitary and stimulates uterine
contractions
– thyrotropin-releasing factor (three residues), which is
formed in the hypothalamus and stimulates the release
of another hormone, thyrotropin, from the anterior
pituitary gland
• Some extremely toxic mushroom poisons, such as
amanitin, are also small peptides, as are many
antibiotics
• At the other extreme is titin, a
constituent of vertebrate muscle,
which has nearly 27,000 amino acid
residues and a molecular weight of
about 3,000,000
• The vast majority of naturally
occurring proteins are much smaller
than this, containing fewer than
2,000 amino acid residues
• Some proteins consist of a single polypeptide
chain, but others, called multisubunit
proteins, have two or more polypeptides
associated noncovalently
• The individual polypeptide chains in a
multisubunit protein may be identical or
different
• If at least two are identical the protein is said
to be oligomeric, and the identical units
(consisting of one or more polypeptide chains)
are referred to as protomers
• Hemoglobin, for
example, has four
polypeptide subunits
all held together by
noncovalent
interactions:
– two identical α
chains
– two identical β
chains
• Each α subunit is paired
in an identical way with
a β subunit within the
structure of this
multisubunit protein, so
that hemoglobin can be
considered either a
tetramer of four
polypeptide subunits or
a dimer of αβ
protomers
• A few proteins contain two or more
polypeptide chains linked covalently
• For example, the two polypeptide chains of
insulin are linked by disulfide bonds
• In such cases, the individual polypeptides
are not considered subunits but are
commonly referred to simply as chains
• The amino acid composition of proteins
is also highly variable
• The 20 common amino acids almost
never occur in equal amounts in a
protein
• Some amino acids may occur only once
or not at all in a given type of protein;
others may occur in large numbers
 Classification of Proteins
Different classifications of proteins are
based on their:
1. Shape and Size
2. Biological actions
3. Solubility and physical properties
4. Quality
 i. Classification of Proteins on Shape and Size
Basis
• This classification is based on their Axial Ratio
[The ratio of molecular length to molecular
width]
i. Fibrous Proteins: Axial Ratio > 10. e.g. Keratins,
collagens, fibrinogen, fibrin and elastin etc
ii. Globular Proteins: Axial Ratio < 10 [usually not
> 3 or 4] e.g. myoglobin, hemoglobin,
ribonuclease and most of the plasma proteins
i.e. hormones, enzymes, immunoglobulins etc
ii. Functional Classification of Proteins
1. Catalytic Proteins:
These specialized proteins are called enzymes which
catalyze the biochemical reactions
2. Protective Proteins
a) Immunoglobins (Igs)
• These freely circulating proteins protect the body
from invading bacteria or viruses by engulfing them
• In acquired immunodeficiency syndrome (AIDS) Igs
synthesized have no immunity against antigens
b) Fibrinogen
• This forms fibrin clot and stops bleeding from wounds
3. Regulatory Proteins:
• Hormones control biochemical reactions
catalyzed by enzymes either activating or
inactivating them through modification
of their molecules
• Protein hormones are
– growth hormone
– Insulin
– Glucagon
– somatostatin
4. Structural Proteins:
• These proteins form various body structures e.g.
– Collagen
– Elastin
– Keratin

5. Transport Proteins:
• These proteins transport various substances
from one part of the body to the other e.g.
– Hemoglobin transport O2 from lungs to tissues and
CO2 from tissues to lungs
– Transferrin transports iron
6. Contractile Proteins:
• These proteins are involved in muscle
contraction and relaxation
– Myosin of thick filaments
– Actin of thin filaments of skeletal muscles
7. Respiratory Proteins:
• Heme containing proteins are involved in
the function of respiration e.g.
– Hemoglobin
– Myoglobin
– cytochromes
8. Digestive Proteins:
• These proteins are digestive enzymes
which digest our food materials such as
carbohydrates, proteins, lipids and
include
– Amylase

– Pepsin

– Lipases etc
9. Toxin Proteins:
• These proteins are hydrolytic enzymes
found in the venom of poisonous snakes,
sting of bees and insects and hydrolyze
the compounds forming the structure of
the cell membrane
• The poisonous mushrooms also have such
toxins
10. Storage Proteins:
• These proteins store some specific
elements or compounds with them
• This is because of the presence of the
many binding sites in them for the
particular element e.g.
–ferritin stores iron

–ceruloplasmin stores copper

 iii. Classification of Proteins on Solubility and
Physicals Properties Basis
• The following classification is based upon
physiochemical properties of proteins
• A protein may belong to one of the three
types, i.e.
A. Simple Proteins
B. Compound or Conjugated Proteins
C. Derived Proteins
A. Simple Proteins:
• On hydrolysis, these proteins yield only
amino acids or their derivatives
• These consist of the following types
1. Albumins: Soluble in water and dilute salt
solution
– Animal Sources: Egg albumin, lactalbumin,
serum albumin

– Vegetable Sources: Legumelin of peas, leucosine

of wheat
2. Globulins:
• Sparingly soluble in water but soluble in dilute salt solution
• Heat coagulable
• Examples include:

– lactoglbulins,

– myosin in muscle,

– ovoglobulin of egg yolk,

– lactglobulin,

– serum globulin

– legumin
3. Globins
• They unite with heme to form hemoglobin
• Hemoglobins of different species differ only with
respect to globin, and the heme part is the same
in all cases
4. Prolamines or Gliadins:
• Insoluble in water, soluble in 70-80% ethanol
• These are rich in proline but deficient in lysine
• Examples: Gliadin of wheat,
Zein of maize
5. Histones:
• Very strongly basic proteins as they are
rich in arginine
• Soluble in water but insoluble in dilute
ammonia
• Readily heat coagulable
• Examples: nucleohistones, chromosomal
nucleoproteins,
6. Protamines:
• Basic proteins
• Soluble in water
• Examples:
– Salmine,

– Sardinine,

– sturine of fish sperms

7. Albuminoids/Sclero Proteins:
• Insoluble in all solvents and
include
i. Collagen:
i. Deficient in trp but rich in gly
ii. Present in bones, skin, cornea and
all body structure
i. Elastin:
i. Rich in nonpolar amino acid ala,
leu, val and pro

ii. Formed in excess during pregnancy

in uterine elastic tissue
ii. Keratin:
i. Rich in cys
B. conjugated proteins
• These proteins contain
permanently associated chemical
components in addition to amino
acids
• The non-amino acid part of a
conjugated protein is usually
called its prosthetic group
• Conjugated proteins are classified on
the basis of the chemical nature of
their prosthetic groups for example,
–Lipoproteins contain lipids,
–glycoproteins contain sugar groups
–metalloproteins contain a specific
metal
• Some proteins contain more than
one prosthetic group

• Usually the prosthetic group

plays an important role in the
protein's biological function
C. Derived Proteins:
• This class of proteins includes
substances which are derived from
simple and conjugated proteins
• These proteins are subdivided into
primary and secondary derived
proteins
iv. Quality of Proteins
• Proteins containing all 20 amino acids
that can be absorbed are called
Complete or Grade-I proteins
• Other proteins, deficient in some
essential amino acids are called
Incomplete or Grade-II proteins
• FOUR ORDERS OF
PROTEIN STRUCTURE
The modular nature of protein synthesis and
folding are
embodied in the concept of orders of protein
structure: primary
structure—the sequence of amino acids in a
polypeptide chain;
secondary structure—the folding of short (3-30
residue), contiguous segments of polypeptide into
geometrically ordered
• units; tertiary structure—the assembly of
secondary structural
units into larger functional units such as the
mature polypeptide
and its component domains; and quaternary
structure—the
number and types of polypeptide units of
oligomeric proteins
and their spatial arrangement.
• SECONDARY STRUCTURE
peptide Bonds restrict possible
Secondary Conformations
Free rotation is possible about only two of the three covalent
bonds of the polypeptide backbone: the bond linking the
a-carbon (Ca) to the carbonyl carbon (Co) and the bond linking Ca to nitrogen (see Figure 3–
9). The partial double-bond
character of the peptide bond that links Co to the a-nitrogen
requires that the carbonyl carbon, carbonyl oxygen, and
a-nitrogen remain coplanar, thus preventing rotation. The
angle about the Ca[N bond is termed the phi (Φ) angle, and
that about the Co[Ca bond the psi (Ψ) angle. In peptides, for
amino acids other than glycine, most combinations of phi and psi
angles are disallowed because of steric hindrance (Figure 5–1).
The conformations of proline are even more restricted as its
cyclic structure prevents free rotation of the N[Ca bond.
Regions of ordered secondary structure arise when a
series of aminoacyl residues adopt similar phi and psi angles.
Extended segments of polypeptide (eg, loops) can possess a
variety of such angles. The angles that define the two most
common types of secondary structure, the ` helix and the
a sheet, fall within the lower and upper left-hand quadrants of
a Ramachandran plot, respectively (Figure 5–1
• Alpha Helix
The polypeptide backbone of an a helix is twisted by an equal
amount about each a-carbon with a phi angle of approximately
-57° and a psi angle of approximately -47°
• A complete turn of the helix contains an average of 3.6 aminoacyl residues, and
the distance it rises per turn (its pitch) is 0.54 nm (Figure 5–2).
The R groups of each aminoacyl residue in an a helix face outward (Figure 5–3). Proteins contain only l-
amino acids, for
which a right-handed a helix is by far the more stable, and
only right-handed a helices are present in proteins. Schematic
diagrams of proteins represent a helices as coils or cylinders.
The stability of an a helix arises primarily from hydrogen bonds formed between the oxygen of the
peptide bond
carbonyl and the hydrogen atom of the peptide bond nitrogen
of the fourth residue down the polypeptide chain (Figure 5–4).
The ability to form the maximum number of hydrogen bonds,
supplemented by van der Waals interactions in the core of this
tightly packed structure, provides the thermodynamic driving force for the formation of an a helix
• Since the peptide bond nitrogen of proline lacks a hydrogen atom, it is incapable of forming a hydrogen
bond with a carbonyl oxygen
• Consequently, proline can only be stably accommodated within the first turn of an a helix
• When present elsewhere, proline disrupts the conformation of the helix, producing a
bend
• Because it possesses such a small R group, glycine also frequently induces bends within a helices.
• Many a helices have predominantly hydrophobic
R-groups projecting from one side of the axis of
the helix and predominantly hydrophilic R-groups
projecting from the other side.
These amphipathic helices are well adapted to
the formation of interfaces between polar and
nonpolar regions such as the hydrophobic interior
of a protein and its aqueous environment
• Clusters of amphipathic helices can create
channels, or pores, through hydrophobic cell
membranes that permit specific polar molecules
to pass.
• Loops & Bends
Roughly half of the residues in a “typical” globular protein
reside in a helices or β sheets, and half in loops, turns, bends,
and other extended conformational features. Turns and bends
refer to short segments of amino acids that join two units of
the secondary structure, such as two adjacent strands of an
antiparallel β sheet. A β turn involves four aminoacyl residues,
in which the first residue is hydrogen-bonded to the fourth,
resulting in a tight 180° turn (Figure 5–7). Proline and glycine
often are present in β turns.
Loops are regions that contain residues beyond the
minimum number necessary to connect adjacent regions of
secondary structure. Irregular in conformation, loops nevertheless serve key biologic roles. For
many enzymes, the loops
that bridge domains responsible for binding substrates often
contain aminoacyl residues that participate in catalysis. Helixloop-helix motifs provide the
oligonucleotide-binding portion of many DNA-binding proteins such as repressors and
transcription factors. Structural motifs such as the helix-loophelix motif or the E-F hands of
calmodulin (see Chapter 51)
that are intermediate in scale between secondary and tertiary
structures are often termed supersecondary structures. Since
many loops and bends reside on the surface of proteins, and
are thus exposed to solvent, they constitute readily accessible
sites, or epitopes, for recognition and binding of antibodies.
• While loops lack apparent structural regularity, many adopt
a specific conformation stabilized through hydrogen bonding,
salt bridges, and hydrophobic interactions with other portions
of the protein. However, not all portions of proteins are
necessarily ordered. Proteins may contain “disordered”
regions,
often at the extreme amino or carboxyl terminal, characterized
by high conformational flexibility. In many instances, these
disordered regions assume an ordered conformation upon
binding of a ligand. This structural flexibility enables such
regions
to act as ligand-controlled switches that affect protein
structure
and function
• Tertiary & Quaternary Structure
The term “tertiary structure” refers to the entire three-dimensional
conformation of a polypeptide. It indicates, in three-dimensional
space, how secondary structural features—helices, sheets, bends,
turns, and loops—assemble to form domains and how these domains
relate spatially to one another. A domain is a section of
the protein structure sufficient to perform a particular chemical or
physical task such as binding of a substrate or other ligand. Most
domains are modular in nature, and contiguous in both primary
sequence and three-dimensional space (Figure 5–8). Simple proteins,
particularly those that interact with a single substrate, such as
lysozyme or triose phosphate isomerase (Figure 5–6) and the oxygen
storage protein myoglobin (see Chapter 6), often consist of a single
domain. By contrast, lactate dehydrogenase is comprised of two
domains, an N-terminal NAD+-binding domain and a C-terminal
binding domain for the second substrate, pyruvate (Figure 5–8)
• Not all domains bind substrates. Hydrophobic membrane
domains anchor proteins to membranes or enable them to
span membranes. Localization sequences target proteins to specific subcellular or extracellular
locations such as the nucleus,
mitochondria, secretory vesicles, etc. Regulatory domains trigger changes in protein function in
response to the binding of
allosteric effectors or covalent modifications (see Chapter 9).
Combining the genetic material coding for individual domain
modules provides a facile route for generating proteins of great
structural complexity and functional sophistication (Figure 5–9).
Proteins containing multiple domains can also be assembled through the association of multiple
polypeptides, or
protomers. Quaternary structure defines the polypeptide composition of a protein and, for an
oligomeric protein, the spatial
relationships between its protomers or subunits. Monomeric
proteins consist of a single polypeptide chain. Dimeric proteins contain two polypeptide chains.
Homodimers contain
two copies of the same polypeptide chain, while in a heterodimer the polypeptides differ. Greek
letters (a, β, γ, etc) are
used to distinguish different subunits of a hetero-oligomeric
protein, and subscripts indicate the number of each subunit
type. For example, a4 designates a homotetrameric protein,
and a
2β2γ, a protein with five subunits of three different types
• Since even small proteins contain many thousands of
atoms, depictions of protein structure that indicate the position
of every atom are generally too complex to be readily
interpreted. Simplified schematic diagrams thus are used to
depict the key features of a protein’s tertiary and quaternary
structure. Ribbon diagrams (Figures 5–6 and 5–8) trace the
conformation of the polypeptide backbone, with cylinders and
arrows indicating regions of a helix and β sheet, respectively.
In an even simpler representation, line segments that link the
a carbons of each amino acid residue indicate the path of the
polypeptide backbone. In order to emphasize specific structure-
function relationships, these schematic diagrams often
depict the side chains of selected amino acids.
• MULTIPLE FACTORS STABILIZE
TERTIARY & QUATERNARY
STRUCTURE
Higher orders of protein structure are stabilized primarily—
and often exclusively—by noncovalent interactions. Principal
among these are hydrophobic interactions that drive most
hydrophobic amino acid side chains into the interior of the
protein away from the surrounding water. Other significant
contributors include hydrogen bonds and salt bridges between
the carboxylates of aspartic and glutamic acid and the oppositely charged
side chains of protonated lysyl, argininyl, and
histidyl residues. These interactions are individually weak—1
to 5 kcal/mol relative to 80 to 120 kcal/mol for a covalent
bond. However, just as a Velcro fastener harnesses the cumulative strength
of a multitude of tiny plastic loops and hooks,
collectively these individually weak but numerous interactions
confer a high degree of stability to the biologically functional
conformation of a protein.
• Some proteins contain covalent disulfide (S[S) bonds
that link the sulfhydryl groups of cysteinyl residues.
Formation
of disulfide bonds involves oxidation of the cysteinyl
sulfhydryl groups and requires oxygen.
Intrapolypeptide disulfide
bonds further enhance the stability of the folded
conformation
of a peptide, while interpolypeptide disulfide bonds
stabilize
the quaternary structure of certain oligomeric
proteins.
 The Structure of Proteins
• Four levels of protein structure are
commonly defined
• A description of all covalent bonds
(mainly peptide bonds and disulfide
bonds) linking amino acid residues in a
polypeptide chain is its primary structure
• The most important element of primary
structure is the sequence of amino acid
residues
• Secondary structure
refers to the folding of
short (3- to 30-
residue), contiguous
segments of a
polypeptide into
particularly stable
arrangements of amino
acid residues giving
rise to geometrically
ordered recurring
structural patterns
• Tertiary structure
describes the assembly of
secondary structural units
into larger functional
units such as the mature
polypeptide and its
component domains
• Tertiary structure hence
describes all aspects of
the three-dimensionl
folding of a poypeptide
• When a
protein has
two or more
polypeptide
subunits,
their
arrangement
in space is
referred to as
quaternary
structure
• The primary structure of a protein
determines how it folds up into its unique
three-dimensional structure, and this in
turn determines the function of the
protein
• The function of a protein therefore
depends on its amino acid sequence
• Thousands of human genetic diseases
have been traced to the production of
defective proteins
• The defect can range from a single
change in the amino acid sequence
(as in sickle cell anemia) to deletion
of a larger portion of the polypeptide
chain (as in most cases of Duchenne
muscular dystrophy)
• Thus we know that if the primary
structure is altered, the function of
the protein may also be changed
• Finally, on comparing functionally similar
proteins from different species, we find
that these proteins often have similar
amino acid sequences
• An extreme case is ubiquitin, a 76-
residue protein involved in regulating the
degradation of other proteins
• The amino acid sequence of ubiquitin is
identical in species as disparate as fruit
flies and humans
• We will examine how a sequence of amino
acids in a polypeptide chain is translated into
a discrete, three-dimensional protein
structure
• We emphasize five themes

– First, the three-dimensional structure of a

protein is determined by its amino acid
sequence

– Second, the function of a protein depends

on its structure
–Third, an isolated protein usually exists in one
or a small number of stable structural forms

–Fourth, the most important forces stabilizing

the specific structures maintained by a given
protein are noncovalent interactions

–Finally, amid the huge number of unique

protein structures, we can recognize some
common structural patterns that help to
organize our understanding of protein
architecture
• The spatial arrangement of atoms in a
protein is called its conformation
• The possible conformations of a protein
include any structural state it can achieve
without breaking covalent bonds
• Of the many conformations that are
theoretically possible in a protein
containing hundreds of single bonds one
(more commonly) or a few generally
predominate under biological conditions
• The conformations existing under
a given set of conditions are
usually the ones that are
thermodynamically the most
stable

• Proteins in any of their functional,

folded conformations are called
native proteins
• The chemical interactions that
stabilize the native conformation
include disulfide (covalent) bonds
and the weak (noncovalent)
interactions:
–Hydrogen bonds
–Hydrophobic
• Many proteins do not have
disulfide bonds

• Disulfide bonds are found

primarily in secreted, extracellular
proteins (for example, the
hormone insulin)
• For the intracellular proteins of most
organisms, weak interactions are
especially important in the folding of
polypeptide chains into their
secondary and tertiary structures
• The association of multiple
polypeptides to form quaternary
structures also relies on these weak
interactions
• About 200 to 460 kJ/mol are required
to break a single covalent bond,
whereas weak interactions can be
disrupted by a mere 4 to 30 kJ/mol

• Individual covalent bonds, such as

disulfide bonds linking separate parts
of a single polypeptide chain, are
clearly much stronger than individual
weak interactions
• Yet, because they are so numerous,
it is the weak interactions that
predominate as a stabilizing force
in protein structure

• In general, the protein

conformation that is most stable is
the one with the maximum
number of weak interactions
• On carefully examining the contribution of
weak interactions to protein stability, we find
that hydrophobic interactions generally
predominate
• The interior of a protein is generally a densely
packed core of hydrophobic amino acid side
chains
• It is also important that any polar or charged
groups in the protein interior have suitable
partners for hydrogen bonding or ionic
interactions
• Salt bridges, especially those that
are partly or entirely buried, can
thus provide significant stabilization
to a protein structure.
• Ionic interactions also Limit
structural flexibility and confer a
uniqueness to protein structure that
nonspecific hydrophobic
interactions cannot provide
• Most of the structural patterns
outlined so far reflect two simple
rules:
–(1) hydrophobic residues are
largely buried in the protein
interior, away from water
– (2) the number of hydrogen
bonds and ionic interactions
within the protein is maximum
• Protein structure is stabilized by
multiple weak interactions
• Hydrophobic interactions are the
major contributors to stabilizing the
globular form of most soluble
proteins
• Hydrogen bonds and ionic
interactions are optimized in the
thermodynamically stable structures
• The nature of the covalent bonds
in the polypeptide backbone
places constraints on structure
• The peptide bond has a partial
double-bond character that keeps
the entire six-atom
peptide group in a rigid
planar configuration
Requirements of the Three-
Dimensional Structure

• The overall three-dimensional

structure of a protein must meet
certain requirements to enable the
protein to function in the cell or
extracellular medium of the body
• The first requirement is the creation
of a binding site that is specific for
just one molecule, or a group of
molecules with similar structural
properties

• The specific binding sites of a protein

usually define its role
• The three-dimensional structure also
must exhibit the degrees of flexibility
and rigidity appropriate to its
function
• Some rigidity is essential for the
creation of binding sites and for a
stable structure (i.e., a protein that
just flopped all over the place could
not accomplish anything)
• However, flexibility and mobility in
structure enables the protein to fold as it
is synthesized, and to adapt as it binds
other proteins and small molecules
• The three-dimensional structure must
have an external surface appropriate for
its environment (e.g., cytoplasmic
proteins need to keep polar amino acids
on the surface to remain soluble in an
aqueous environment)
• In addition, the conformation must
also be stable, with little tendency to
undergo refolding into a form that
cannot fulfill its function or that
precipitates in the cell
• Finally, the protein must have a
structure that can be degraded when
it is damaged or no longer needed in
the cell
Requirements of a Protein Structure
• Function
• Binding specificity
• Flexibility
• Solubility or lipophilicity
• Stability
• Degradability
SECONDARY STRUCTURE
• Secondary structure, the folding of short
(3- to 30-residue), contiguous segments of
polypeptide into geometrically ordered
units
• The term secondary structure refers to any
chosen segment of a polypeptide chain and
describes the local spatial arrangement of
its main-chain atoms, without regard to its
relationship to other segments
• The angle about
the C α—N bond
is termed the phi
(ф) angle, and
that about the
Co—Cα bond the
psi (ψ) angle
Peptide Bonds Restrict Possible Secondary Conformations
• Free rotation is possible about only two of the three
covalent bonds of the polypeptide backbone:

– the α-carbon (Cα) to the carbonyl carbon (Co)

bond, {the psi (ψ) angle}

– the Cα to nitrogen bond {the phi (ф) angle}

• There thus is no freedom of rotation
about the bond that connects the
carbonyl carbon and the nitrogen of a
peptide bond
• The imposed semi-rigidity of the
peptide bond has important
consequences for the manner in
which peptides and proteins fold to
generate higher orders of structure
• Regions of ordered secondary structure arise
when a series of aminoacyl residues adopt
similar phi and psi angles

• Extended segments of polypeptide (eg, loops)

can possess a variety of such angles
• There are a few types of secondary
structure that are particularly stable and
occur widely in proteins
• The most prominent are the α helix and
β conformations
• Another common type is the β turn
• Where a regular pattern is not found, the
secondary structure is sometimes
referred to as undefined or as a random
coil
 The α Helix is a common protein secondary
structure
• The simplest arrangement the polypeptide
chain can assume, given its rigid peptide
bonds (but free rotation around its other,
single bonds), is a helical structure, called the
α helix
• Generally, about one fourth of all amino acid
residues in proteins are found in α helices, the
exact fraction varying greatly from one
protein to another
• The polypeptide
backbone of an α
helix is twisted by an
equal amount about
each α -carbon with
a phi angle of
approximately –57
degrees and a psi
angle of
approximately –47
degrees
• In this structure
the polypeptide
backbone is tightly
wound around an
imaginary axis
drawn
longitudinally
through the
middle of the helix
• The R
groups of
each
aminoacyl
residue in
an α helix
face
outward
• Proteins contain
only L-amino
acids, for which a
right-handed
helix is by far the
more stable, and
only right-handed
helices are
present in
proteins
• The repeating
unit is a single
turn of the helix,
and each helical
turn includes 3.6
amino acid
residue and the
distance it rises
per turn (its
pitch ) is 0.54 nm
• The stability of an α
helix arises primarily
from hydrogen bonds
formed between the
oxygen of the peptide
bond carbonyl and
the hydrogen atom of
the peptide bond
nitrogen of the fourth
residue down the
polypeptide chain
• Within the α
helix, every
peptide bond
(except those
close to each
end of the
helix)
participates
in such
hydrogen
bonding
• Each successive turn of the α helix is held
to adjacent turns by three to four
hydrogen bonds, conferring significant
stability on the overall structure

• The ability to form the maximum number

of hydrogen bonds, supplemented by van
der Waals interactions in the core of this
tightly packed structure, provides the
stability for the helix
• Many helices have
predominantly hydrophobic R
groups on one side of the axis of
the helix and predominantly
hydrophilic ones on the other
• These amphipathic helices are
suitable for the formation of
interfaces between polar and
nonpolar regions such as the
hydrophobic interior of a protein
and its aqueous environment
• Clusters of amphipathic helices
can create a channel, or pore
• Not all polypeptides can form a stable α
helix
• Each amino acid residue in a polypeptide
has an intrinsic propensity to form an a
helix, reflecting the properties of the R
group and how they affect the capacity of
the adjoining main-chain atoms to take up
the characteristic angles
• Alanine shows the greatest tendency to
form α helices in most experimental model
systems
• The position of an amino acid
residue relative to its neighbors is
also important

• Interactions between amino acid

side chains can stabilize or
destabilize the α-helical structure
• For example, if a polypeptide chain
has a long block of Glutamine
residues, this segment of the chain
will not form an α helix at pH 7.0
• The negatively charged carboxyl
groups of adjacent GIutamine
residues repel each other so strongly
that they prevent formation of the α
helix
• For the same reason, if there are many
adjacent Lys and/or Arg residues, with
positively charged R groups at pH 7.0,
they also repel each other and prevent
formation of the α helix

• The bulk and shape of Ser, Thr, and Cys

residues can also destabilize an α helix
if they are close together in the chain
• Another constraint on the formation of the α
helix is the presence of Proline or Glycine
residues
• Since the peptide bond nitrogen of proline lacks
a hydrogen atom, it is incapable of forming a
hydrogen bond with a carbonyl oxygen
• Consequently, proline can only be stably
accommodated within the first turn of an a helix
• When present elsewhere, proline disrupts the
conformation of the helix, producing a
bend
• Glycine occurs infrequently in α
helices for a different reason
• Because it possesses such a small R
group, glycine also frequently
induces bends within a helices
• Polymers of glycine tend to take up
coiled structures quite different
from an α helix
• In summary, four types of constraints affect the
stability of an α helix:
– (1) The intrinsic propensity of an amino acid residue to
form an α helix
– (2) The interactions between R groups, particularly
those spaced three (or four) residues apart
– ( 3) The bulkiness of adjacent R groups
– ( 4) The occurrence of Pro and Gly residues

• The tendency of a given segment of a polypeptide

chain to form an α helix therefore depends on the
identity and sequence of amino acid residues within
the segment
 The β Conformation organizes polypeptide
Chains in β sheets
•  Sheets are a second type of regular
secondary structure that maximizes hydrogen
bonding between the peptide backbones while
maintaining the allowed torsion angles
• The backbone of the polypeptide chain is
extended into a zigzag rather than helical
structure
 Beta Sheet
• The second (hence “beta”) recognizable
regular secondary structure in proteins is the
β sheet
• The amino acid residues of a β sheet, when
viewed edge-on, form a zigzag or pleated
pattern in which the R groups of adjacent
residues project in opposite directions
• Unlike the compact backbone of the α helix,
the peptide backbone of the β sheet is highly
extended
• Beta Sheet
The second (hence “beta”) recognizable regular secondary
structure in proteins is the β sheet
• The amino acid residues of a β sheet, when viewed edge-on, form a zigzag or pleated pattern in
which the R groups of adjacent residues project in opposite
directions
• Unlike the compact backbone of the a helix, the peptide backbone of the β sheet is highly extended
• But like the α helix, β sheets derive much of their stability from hydrogen
bonds between the carbonyl oxygens and amide hydrogens of
peptide bonds
• However, in contrast to the a helix, these bonds are formed with adjacent segments of the β sheet
(Figure 5–5)
• Interacting β sheets can be arranged either to form a parallel β sheet, in which the adjacent
segments of the polypeptide chain proceed in the same direction amino to carboxyl, or an
antiparallel sheet, in which they proceed in opposite
directions (Figure 5–5)
• Either configuration permits the maximum number of hydrogen bonds between segments, or
strands, of the sheet
• Most β sheets are not perfectly flat but tend to have a right-handed twist. Clusters of twisted strands
of β sheet, sometimes referred to as β barrels, form the core
of many globular proteins (Figure 5–6)
• Schematic diagrams represent β sheets as arrows that point in the amino to the
carboxyl terminal direction
• The zigzag polypeptide chains can be
arranged side by side to form a
structure resembling a series of pleats

• In this arrangement called a β sheet,

hydrogen bonds form between
adjacent segments of polypeptide
chain
• The β-pleated sheet is described as
parallel if the polypeptide strands run in
the same direction (as defined by their
amino and carboxy terminals) and anti-
parallel if they run in opposite direction

• Antiparallel strands are often the same

polypeptide chain folded back on itself,
with simple hairpin turns or long runs of
polypeptide chain connecting the strands
• The amino acid side chains of each
polypeptide strand alternate
between extending above and
below the plane of the β-sheet

• Most sheets are not perfectly flat

but tend to have a right-handed
twist
• The individual segments that form a β
sheet are usually nearby on the
polypeptide chain, but can also be
quite distant from each other in the
linear sequence of the polypeptide
and may even be in different
polypeptide chains
• Clusters of twisted strands of sheet
form the core of many globular
proteins
C. Nonrepetitive Secondary Structures
• α-Helices and β-pleated sheets are
patterns of regular structure with a
repeating element, the turn of α - helix
or β - pleat

• In contrast, bends, loops, and turns are

nonregular secondary structures that do
not have a repeating element
• Roughly half of the residues in a "typical" globular
protein reside in α helices and β sheets and half in
loops, turns, bends, and other extended
conformational features

• They are characterized by an abrupt change of

direction and are often found on the protein
surface

• For example, β-turns are short regions usually

involving four successive amino acid residues
 β Turns are common in Proteins
• In globular proteins, which have a
compact folded structure, nearly one-
third of the amino acid residues are in
turns or loops where the polypeptide
chain reverses direction

• These are the connecting elements that

link successive runs of α helix or β sheets
• The structure is a 180O turn involving
four amino acid residues, with the
carbonyl oxygen of the first residue
forming a hydrogen bond with the
amino-group hydrogen of the fourth
• The peptide groups of the central two
residues do not participate in any
inter-residue hydrogen bonding
• Glycine and Proline residues often
occur in β turns
• Glycine because it is small and
flexible
• Proline because peptide bonds
involving the imino nitrogen readily
assume the cis configuration, a form
that is particularly amenable to a
tight turn
• Loops or random coils are irregular in
conformation, and serve key biologic
roles

• While loops lack apparent structural

regularity, they are neither truly
disordered nor random
• They are stabilized through specific
hydrogen bonds dictated by the
primary sequence of the protein

• The nonregular coils, loops, and

other segments are usually more
flexible than the relatively rigid α-
helices and β-pleated sheets
Supersecondary structure
• A motif also called a supersecondary
structure or fold is simply a recognizable
folding pattern involving two or more
elements of secondary structure and
the connection(s) between them

• Structural motifs are intermediate

between secondary and tertiary
structures
• A motif can be very simple, such as
two elements of secondary structure
folded against each other, and
represent only a small part of a protein
• An example is a β-α-β loop
• A motif can also be a very elaborate
structure involving scores of protein
segments folded together, such as the
β barrel
The tertiary structure
• The tertiary structure of a protein is the
folding pattern of the secondary structural
elements into a three-dimensional
conformation
• It indicates, in three-dimensional space, how
secondary structural features—helices,
sheets, bends, turns, and loops—assemble
to form domains and how these domains
relate spatially to one another
 Domains in the Tertiary Structure
• The tertiary structure of large complex
proteins is often described in terms of
physically independent regions called
structural domains

• You can usually identify domains from

visual examination of a three-
dimensional figure of a protein
• Each domain is formed from a
continuous sequence of amino acids
in the polypeptide chain that are
folded into a three-dimensional
structure independently of the rest of
the protein

• Two domains are connected through

a simpler structure like a loop
• The structural features of each domain
can be discussed independently of
another domain in the same protein,
and the structural features of one
domain may not match that of other
domains in the same protein
• A domain is a section of protein
structure sufficient to perform a
particular chemical or physical task such
as binding of a substrate or other ligand
Quaternary structure
• Some proteins contain two or
more separate polypeptide chains,
or subunits, which may be
identical or different
• The arrangement of these protein
subunits in three-dimensional
complexes constitutes quaternary
structure
• Many proteins have multiple polypeptide
subunits (from two to hundreds)
• The association of polypeptide chains
can serve a variety of functions
• A multisubunit protein is also referred to
as a multimer, with the prefixes “homo”
or “hetero” used to describe identical or
different subunits, respectively
• A multimer with just a few subunits is
often called an oligomer
• If a multimer has nonidentical subunits,
the overall structure of the protein can be
asymmetric and quite complicated

• However, most multimers have identical

subunits or repeating groups of
nonidentical subunits, usually in
symmetric arrangements

• The repeating structural unit in such a

• Insulin is composed of two nonidentical
polypeptide chains attached to each
other through disulfide bonds between
the chains

• The subunits of globular proteins are

generally not held together by disulfide
bonds, but regions of the same chain may
be connected by disulfide bonds that
form as the chain folds
• Insulin actually fits this generalization because
it is synthesized as a single polypeptide chain,
which forms the disulfide bonds

• Subsequently, a proteolytic enzyme in

secretory vesicles clips the polypeptide chain
into two nonidentical subunits

• Generally, each subunit of most protomers

and oligomers is synthesized as a separate
polypeptide chain
 Protein Folding
• Proteins are dynamic molecules that can fold
into their functionally competent conformation
in milliseconds, and sometimes can refold if their
conformation becomes disrupted, or denatured
• How is this remarkable process of folding
achieved?
• Folding into the native state does not involve a
haphazard search of all possible structures
• Denatured proteins are not just random coils
The Native Conformation of a Protein Is
Thermodynamically Favored
• The number of potential conformations of even a
relatively small—15-kDa—polypeptide is
unbelievably vast
• Proteins are guided through this vast labyrinth of
possibilities by thermodynamics
• Since the biologically relevant—or native—
conformation of a protein generally is that which
is most energetically favored, knowledge of the
native conformation is specified in the primary
sequence
• However, if one were to wait for a
polypeptide to find its native
conformation by random
exploration of all possible
conformations, the process would
require billions of years to complete
• Clearly, protein folding in cells takes
place in a more orderly and guided
fashion
• Protein folding generally occurs
via a stepwise process
• In the first stage, as the newly
synthesized polypeptide emerges
from the ribosome, short
segments fold into secondary
structural units that provide local
regions of organized structure
• Then, each element of secondary or
super-secondary structure facilitates
proper folding by directing the folding
process toward the native conformation
and away from unproductive
alternatives

• For oligomeric proteins, individual

protomers tend to fold before they
associate with other subunits
 Auxiliary Proteins Assist Folding
• Under appropriate laboratory
conditions, many proteins will
spontaneously refold after being
denatured (i.e., unfolded) by
treatment with acid or base,
chemotropic agents, or detergents
• However, refolding under these
conditions is slow—minutes to hours
• A loss of three-dimensional structure
sufficient to cause loss of function is
called denaturation
• The denatured state does not
necessarily equate with complete
unfolding of the protein and
randomization of conformation
• Under most conditions, denatured
proteins exist in a set of partially folded
state
• Certain proteins denatured by heat,
extremes of pH, or denaturing reagents
will regain their native structure and their
biological activity if returned to
conditions in which the native
conformation is stable

• This process is called renaturation

• A classic example is the denaturation and

• Purified ribonuclease A denatures
completely in a concentrated urea solution
in the presence of a reducing agent
• Denaturation of ribonuclease is
accompanied by a complete loss of catalytic
activity
• When the urea and the reducing agent are
removed, the randomly coiled, denatured
ribonuclease spontaneously refolds into its
correct tertiary structure, with full
restoration of its catalytic activity
• Moreover, some proteins fail to
spontaneously refold in vitro, often
forming insoluble aggregates,
disordered complexes of unfolded or
partially folded polypeptides held
together by hydrophobic interactions

• Cells employ auxiliary proteins to speed

the process of folding and to guide it
toward a productive conclusion
• Chaperone proteins participate in the folding
of over half of mammalian proteins
• The hsp70 (70-kDa heat shock protein) family
of chaperones binds short sequences of
hydrophobic amino acids in newly synthesized
polypeptides
• Chaperones prevent aggregation, thus
providing an opportunity for the formation of
appropriate secondary structural elements
and their subsequent coalescence into a
molten globule
• The hsp60 family of chaperones, sometimes
called chaperonins, differ in sequence and
structure from hsp70 and its homologs
• Hsp60 acts later in the folding process,
often together with an hsp70 chaperone
• The central cavity of the donut-shaped
hsp60 chaperone provides a sheltered
environment in which a polypeptide can fold
until all hydrophobic regions are buried in its
interior, eliminating aggregation
PROTEIN MISFOLDING

• Protein folding is a complex process

that can sometimes result in
improperly folded molecules

• These misfolded proteins are usually

tagged and degraded within the cell
• However, this quality control system is
not perfect, and intracellular or
extracellular aggregates of misfolded
proteins can accumulate, particularly as
individuals age

• Deposits of these misfolded proteins

are associated with a number of
diseases
 Amyloid disease
• Misfolding of
proteins may
occur
spontaneously,
or be caused by
a mutation in a
particular gene,
which then
produces an
altered protein
 Amyloid disease
• In addition, some
apparently normal
proteins can, after
abnormal proteolytic
cleavage, take on a
unique conformational
state that leads to the
formation of long,
fibrillar protein
assemblies consisting
of β-pleated sheets
• Accumulation of
these insoluble,
spontaneously
aggregating proteins,
called amyloids, has
been implicated in
many degenerative
diseases—
particularly in the
age-related
neurodegenerative
disorder, Alzheimer
disease
• The dominant
component of
the amyloid
plaque that
accumulates in
Alzheimer
disease is
amyloid β (Aβ),
a peptide
containing 40–
42 amino acid
residues
• X-ray
crystallography
demonstrates
a characteristic
β-pleated
sheet
conformation
in
nonbranching
fibrils
• This peptide, when aggregated in a β-pleated
sheet configuration, is neurotoxic, and is the
central pathogenic event leading to the
cognitive impairment characteristic of the
disease

• The Aβ that is deposited in the brain in

Alzheimer disease is derived by proteolytic
cleavages from the larger amyloid precursor
protein—a single transmembrane protein
expressed on the cell surface in the brain and
other tissues
• The Aβ peptides aggregate, generating
the amyloid that is found in the brain
parenchyma and around blood vessels

• Most cases of Alzheimer disease are not

genetically based, although at least 5–
10% of cases are familial
• A second biologic factor involved in the
development of Alzheimer disease is the
accumulation of neurofibrillary tangles inside
neurons

• A key component of these tangled fibers is an

abnormal form of the tau (τ) protein, which in
its healthy version helps in the assembly of
the microtubular structure

• The defective τ, however, appears to block the

Prion disease

• The prion protein (PrP) has been

strongly implicated as the causative
agent of transmissible spongiform
encephalopathies (TSEs), including
Creutzfeldt-Jakob disease in humans,
scrapie in sheep, and bovine spongiform
encephalopathy in cattle (popularly
called “mad cow disease”)
• After an extensive series of purification
procedures, scientists were astonished
to find that the infectivity of the agent
causing scrapie in sheep was associated
with a single protein species that was
not complexed with detectable nucleic
acid

• This infectious protein is designated

PrPSc (Sc = scrapie)
• It is highly
resistant to
proteolytic
degradation, and
tends to form
insoluble
aggregates of
fibrils, similar to
the amyloid
found in some
other diseases of
the brain
• A noninfectious
form of PrPC (C =
cellular), encoded
by the same gene
as the infectious
agent, is present
in normal
mammalian
brains on the
surface of
neurons and glial
cells
• Thus, PrPC is a host protein

• No primary structure differences or

alternate posttranslational modifications
have been found between the normal
and the infectious forms of the protein

• The key to becoming infectious

apparently lies in changes in the three-
dimensional conformation of PrPC
• The infective
agent is thus an
altered version
of a normal
protein, which
acts as a
“template” for
converting the
normal protein
to the
pathogenic
conformation
• It has been observed that a number of α-helices
present in noninfectious PrPC are replaced by β-
sheets in the infectious form

• It is presumably this conformational difference

that confers relative resistance to proteolytic
degradation of infectious prions, and permits
them to be distinguished from the normal PrPC
in infected tissue

• The TSEs are invariably fatal, and no treatment is

currently available that can alter this outcome.
• In considering higher levels of
structure, it is useful to classify
proteins into two major groups:
– fibrous proteins, with polypeptide
chains arranged in long strands or
sheets
–globular proteins, with polypeptide
chains folded into a spherical or
globular shape
• Fibrous proteins usually consist largely of a
single type of secondary structure, and their
tertiary structure is relatively simple
• Globular proteins often contain several types
of secondary structure
• The two groups also differ functionally:
– the structures that provide support, shape, and
external protection to vertebrates are made of
fibrous proteins
– Whereas most enzymes and regulatory proteins
are globular proteins
• Fibrous proteins are adapted for
a Structural function and possess
axial ratios (the ratio of their
shortest to longest dimensions)
of 10 or more
• Fibrous proteins share
properties that give strength and
or flexibility to the structures in
which they occur
• All fibrous proteins are
insoluble in water, a property
conferred by a high
concentration of hydrophobic
amino acid residues both in
the interior of the protein and
on its surface
• Tertiary Structure of Fibrous
Protein collagen, nicely illustrates
the relationship between protein
structure and biological function

• In this case, the fundamental

structural unit is a simple
repeating element of secondary
structure
• Collagen has evolved to provide
strength
• It is found in connective tissue such
as tendons, cartilage, the organic
matrix of bone, and the cornea of
the eye
• Most abundant protein in the
body- 25% of the body protein mass
• The collagen super family of proteins
includes more than 20 collagen types

• Collagens can be organized into three

groups based on their location and
functions in the body
Fibril forming collagens
–Type I, II, and III are the fibrillar
collagen
• Type I are found in skin, bone,
tendons, blood vessels, and cornea
• Type II are found in cartilages, inter
vertebral discs, and vitreous body
• Type III in blood vessels and fetal
skin
• Types I, II, and III are the fibrillar
collagens, and have the rope-like
structure for a typical collagen
molecule

• Type I collagen fibers are found in

supporting elements of high tensile
strength (for example, tendon and
cornea)
• Fibers formed from type II collagen
molecules are restricted to
cartilaginous structures

• The fibers derived from type III

collagen are prevalent in more
distensible tissues, such as blood
vessels
• In the electron microscope, these linear
polymers of fibrils have characteristic banding
patterns, reflecting the regular staggered
packing of the individual collagen molecules
in the fibril
 Net work forming collagens
• They form a three dimensional
mesh, rather than distinct fibrils
–Types IV and VII are included in
this group
• Type IV present in basement
membranes
• Type VII present beneath
epithelia
Electron micrograph of a • Types IV and VII form a
polygonal network formed by
association of collagen type IV three-dimensional
monomers. mesh, rather than
distinct fibrils

• For example, type IV

molecules assemble
into a sheet or
meshwork that
constitutes a major
part of basement
membranes
 Fibril associated
• Type IX and XII bind to the surface of
collagen fibrils, linking these fibrils to
one another and to other components
in the extracellular matrix
–Type IX is present in cartilage
–Type XII is present in tendons,
ligaments, and some other tissues
• 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
• A typical collagen molecule is a long
rigid right handed triple helical
structure
• Each of the polypeptides in the triple helix
is known as α chain(different from α-helix)
• Each α chain has three amino acids per
turn and is a left handed helix
• The three polypeptide α chains are
held together by hydrogen bonds
between the chains
• Variations in the amino acid
sequence of the α chains result in
structural components that are
about the same size (approx. 1000
amino acid long), but with different
properties
 Structure of collagen
• Collagen is rich in proline and glycine
– Glycine – 35%
– Alanine – 11%
– Proline and hydroxyproline – 21%
• The glycine residues are part of a repeating
sequence, -Gly-X-Y-,
– X is frequently proline
– Y is often hydroxyproline (but can be hydroxylysine)
• Proline facilitates the formation of the
helical conformation of each α-chain
because its ring structure causes "kinks"
in the peptide chain
• 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
• Collagen contains hydroxyproline and
hydroxylysine which are not present in
most other proteins
• These residues result from hydroxylation
of some of the proline and lysine residues
as a post translational modification
• Hydroxyproline is important in stabilising
the triple helical structure by maximizing
inter chain hydrogen bonding
• 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
Biosynthesis of collagen

• The polypeptide precursors of the collagen

molecule are formed in fibroblasts (or in the
related osteoblasts of bone and
chondroblasts of cartilage), and are secreted
into the extracellular matrix

• After enzymic modification, the mature

collagen monomers aggregate and become
cross-linked to form collagen fibrils
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(pre-pro-collagen) contain a special
amino acid sequence at their N-terminal
ends
• This acts as a signal that the polypeptide
being synthesized is destined to leave the
cell
• The signal sequence facilitates the
binding of ribosomes to the rough
endoplasmic reticulum (RER), and
directs the passage of the polypeptide
chain into the cisternae of the RER

• The signal sequence is rapidly cleaved in

the endoplasmic reticulum to yield a
precursor of collagen called a pro-α-
chain
Hydroxylation:
• The pro α chains are processed by a
number of 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 hydroxylation
reactions require
molecular oxygen
and the reducing
agent vitamin C
(ascorbic acid),
without which the
hydroxylating
enzymes, prolyl
hydroxylase and
hydroxylase, are
unable to function
• In the case of ascorbic acid
deficiency (therefore, a lack of
prolyl and lysyl hydroxylation),
collagen fibers cannot be cross-
linked, greatly decreasing the tensile
strength of the assembled fiber

• One 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 of
blood due to
capillary fragility
 Glycosylation:
• Glucosyl and galactosyl transferase attach
glucosyl or galactosyl residues to the hydroxyl
groups of specific hydroxylysyl residues
Assembly and secretion:
• After hydroxylation and glycosylation, pro α
chains form procollagen, a precursor of
collagen that has a central region of triple
helix flanked by the non-helical amino- and
carboxy- 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 are translocated to
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
 Extracellular cleavage of procollagen molecules
• After their release, the procollagen molecules are
cleaved by N- and C-pro-collagen peptidases, which
remove the terminal propeptides, releasing triple-
helical collagen molecules
 Formation of collagen fibrils:
• Individual collagen molecules spontaneously associate
to form fibrils by forming an ordered, overlapping,
parallel array, with adjacent collagen molecules, each
overlapping its neighbor by a length approximately
three-quarters of a molecule
Cross-link formation:
• The fibrillar array of
collagen molecules
serves as a
substrate for lysl
oxidase (copper is a
co factor)
• This extracellular
enzyme oxidatively
deaminates some of
the lysyl and
hydroxylysyl
residues in collagen
• The aldehydes that
result (allysine and
hydroxyallysine)
can condense with
lysyl or hydroxylysyl
residues in
neighboring
collagen molecules
to form covalent
cross-links and thus
mature collagen
• This cross-linking is essential for
achieving the tensile strength
necessary for the proper functioning
of connective tissue
• Therefore, any mutation that
interferes with the ability of
collagen to form cross-linked fibrils
almost certainly affects the stability
of the collagen
• 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 to their constituent amino
acids
 Nutritional & Genetic Disorders
Can Impair Collagen Maturation
• The complex series of events in
collagen maturation provide a
model that illustrates the
biologic consequences of
incomplete polypeptide
maturation
• The best-known defect in collagen
biosynthesis is scurvy, a result of a
dietary deficiency of vitamin C required
by prolyl and lysyl hydroxylases.
• The resulting deficit in the number of
hydroxyproline and hydroxylysine
residues undermines the conformational
stability of collagen fibers, leading to
bleeding gums, swelling joints, poor
wound healing, and ultimately death
• Menkes' syndrome,
characterized by kinky hair and
growth retardation, reflects a
dietary deficiency of the copper
required by lysyl oxidase, which
catalyzes a key step in formation
of the covalent cross-links that
strengthen collagen fibers
• Ehlers-Danlos syndrome (formerly called
Cutis hyperelastica), comprises a group of
inherited disorders whose principal
clinical features are hyperextensibility of
the skin, abnormal tissue fragility, and
increased joint mobility

• The clinical picture is variable, reflecting

underlying extensive genetic
heterogeneity
• A number of forms of the disease
caused by genetic defects in proteins
involved in the synthesis and assembly
of collagens type I, III and V are known,
and since 1997 the Villefranche
classification of 6 subtypes based on
their phenotype and molecular defects
has been used
• The hypermobility, vascular and classical
subtypes are more common, while the
other three, kyphoscoliosis, arthrochalasis
and dermatosparaxis are extremely rare

• The vascular subtype is the most serious

because of its tendency for spontaneous
rupture of arteries or the bowel, reflecting
abnormalities in type III collagen
• Patients with kyphoscoliosis exhibit
progressive curvature of the spine
(scoliosis) and a tendency to ocular
rupture due to a deficiency of lysyl
hydroxylase

• A deficiency of procollagen N-proteinase,

causing formation of abnormal thin,
irregular collagen fibrils, results in
dermatosparaxis, manifested by markedly
fragile and sagging skin
Osteogenesis imperfecta (OI)
• This disease, known as brittle bone syndrome, is also
a heterogeneous group of inherited disorders
distinguished by bones that easily bend and fracture
• Retarded wound healing and a rotated and twisted
spine leading to a "humped-back" appearance are
common features of the disease
• Type I osteogenesis imperfecta is called osteogenesis
imperfecta tarda
• This disease presents in early infancy with fractures
secondary to minor trauma, and may be suspected if
prenatal ultrasound detects bowing or fractures of
long bones
• Type II osteogenesis imperfecta congenita, is more
severe, and patients die in utero or in the neonatal
period of pulmonary hypoplasia
• Most patients with severe OI have mutations in the
gene for either the pro1- or pro 2- α- chains of type
I collagen
• The most common mutations cause the
substitution of single amino acids with bulky side
chains for the glycine residues that appear as every
third amino acid in the triple helix
• The structurally abnormal pro α chains can prevent
folding of the protein into a triple-helical
conformation
• Chondrodysplasias are caused by
mutations in genes encoding type ii
collagen & fibroblast growth factor
receptors

• Chondrodysplasias are a mixed group of

hereditary disorders affecting cartilage

• They are manifested by short-limbed

dwarfism and numerous skeletal
deformities
• A number of them are due to a variety
of mutations, leading to abnormal
forms of type II collagen

• One example is the Stickler syndrome,

manifested by degeneration of the joint
cartilage and of the vitreous body of the
eye
• The Alport syndrome (hereditary nephritis)
is the designation applied to a number of
genetic disorders (both X-linked and
autosomal) affecting type IV collagen, a
network-like collagen which forms part of
the structure of the basement membranes
of the renal glomeruli, inner ear and eye

• Mutations in several genes encoding type IV

collagen fibers have been demonstrated
•The main presenting sign is hematuria,
accompanied by ocular lesions and
hearing loss, and patients may eventually
develop endstage renal disease

•Electron microscopy reveals

characteristic abnormalities of the
structure of the basement membrane
and lamina densa
• In epidermolysis bullosa, the skin breaks
and blisters as a result of minor trauma

• The dystrophic form is due to mutations,

affecting the structure of type VII
collagen

• This collagen forms delicate fibrils that

anchor the basal lamina to collagen
fibrils in the dermis
• These anchoring fibrils have been
shown to be markedly reduced in this
form of the disease, probably resulting
in the blistering

• Epidermolysis bullosa simplex, another

variant, is due to mutations in keratin 5
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

• They can be stretched to several times their normal

length, but recoil to their original shape when the
stretching force is relaxed

• Elastic fibers composed of elastin and glycoprotein

microfibrils are found in the lungs, the walls of large
arteries, and elastic ligaments
• Smaller quantities of elastin are also
found in skin, ear cartilage, and
several other tissues

• In contrast to collagen, there

appears to be only one genetic type
of elastin, although variants arise by
alternative splicing
• Elastin is synthesized as a soluble
monomer of ∼70 kDa called
tropoelastin, which is a linear
polypeptide composed of about 700
amino acids that are primarily small and
nonpolar (e.g., glycine, alanine, and
valine)

• 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 prolines of tropoelastin

are hydroxylated to hydroxyproline
by prolyl hydroxylase, though
hydroxylysine and glycosylated
hydroxylysine are not present
• Unlike collagen, tropoelastin is not
synthesized in a pro-form with
extension peptides

• Furthermore, elastin does not

contain repeat Gly-X-Y sequences,
triple helical structure, or
carbohydrate moieties
• After secretion
from the cell,
certain lysyl
residues of
tropoelastin are
oxidatively
deaminated to
aldehydes by lysyl
oxidase, the same
enzyme involved in
this process in
collagen
• However, the major
cross-links formed in
elastin are the
desmosines, which
result from the
condensation of
three of these lysine-
derived aldehydes
with an unmodified
lysine to form a
tetrafunctional cross-
link unique to elastin
• Once cross-linked in its mature,
extracellular form, elastin is highly
insoluble and extremely stable and
has a very low-turnover rate
• Elastin exhibits a variety of random
coil conformations that permit the
protein to stretch and subsequently
recoil during the performance of its
physiologic functions
• Deletions in the elastin gene have been
found in approximately 90% of subjects
with the Williams- Beuren syndrome, a
developmental disorder affecting
connective tissue and the central nervous
system
• Fragmentation or, alternatively, a
decrease of elastin is found in conditions
such as pulmonary emphysema, cutis
laxa, and aging of the skin
• Supravalvular aortic stenosis

• SVAS results from an insufficiency of

elastin in the vessel wall, leading to a
narrowing of the large elastic arteries

• Mutations, by affecting synthesis of

elastin, probably play a causative role in
the supravalvular aortic stenosis often
found in this condition
• Current theory suggests that the
levels of elastin in the vessel walls
may regulate the number of smooth
muscle cell rings that encircle the
vessel

• If the levels of elastin are low, smooth

muscle hypertrophy results, leading to
a narrowing and stenosis of the artery
• FIBRILLINS ARE STRUCTURAL
COMPONENTS OF MICROFIBRILS

• Microfibrils are fine fiber-like strands 10 to

12 nm in diameter which provide a scaffold
for the deposition of elastin in the ECM

• Fibrillins are large glycoproteins (about 350

kDa) that are major structural component
of these fibers
• They are secreted (subsequent to a
proteolytic cleavage) into the ECM by
fibroblasts and become incorporated
into the insoluble microfibrils

• Fibrillin-1 is the main fibrillin present,

but fibrillins-2 and -3 have also been
identified, and fibrillin-2 is thought to
be important in deposition of
microfibrils early in development
• Other proteins including microfibril-
associated proteins (MAGPs are also
associated with microfibrils

• Fibrillin microfibrils are found in elastic

fibers and also in elastin-free bundles in
the eye, kidney, and tendons
• Marfan Syndrome Is Caused by Mutations
in the Gene for Fibrillin-1

• Marfan syndrome is a relatively prevalent

inherited disease affecting connective tissue

• It is inherited as an autosomal dominant

trait

• Abraham Lincoln may have had this

condition
• It affects:

– the eyes (eg, causing dislocation of the lens,

known as ectopia lentis),

– the skeletal system (most patients are tall and

exhibit long digits [arachnodactyly] and
hyperextensibility of the joints),

– and the cardiovascular system (eg, causing

weakness of the aortic media, leading to
dilation of the ascending aorta)
• Most cases are caused by mutations in
the gene (on chromosome 15) for
fibrillin-1

• This results in abnormal fibrillin and/or

lower amounts being deposited in the
ECM
• Since the cytokine TGF-β normally binds
to fibrillin-1, decreased binding in Marfan
syndrome causes disturbances in TGF-β
signaling which contribute to the
pathology found in the condition
• This could potentially lead to the
development of therapies for the
condition using drugs that antagonize
TGF-β (eg, the angiotensin II receptor
antagonist, Losartan)
• Mutations in the fibrillin-1 gene have
also been identified recently as the
cause of acromicric dysplasia and
geleophysic dysplasia, which are
characterized by short stature, skin
thickening, and stiff joints

• Congenital contractural arachnodactyly

is associated with a mutation in the
gene for fibrillin-2
 Plasma Proteins
• The concentration of total protein in
human plasma is approximately
7.0-7.5g/dl
• The proteins of the plasma are actually
a complex mixture that includes not
only simple proteins but also
conjugated proteins such as
glycoproteins and various types of
lipoproteins
• The concentration of proteins in
plasma is important in determining the
distribution of fluid between blood
and tissues

• The osmotic pressure (oncotic

pressure) exerted by the plasma
proteins is responsible for attracting
water back into the circulation from
the tissues
• In hypoprotinemia fluid is
not attracted back into the
intravascular compartment
and accumulates in the
extravascular tissue spaces, a
condition known as edema
• Because of the relative ease with
which they can be obtained,
plasma proteins have been
studied extensively
• One can separate plasma
proteins into three major groups
– fibrinogen, albumin, and
globulins
• The most common method
of analyzing plasma proteins
is by electrophoresis
• It`s use permits resolution,
after staining, into five
bands, designated albumin,
α1, α2, β and γ
• Most plasma proteins are
synthesized in the liver
• However the γ globulins are
synthesized in plasma cells
and certain plasma proteins
are synthesized in other
tissues such as endothelial
cells
• Most plasma proteins are
synthesized as preproteins and
contain amino terminal signal
peptides
• They usually undergo various
posttranslational modifications
as they travel through the cell
• Most plasma proteins are
glycoproteins
• They generally contain either N-
or O- linked oligosacharrides
which have different functions
• Albumin is the major exception
and does not contain sugar
residues
• Each plasma protein has a
characteristic half life in circulation

• In certain diseases, half life of a

plasma protein may be markedly
altered
• The levels of certain plasma
proteins increase during acute
inflammatory states or secondary to
certain types of tissue damage
• These proteins are called acute
phase proteins and include C-
reactive protein, α1-antitrypsin,
haptoglobin, α1- acid glycoprotein,
and fibrinogen
• The elevations of these proteins vary
from as little as 50% to as much as a
1000 fold in the case of CRP
• Their levels are also usually elevated
during chronic inflammatory states and
in patients with cancer
• These proteins are believed to play a
role in the body’s response to
inflammation
 Functions of plasma proteins
• Antiproteases
– Antichymotrypsin
–α1 Antitrypsin
–Antithrombin

• Blood clotting
–Various coagulation factors
–fibrinogen
• Enzymes
– Functions in blood e.g., cholinestrase
– Leakage from cells or tissues e.g.
aminotransferases

• Hormones
– Erythropoitin

• Immune defense
– Immunoglobulins
– Complement proteins
– β2 microglobulins
• Inflammatory responses
– Acute phase proteins e.g. CRP

• Oncofetal
– α1 Fetoprotein

• Transport or Binding proteins

– Albumin binds various ligands e.g. bilirubin, FFA,
ions (Ca2+), metals (Cu2+ , Zn2+), methane,
steroids, other hormones and a variety of drugs
– Cerruloplasmin binds Cu
– Corticosteroid Binding Globulin (transcortin) bind
cortisol
– Haptoglobin binds extracorpuscular
haemoglobin
– Lipoproteins (Chylomicrons, VLDL, LDL,
HDL) transport lipids
– Hemopexin binds heme
– Retinol Binding Protein binds Retinol
– Sex Hormone Binding Globulin binds
testosterone, estradiol
– Thyroid Binding Globulin binds T4, T3
– Transferrin transports iron
 Albumin
• Albumin is the major protein of human
plasma and makes up approximately
60% of the total plasma protein
• It’s molecular weight is 69 kDa and it’s
concentration in plasma is 3.4 to 4.7
g/dl
• About 40% of albumin is present in the
plasma and the other 60% in the extra
cellular space
• The liver produces about 12g of
albumin per day, representing
about 25% of total hepatic protein
synthesis and half of it’s secreted
protein

• Albumin is initially synthesized as a

preproprotein
• It’s signal peptide is removed as
it passes through RER

• A hexapeptide at the resulting

amino terminal is subsequently
cleaved further along the
secretory pathway
• The mature human albumin
consists of one polypeptide chain
of 585 amino acids and contains 17
disulfide bonds

• The synthesis of albumin is

depressed in a variety of diseases,
particularly those of the liver
• The plasma of patients with liver
disease often shows a decreased
albumin : globulin ratio

• The synthesis of albumin

decreases relatively early in
conditions of protein
malnutrition, such as
kwashiorkor
• Because of it's relatively low molecular
weight and high concentration,
albumin is responsible for 75-80% of
the osmotic pressure of the human
plasma

• Electrophoretic studies have shown

that the plasma of certain humans
lacks albumin and are said to exhibit
analbuminemia
• Patients with this condition show only
moderate edema, probably by an increase
in the amounts of other plasma proteins to
compensate for the lack of albumin

• Another important function of albumin is

it’s ability to bind various ligands, including
free fatty acids, calcium, certain steroid
hormones, bilirubin, drugs like
sulfonamides, penicillin G, dicumarol, and
aspirin and some of the plasma tryptophan
• Haptoglobin is a glycoprotein that binds
extracorpuscular hemoglobin in a tight
noncovalent complex (Hb-Hp)
• The amount of haptoglobin in human
plasma ranges from 40mg to 180mg of
Hb binding capacity
• Approximately 10% of Hb that is
degraded each day is released into the
circulation and is thus extracorpuscular
• The other 90% is present in old damaged
RBCs, which are degraded by cells of the
histiocytic system
• The molecular mass of the Hb is 65kDa, and
that of the Hp is approximately 90kDa
• Thus the combined mass of Hb-Hp complex is
155kDa which is too large to pass through the
kidney glomeruli
• Free Hb passes through the glomerulus of the
kidney, enters the tubules, and precipitates
• The function of the Hp thus appears to
prevent loss of free Hb into the kidney,
which not only protects the kidney
glomeruli but also conserves valuable
iron which would otherwise be lost
• The levels of Hp in human plasma vary
and are of some diagnostic use
• Low levels of Hp are found in patients
with hemolytic anemias
• This is explained by the fact that
whereas half life of Hp is 5 days, the half
life of Hp-Hb complex is reduced to
about 90 minutes, the complex being
rapidly removed from the plasma by
hepatocytes

• Hp is an acute phase protein and it’s

plasma levels are elevated in a variety
of inflammatory states
• Transferrin (Tf) is a plasma protein that
plays a central role in transporting iron
around the body to sites where it is needed

• Tf is a β1 globulin with a molecular mass of

76kDa
• It is a glycoprotein and is synthesized in the
liver
• About 20 polymorphic forms of Tf have
been identified
• It plays a central role in the metabolism
of iron because it transports iron
(2molecules of Fe3+ per mole of Tf) in
the circulation to sites where it is
required

• Approximately 200 billion RBCs (about

20ml) are catabolized per day, releasing
about 25mg of iron into the body - most
of which will be transported by Tf
• Cerruloplasmin (about 160 kDa)is an α2-
globulin

• It has a blue color because of it’s high

copper content and carries 90% of the
copper present in plasma

• Each molecule of cerruloplasmin binds

six atoms of copper very tightly, so that
the copper is not readily exchangeable
• Albumin carries the other 10% of the
plasma copper but binds the metal
less tightly than does cerruloplasmin

• Albumin thus donates it’s copper to

tissues more readily than
cerruloplasmin and apears to be
more important than cerruloplasmin
in copper transport in the human
body
• Cerruloplasmin exhibits a copper
dependant oxidase activity, and
is involved in the oxidation of Fe2+
to Fe 3+

• The amount of cerruloplasmin in

plasma is decreased in liver
disease
• In particular, low levels are found
in Wilson Disease, a disease due
to abnormal metabolism of
copper in the human body

• Menkes Disease is another

condition involving abnormal
copper metabolism
• α1 Antiproteinase (about 52kDa)
was formerly called α1 Antitrypsin

• It is a single chain protein, contains

three oligosacharride chains, and is
the major component (>90%) of the
α1 fraction of the human plasma
• It is synthesized by hepatocytes
and macrophages and is the
principal serine protease
inhibitor of human plasma

• It inhibits trypsin, elastase and

certain other proteases by
forming complexes with them
• Most of the α1 antitrypsin is
synthesized by the liver, but some
is synthesized by several tissues,
including monocytes and alveolar
macrophages, which may be
important in preventing local
tissue injury by elastase
Role of α1 antitrypsin in elastin degradation

• α1- antitrypsin 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

• In the normal lung, the alveoli are chronically

exposed to low levels of neutrophil elastase
released from activated and degenerating
neutrophils
Role of α1- antitrypsin in the lungs:

• This proteolytic activity can destroy the

elastin in alveolar walls if unopposed by
the inhibitory action of α1- antitrypsin,
the most important inhibitor of
neutrophil elastase
• Because lung tissue cannot regenerate,
emphysema results from the destruction
of the connective tissue of alveolar walls
• In the United States, approximately two to
five percent of patients with emphysema are
predisposed to the disease by inherited
defects in α1- antitrypsin gene

• A number of different mutations in the gene

are known to cause a deficiency of this
protein, but one single purine base mutation
(resulting in the substitution of lysine for
glutamic acid at position 342 of the protein) is
clinically the most widespread
• An individual must inherit two
abnormal alleles to be at risk for the
development of emphysema

• In a heterozygote, with one normal

and one defective gene, the levels
of α1- antitrypsin are sufficient to
protect the alveoli from damage
• A specific α1-AT methionine is
required for the binding of the
inhibitor to its target proteases

• Smoking causes the oxidation and

subsequent inactivation of that
methionine residue, thereby
rendering the inhibitor powerless to
neutralize elastase
• Smokers with α1-AT deficiency, therefore, have
a considerably elevated rate of lung
destruction and a poorer survival rate than
non smokers with the deficiency
• The deficiency of elastase inhibitor can be
reversed by weekly intravenous administration
of α1-AT

• The α1-AT diffuses from the blood into the

lung, where it reaches therapeutic levels in the
fluid surrounding the lung epithelial cells
• The immunoglobulins are
synthesized mainly in plasma
cells, that are specialized cells of
B cell lineage that synthesize and
secrete immunoglobulins into
the plasma in response to
exposure to a variety of antigens
• Immunoglobulins
contain a
minimum of two
identical light
(L)and two
identical heavy
(H) chains, held
together as a
tetramer (L2H2)
by disulfide
bonds
• The structure of IgG is Y shaped, with
antigen binding occurring at both tips of
the Y
• Each chain
can be
divided
conceptually
into specific
domains or
regions that
have
structural and
functional
significance
• The half of the
light chain (L)
towards the
carboxy terminal is
referred as the
constant region
(CL), while the
amino terminal
half is the variable
region (VL) of the
light chain
• Approximately one-
quarter of the heavy
chain (H) at the
amino terminal is
referred to as it’s
variable region (VH),
and the other three-
quarters of the
heavy chain are the
constant regions
(CH1, CH2, CH3) of
• The portion of
the
immunoglobulin
molecule that
binds the antigen
is formed by the
amino terminal
portions (variable
regions) of both
the H and L
chains, i.e. the VH
and VL domains
• Digestion of an
immunoglobulin by
the enzyme papain,
produces two
antigen binding
fragments (Fab) and
one crystallizable
fragment (Fc),
which is responsible
for functions of
immunoglobulins
other than binding
of antigens
• Because there are two Fab regions, IgG
molecules bind two molecules of antigen
and are termed divalent
• The site on
the antigen
to which an
antibody
binds is
termed an
antigenic
determinant,
or epitope
• The area in
which papain
cleaves the
immuno
globulin
molecule (the
region between
the CH1and CH2
domains) is
referred to as
the hinge
region
• The hinge region confers flexibility and
allows both Fab arms to move
independently, thus helping them to
bind antigenic sites that may be variable
distance apart

• The Fc and hinge regions differ in

different classes of antibodies but
overall model of antibody structure is
the same
• There are two general types of light
chains, kappa (κ) and lambda (λ), which
can be distinguished on the basis of
structural differences in their CL regions

• A given immunoglobulin molecule always

contains two κ and two λ chains, never a
mixture of κ and λ
• In humans κ chains are more frequent
than λ chains in immunoglobulins
• Five classes of H chains have been found
in humans, distinguished by differences in
their CH regions

• They are designated:

–γ,
–α,
–µ,
–δ,
–є
• The type of H chain thus determines the
class immunoglobulins
• There are thus five immunoglobulin
classes:
–IgG, having light chain γ
–IgA, having light chain α
–IgM, having light chain µ
–IgD, having light chain δ
–IgE, having light chain ε
• Some
immunoglobulins
such as IgG exist
only in the basic
monomeric
structure, while
others such as IgA
and IgM can exist in
higher order
polymers of two,
three (IgA), or five
(IgM)
• The major function of IgG
immunoglobulins are:
– Main antibody in secondary response
– Opsonizes bacteria making them easier to
phagocytose
– Fixes complement which enhances bacterial
killing
– Neutralizes bacterial toxins and viruses
– Crosses the placenta
• IgA :
–The only secreted antibody,
–prevents attachment of bacteria and
viruses to mucous membranes

• IgM :
–Produced in the primary response to
an antigen,
–and does not cross the placenta
• IgD :
–Functions are uncertain

• IgE :
–Mediates immediate hypersensitivity
by causing release of mediators from
mast cells upon exposure to antigen
(allergan)
–Defends against worm infestation
• Both over and under production of
immunoglobulins may result in
disease states
• Multiple myeloma is a neoplastic
condition in which there is either a
large increase of one particular
immunoglobulin or one particular
light chain (the latter termed a
Bence jones protein)
• Decreased production may be restricted to
a single class of immunoglobulin molecules
(e.g. IgA, or IgG) or may involve
underproduction of all classes of
immunoglobulins

• A severe reduction in synthesis of an

immunoglobulin class can result in a
serious immunodeficency disease – e.g.
aggamagloblinemia, in which production
of IgG is markedly affected
Extra cellular matrix

• Most mammalian cells are located in

tissues where they are surrounded by a
complex ECM often referred to as
“connective tissue,” which protects the
organs and also provides elasticity
where required (eg, in blood vessels,
lungs, and skin)
• The ECM contains three major classes of
biomolecules:

1. Structural proteins, for example, collagen,

elastin, and fibrillin,

2. certain specialized proteins such as

fibronectin and laminin, which form a
mesh of fibers that are embedded in

3. proteoglycans
• The ECM is not simply a glue that
holds cells together

• It also serves to keep cells from

moving to other locations and to
prevent large molecules and other
particles, such as microorganisms,
from reaching contiguous and
distant cells
• This confining property of the matrix is
medically important
• For example, infections spread, in part,
because the infectious agent alters the
“containing” capacity of the ECM

• Cancer cells that metastasize (migrate

to other tissues) can do so only by
altering the integrity of the matrix
• Diseases such as rheumatoid arthritis
(an autoimmune destruction of
articular and periarticular tissues) and
osteoarthritis (degenerative joint
disease often associated with aging)
involve damage to the functional
capacity of the matrix
• Genetic defects may cause components of the
matrix to be structurally and functionally
abnormal, resulting in connective tissue
disorders such as the

– Ehlers- Danlos syndrome (caused by a

number of mutations that affect specific
collagen genes)

– Marfan’s syndrome (a defect in the protein,

fibrillin)
• Alterations in the structural
characteristics of the matrix of the renal
glomerulus may allow proteins to be
excreted into the urine, an indication of
inexorable decline in renal function

• Deficiencies of lysosomal enzymes

involved in normal degradation of
molecules of the matrix result in diseases
such as the mucopolysaccharidoses
• FIBRONECTIN IS AN IMPORTANT
GLYCOPROTEIN INVOLVED IN CELL
ADHESION & MIGRATION

• Fibronectin is a major glycoprotein of

the extracellular matrix, also found in a
soluble form in plasma
• Fibronectin is also involved in cell
migration, as it provides a binding site
for cells and thus helps them to steer
their way through the ECM

• The amount of fibronectin around many

transformed cells is sharply reduced,
partly explaining their faulty interaction
with the ECM
• LAMININ IS A MAJOR PROTEIN COMPONENT
OF BASAL LAMINAS

• Basal laminas are specialized areas of the ECM

that surround epithelial and some other cells
(eg, muscle cells)

• Laminin (a glycoprotein of about 850 kDa and

70 nm length) consists of three distinct
elongated polypeptide chains (α, β, and γ
chains) linked together to form a complex,
elongated shape
• In basal laminas, laminin forms networks
which are attached to type IV collagen
networks
• The collagen interacts with laminin (rather
than directly with the cell surface), which in
turn interacts with integrins thus anchoring
the lamina to the cells
• In the renal glomerulus, the basal lamina
consists of two separate sheets of cells (one
endothelial and one epithelial), each disposed
on opposite sides of the lamina
• These three layers make up the glomerular
membrane
• This relatively thick basal lamina has an
important role in glomerular filtration,
regulating the passage of large molecules
(most plasma proteins) across the glomerulus
into the renal tubule
• The glomerular membrane allows small
molecules, such as inulin (5.2 kDa), to
pass through as easily as water

• On the other hand, only a small amount

of the protein albumin (69 kDa), the
major plasma protein, passes through
the normal glomerulus
• This is explained by two sets of facts:
1. The pores in the glomerular membrane
are large enough to allow molecules up
to about 8 nm to pass through
2. Albumin is smaller than this pore size,
but it is prevented from passing
through easily by the negative charges
of heparan sulfate and of certain sialic
acid-containing glycoproteins present in
the lamina
• These negative charges repel albumin
and most plasma proteins, which are
negatively charged at the pH of blood

• The normal structure of the glomerulus

may be severely damaged in certain
types of glomerulonephritis (eg, caused
by antibodies directed against various
components of the glomerular
membrane)
• This alters the pores and the amounts
and dispositions of the negatively
charged macromolecules referred to
above, and relatively massive amounts
of albumin (and of certain other plasma
proteins) can pass through into the
urine, resulting in severe albuminuria
PROTEOGLYCANS &
GLYCOSAMINOGLYCANS

• The Glycosaminoglycans Found in

Proteoglycans Are Built Up of
Repeating Disaccharides

• Proteoglycans are proteins that

contain covalently linked
• The proteins bound covalently to
glycosaminoglycans are called “core
proteins”

• The amount of carbohydrate in a

proteoglycan is usually much greater
than that found in a glycoprotein and
may comprise up to 95% of its weight
• Figure show
the general
structure of
one particular
proteoglycan,
aggrecan, the
major type
found in
cartilage
• It is very large
(about 2 ×
103 kDa),
with its
overall
structure
resembling
that of a
bottlebrush
• It contains a
long strand of
hyaluronic acid
(one type of
GAG) to which
link proteins
are attached
noncovalently
• In turn, the link
proteins
interact
noncovalently
with core
protein
molecules from
which chains of
other GAGs
(keratan sulfate
and chondroitin
sulfate in this
case) project
• There are at least seven GAGs: hyaluronic
acid (hyaluronan), chondroitin sulfate,
keratan sulfates I and II, heparin,
heparan sulfate, and dermatan sulfate

• GAGs are unbranched polysaccharides

made up of repeating disaccharides, one
component of which is always an amino
sugar (hence, the name GAG), either d-
glucosamine or d-galactosamine
• The other component of the repeating
disaccharide (except in the case of
keratan sulfate) is a uronic acid, either l-
glucuronic acid (GlcUA) or its 5′-epimer,
l-iduronic acid (IdUA)

• With the exception of hyaluronic acid,

all the GAGs contain sulfate groups,
either as O-esters or as N-sulfate (in
heparin and heparan sulfate)
• Hyaluronic acid is also exceptional
because it appears to exist as a
polysaccharide in the ECM, with no
covalent attachment to protein, as the
definition of a proteoglycan given above
specifies
• Both GAGs and proteoglycans have
proved difficult to work with, partly
because of their complexity

• However, since they are major

components of the ECM and have a
number of important biologic roles as
well as being involved in a number of
disease processes, interest in them has
increased greatly in recent years
• Proteoglycans Are Important in the Structural
Organization of the Extracellular Matrix

• Proteoglycans are found in every tissue of the

body, mainly in the ECM or “ground substance”

• There they are associated with each other and

also with the other major structural
components of the matrix, collagen and
elastin, in specific ways
• Some proteoglycans bind to collagen
and others to elastin

• These interactions are important in

determining the structural organization
of the matrix

• Some proteoglycans (eg, decorin) can

also bind growth factors, modulating
their effects on cells
• In addition, some of them interact with
certain adhesive proteins such as fibronectin
and laminin , also found in the matrix

• The GAGs present in the proteoglycans are

polyanions and hence bind polycations and
cations such as Na+ and K+

• This latter ability attracts water by osmotic

pressure into the extracellular matrix and
contributes to its turgor
• GAGs also gel at relatively low concentrations

• Because of the long extended nature of the

polysaccharide chains of GAGs and their ability to
gel, the proteoglycans can act as sieves, restricting
the passage of large macromolecules into the ECM
but allowing relatively free diffusion of small
molecules

• Again, because of their extended structures and

the huge macromolecular aggregates they often
form, they occupy a large volume of the matrix
relative to proteins
• Proteoglycans Are Associated With
Major Diseases & With Aging

• Hyaluronic acid may be important in

permitting tumor cells to migrate
through the ECM
• Tumor cells can induce fibroblasts to
synthesize greatly increased amounts of
this GAG, thereby perhaps facilitating
their own spread

• Some tumor cells have less heparan

sulfate at their surfaces, and this may
play a role in the lack of adhesiveness
that these cells display
• The intima of the arterial wall contains hyaluronic
acid and chondroitin sulfate, dermatan sulfate,
and heparan sulfate proteoglycans
• Of these proteoglycans, dermatan sulfate binds
plasma low-density lipoproteins
• In addition, dermatan sulfate appears to be the
major GAG synthesized by arterial smooth muscle
cells
• Because it is these cells that proliferate in
atherosclerotic lesions in arteries, dermatan
sulfate may play an important role in
development of the atherosclerotic plaque
• In various types of arthritis,
proteoglycans may act as autoantigens,
thus contributing to the pathologic
features of these conditions

• The amount of chondroitin sulfate in

cartilage diminishes with age, whereas
the amounts of keratan sulfate and
hyaluronic acid increase
• These changes may contribute to the
development of osteoarthritis, as may
increased activity of the enzyme
aggrecanase, which acts to degrade
Aggrecan

• Changes in the amounts of certain GAGs

in the skin are also observed with aging
and help to account for the characteristic
changes noted in this organ in the elderly
• In the past few years it has become
clear that in addition to their structural
role in the ECM, proteoglycans function
as signaling molecules which influence
cell behavior, and they are now believed
to play a part in diverse diseases such as
fibrosis, cardiovascular disease and
cancer
Functions of GAGS and proteoglycans
• In a globular protein, different
segments of the polypeptide chain
(or multiple polypeptide chains)
fold back on each other, generating
a more compact shape than is seen
in the fibrous proteins
• Globular proteins are compact,
roughly spherical molecules that
have axial ratios of not more than 3
• The folding also provides the
structural diversity necessary for
proteins to carry out a wide array of
biological functions
• Globular proteins include enzymes,
transport proteins, motor proteins,
regulatory proteins,
immunoglobulins, and proteins with
many other functions
• PROTEIN TURNOVER OCCURS IN ALL FORMS OF
LIFE
• The continuous degradation and synthesis of
cellular proteins occur in all forms of life
• Each day, humans turn over 1–2% of their total
body protein, principally muscle protein
• High rates of protein degradation occur in tissues
that are undergoing structural rearrangement,
for example, uterine tissue during pregnancy,
skeletal muscle in starvation, and tadpole tail
tissue during metamorphosis
• Of the liberated amino acids,
approximately 75% are reutilized

• Since excess free amino acids are

not stored, those not immediately
incorporated into new protein are
rapidly degraded
• The major portion
of the carbon
skeletons of the
amino acids is
converted to
amphibolic
intermediates,
while the amino
nitrogen is
converted to urea
and excreted in
the urine
• PROTEASES & PEPTIDASES DEGRADE PROTEINS TO
AMINO ACIDS
• The relative susceptibility of a protein to degradation
is expressed as its half-life (t 1/2), the time required to
lower its concentration to half the initial value
• Half-lives of liver proteins range from under 30 min to
over 150 h
• Typical "housekeeping" enzymes have t 1/2 values of
over 100 h
• By contrast, many key regulatory enzymes have t½
values as low as 0.5–2h
• This is the basis of the following
classification:

• Short-lived proteins (for example,

many regulatory proteins and
misfolded proteins) are rapidly
degraded, having half-lives
measured in minutes or hours
• Long-lived proteins, with half-lives
of days to weeks, constitute the
majority of proteins in the cell

• Structural proteins, such as

collagen, are metabolically stable,
and have half-lives measured in
months or years
• PEST sequences, regions rich in
proline (P), glutamate (E), serine
(S), and threonine (T), target some
proteins for rapid degradation

• Intracellular proteases hydrolyze

internal peptide bonds
• The resulting peptides are then degraded
to amino acids by

–endopeptidases that cleave internal

peptide bonds,

–and by aminopeptidases and

carboxypeptidases that remove amino
acids sequentially from the amino and
carboxyl terminals respectively
• Degradation of circulating peptides such
as hormones follows loss of a sialic acid
moiety from the nonreducing ends of
their oligosaccharide chains

• Asialoglycoproteins are internalized by

liver cell asialoglycoprotein receptors and
degraded by lysosomal proteases termed
cathepsins
• Extracellular, membrane-associated,
and long-lived intracellular proteins
are degraded in lysosomes by ATP
independent processes
• By contrast, degradation of
regulatory proteins with short half-
lives and of abnormal or misfolded
proteins occurs in the cytosol, and
requires ATP and ubiquitin
• Ubiquitin, so named because it is present
in all eukaryotic cells, is a small (8.5 kDa,
76 residues) polypeptide that targets
many intracellular proteins for
degradation
• The primary structure of ubiquitin is
highly conserved
• Only 3 of 76 residues differ between
yeast and human ubiquitin
• Ubiquitin
molecules are
attached by non-
α-peptide bonds
formed between
the carboxyl
terminal of
ubiquitin and the
ε-amino groups
of lysyl residues
in the target
protein
 Reactions involved in the attachment of
ubiquitin (Ub) to proteins
• Three enzymes are
involved
• E1 is an activating
enzyme, E2 is a
ligase, and E3 is a
transferase
• The terminal
COOH of ubiquitin
first forms a
thioester
 Reactions involved in the attachment of
ubiquitin (Ub) to proteins
• The coupled
hydrolysis of PPi by
pyrophosphatase
ensures that the
reaction will proceed
readily
• A thioester exchange
reaction now
transfers activated
ubiquitin to E2
 Reactions involved in the attachment of
ubiquitin (Ub) to proteins
• E3 then catalyzes
the transfer of
ubiquitin to the ε-
amino group of a
lysyl residue of the
target protein
• Additional rounds of
ubiquitination result
in subsequent
polyubiquitination
• The residue present at its amino terminal
affects whether a protein is ubiquitinated
• Amino terminal Met or Ser retards,
whereas Asp or Arg accelerates
ubiquitination
• Attachment of a single ubiquitin molecule
to transmembrane proteins alters their
subcellular localization and targets them
for degradation
• Soluble proteins undergo polyubiquitination,
further ligase-catalyzed attachment of four or
more additional ubiquitin molecules
• Subsequent degradation of ubiquitin-tagged
proteins takes place in the proteasome, a
macromolecule with multiple different subunits
that also is ubiquitous in eukaryotic cells
• Metabolic diseases associated with defects of
ubiquitination include the Angelman syndrome
and the von Hippel-Lindau syndrome in which
there is a defect in the ubiquitin E3 ligase
• Proteins tagged
with ubiquitin are
then recognized
by a large, barrel-
shaped,
proteolytic
molecule called a
proteosome
which functions
like a garbage
disposal
• The proteosome cuts the
target protein into
fragments that are then
degraded to amino acids,
which enter the amino acid
pool
• It is noteworthy that the
selective degradation of
proteins by the ubiquitin
proteosome complex
(unlike simple hydrolysis by
proteolytic enzymes)
requires ATP
Chemical signals for protein degradation:
• Because proteins have different half-lives,
it is clear that protein degradation cannot
be random, but rather is influenced by
some structural aspect of the protein
• For example, some proteins that have
been chemically altered by oxidation or
tagged with ubiquitin are preferentially
degraded
• The half-life of a protein is
influenced by the nature of the N-
terminal residue

• For example, proteins that have

serine as the N-terminal amino
acid are long-lived, with a half-life
of more than twenty hours
• In contrast, proteins with aspartate as the
N-terminal amino acid have a half-life of
only three minutes
• Further, proteins rich in sequences
containing proline, glutamate, serine, and
threonine (called PEST sequences after
the one-letter designations for these
amino acids) are rapidly degraded and,
therefore, exhibit short intracellular half-
lives
• Proteins are subject to physical and functional
changes that mirror the life cycle of the
organisms in which they reside
• A typical protein is:
–born at translation,
–matures through posttranslational processing
events such as partial proteolysis,
–alternates between working and resting states
through the intervention of regulatory factors ,
–ages through oxidation, deamidation, etc ,
–and dies when it is degraded to its component
amino acids
The life cycle of a hypothetical protein
• (1) The life
cycle begins
with the
synthesis on a
ribosome of a
polypeptide
chain, whose
primary
structure is
dictated by an
mRNA
• (2) As
synthesis
proceeds,
the
polypeptid
e begins
to fold
into its
native
conforma
tion (blue)
• (3) Folding
may be
accompanied
by processing
events such as
proteolytic
cleavage of an
N -terminal
leader
sequence
(Met-Asp-Phe-
Gln-Val)) or
the formation
of disulfide
bonds (S—S)
• (4)
Subsequent
covalent
modification
may, for
example,
attach a fatty
acid molecule
(yellow) for (5)
translocation
of the
modified
protein to a
membrane
• (6) Binding an
allosteric
effector (red)
may trigger
the adoption
of a
catalytically
active
conformation
• (7) Over time,
proteins become
damaged by
chemical attack,
deamidation, or
denaturation,
and (8) may be
"labeled" by the
covalent
attachment of
several ubiquitin
molecules (Ub)
• (9) The
ubiquitinated
protein is
subsequently
degraded to
its component
amino acids,
which become
available for
the synthesis
of new
proteins
• α1 Antitrypsin has the important
physiological 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
• In the normal lung, the alveoli are
chronically exposed to low levels of
neutrophil elastase, released from activated
and degenerating neutrophils
• This proteolytic activity can destroy the
elastin in alveolar walls if unopposed by the
inhibitory action of α1 antitrypsin

• Lung tissue cannot regenerate and so

emphysema results from the destruction of
the connective tissue of alveolar walls
• When the amount of α1
Antitrypsin is deficient and
polymorphonuclear white blood
cells increase in the lung (e.g.
during pneumonia), the affected
individuals lack a check to
proteolytic damage of the lungs
by proteases such as elastase
• A particular methionine of α1
antitrypsin is involved in it’s binding
to proteases
• Smoking oxidizes this methionine
and thus inactivates it

• As a result affected molecules of α1

antitrypsin no longer neutralize
proteases
• This is particularly devastating in
patients who already have low
levels of α1 antitrypsin

• Intravenous administration of α1
antitrypsin has been used as
augmentation therapy