Proteins and their biological structures

Catalyzing substrates
1. Chemical evolution, in
which biopolymers were
formed from small molecules
Proteins and
nucleic acids
2. Self organization, in which
biopolymers developed the
capacity for self-replication
3. Biological evolution, in which
primitive living cells generated
sophisticated metabolic systems
and eventually the ability to form
multicellular organisms.

1. Enzymes (more 2000 proteins) - very specific catalysis up to 1020
times
2. Regulatory proteins (hormones, gene expression)
3. Transport proteins (Hb, HSA, membrane proteins)
4. Storage proteins (ovalbumin, casein, ferritin - 460kD -4500 atoms of
iron)
5. Contractile and motile proteins (actin, myosin, tubulin, dynein,
kinesin)
6. Structural proteins (keratins, collagen -1/3 of total)
7. Protective and exploitive proteins (immunoglobulins, thrombin,
fibrinogen)
8. Exotic proteins (antifreeze proteins, monellin – sweetener,
resilin – elastics, glue proteins)
BIOLOGICAL FUNCTIONS OF PROTEINS

Although more than 300 different
amino acids have been described in nature,
only 20 are commonly found as constituents
of mammalian proteins. These are the only
amino acids that are coded for by DNA, the
genetic material in the cell. At physiologic
pH, the carboxyl group is dissociated,
forming the negatively charged carboxylate
ion (–COO-
), and the amino group is
protonated (–NH3
+
). In proteins, almost all
carboxyl and amino groups are combined
through peptide linkage and, in general, are
not available for chemical reaction except
for hydrogen bond formation.

UNCHARGED POLAR SIDE CHAINS
ACIDIC SIDE CHAINS
BASIC SIDE CHAINS

Protein function can be understood only in terms of protein structure
1. Primary structure is the amino acid sequence of its polypeptide chain
2. Secondary structure in the local spatial arrangement of a polypeptide’s
backbone atoms without regards to the conformations f its side chain
3. Tertiary structure refers to the three-dimensional structure of an entire
polypeptide
4. Quaternary structure refers to the spatial arrangement of the subunits of the
protein associated through noncovalent interactions and disulfide bonds.
(a) – Lys – Ala – His – Gly – Lys – Lys – Val – Leu – Gly – Ala –
Primary structure (amino acid sequence in a polypeptide chain)
(b)
Secondar
y
structure
(helix)
(c) (d) β2
β
Tertiary structure: one
complete protein chain
(β chain of
hemoglobin)
β1
α2 α1
Quatemary structure:
the four separate chains
of hemoglobin
assembled into an
oligomeric protein
The structural hierarchy in proteins: (a) primary structure, (b) secondary structure, (c) tertiary
structure, and (d) quaternary structure.

Interactions between the side chains of amino acid residues in proteins.
1- electrostatic interactions; 2 – hydrogen bonds; 3 – hydrophobic
interactions; 4 – disulfide bonds.

SECONDARY STRUCTURE OF PROTEINS
The polypeptide backbone forms regular
arrangements of amino acids that are located
near to each other in the linear sequence.
These arrangements are termed the secondary
structure of the polypeptide. The α-helix, β-
sheet, and β-bend (β-turn) are examples of
secondary structures frequently encountered
in proteins.

The 20 amino acids commonly
found in proteins are joined together by
peptide bonds. The linear sequence of
the linked amino acids contains the
information necessary to generate a
protein molecule with a unique three-
dimensional shape. The complexity of
protein structure is best analyzed by
considering the molecule in terms of
four organizational levels, namely,
primary, secondary, tertiary, and
quaternary.

Interactions stabilizing tertiary structure
The unique three-dimensional structure
of each polypeptide is determined by its
amino acid sequence. Interactions between
the amino acid side chains guide the folding
of the polypeptide to form a compact
structure.
1. Disulfide bonds
2. Hydrophobic interactions
3. Hydrogen bonds
4. Ionic interactions

Size of Protein Molecules
Protein Mr Number of Residues per Chain Subunit Organization
Insulin (bovine) 5,733 21 (A)
30 (B)
αβ
Cytochrome c (equine) 12,500 104 α1
Ribonuclease A (bovine pancreas) 12,640 124 α1
Lysozyme (egg white) 13,930 129 α1
Myoglobin (horse) 16,980 153 α1
Chymotrypsin (bovine pancreas) 22,600 13 (α)
132 (β)
97 (γ)
αβγ
Hemoglobin (human) 64,500 141 (α)
146 (β)
α2β2
Serum albumin (human) 68,500 550 α1
Hexokinase (yeast) 96,000 200 α4
γ-Glubulin (horse) 149,900 214 (α)
446 (β)
α2β2
Glutamate dehydrogenase (liver) 332,694 500 α6
Myosin (rabbit) 470,000 1800 (heavy, h)
190 (α)
149 (α’)
160 (β)
h2α1α’2 β2
Ribulose bisphosphate carboxylase (spinach) 560,000 475 (α)
123 (β)
α8 β8
Glutamine synthetase (E. coli) 600,000 468 α12
Insulin Cytochrome c
Ribonuclease Lysozyme Myoglobin
Hemoglobin
Immunoglobulin Glutamine synthetase

PROTEIN FOLDING
Protein folding, which occurs within the cell in seconds to minutes, employs
a shortcut through the maze of all folding possibilities. For example, positively
and negatively charged side chains attract each other, conversely, similarly
charged side chains repel each other. In addition, interactions involving hydrogen
bonds, hydrophobic interactions, and disulfide bonds all exert an influence on the
folding process. This results in a correctly folded protein with a low-energy state.
DENATURATION OF PROTEINS
Protein denaturation results in the unfolding and disorganization of the
protein’s secondary and tertiary structures, which are not accompanied by
hydrolysis of peptide bonds. Denaturing agents include heat, organic solvents,
mechanical mixing, strong acids or bases, detergents, and ions of heavy metals
such as leas and mercury. Most proteins, once denatured, remain permanently
disordered. Denatured proteins are often insoluble and, therefore, precipitate from
solution.

Structure and function of hemoglobin
Hemoglobin is found exclusively in red blood cells (RBCs), where its
main function is to transport oxygen (O2) from the lungs to the capillaries of
the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of
four polypeptide chains – two α chains and two β chains – held together by
noncovalent interactions. Each subunit has stretches of α-helical structure, and
a heme-binding pocket.

Amino acid substitution in Hb S β chains:
A molecule of Hb S contains two normal α-
globin chains and two mutant β-globin chains
(βS
), in which glutamate at position six has
been replaced with valine.
The replacement of the charged
glutamate with the nonpolar valine forms a
protrusion on the β-globin that fits into a
complementary site on the β chain of another
hemoglobin molecule in the cell. At low
oxygen tension, deoxyhemoglobin S
polymerizes inside the RBC, forming a
network of fibrous polymers that stiffen and
distort the cell, producing rigid, misshapen
erythrocytes. Such sickled cells frequently
block the flow of blood in the narrow
capillaries. This interruption in the supply of
oxygen leads to localized anoxia (oxygen
deprivation) in the tissue, causing pain and
eventually death (infarction) of cells in the
vicinity of the blockage.

1. Amino acid substitution in Hb S β chains:
A molecule of Hb S contains two normal α-globin chains
and two mutant β globin chains (βS
), in which glutamate
at position six has been replaced with valine.
2. Sickling and tissue anoxia: The replacement
of the charged glutamate with the nonpolar valine
forms a protrusion on the β-globin that fits into a
complementary site on the β chain of another
hemoglobin molecule in the cell. At low oxygen tension,
deoxyhemoglobin S polymerizes inside the RBC,
forming a network of fibrous polymers that stiffen and
distort the cell, producing rigid, misshapen
erythrocytes. Such sickled cells frequently block the
flow of blood in the narrow capillaries. This
interruption in the supply of oxygen leads to localized
anoxia (oxygen deprivation) in the tissue, causing pain
and eventually death (infarction) of cells in the vicinity
of the blockage.

Hemeproteins are a group of
specialized proteins that contain heme as a
tightly bound prosthetic group. The role of
the heme group is dictated by the
environment created by the three-
dimensional structure of the protein. In
hemoglobin and myoglobin, the two most
abundant heme-proteins in humans, the
heme group serves to reversibly bind
oxygen.
Heme is a complex of protoporphyrin
IX and ferrous iron (Fe2+
). The iron is held
in the center of the heme molecule by bonds
to the four nitrogens of the porphyrin ring.
The heme Fe2+
can form two additional
bonds, one on each side of the planar
porphyrin ring. In myoglobin and
hemoglobin, one of these positions is
coordinated to the side chain of a histidine
residue of the globin molecule, whereas the
other position is available to bind oxygen.

Structure and function of myoglobin
Myoglobin, a hemeprotein present in heart and skeletal
muscle, functions both as a reservoir for oxygen, and as an
oxygen carrier. Myoglobin consists of a single polypeptide
chain that is structurally similar to the individual subunit
polypeptide chains of the hemoglobin molecule.
Myoglobin is a compact molecule, with approximately 80
% of its polypeptide chain folded into eight stretches of α-
helix.

An artist's rendering of sperm whale myoglobin. One of the heme group's propionic
acid side chains has been displaced for clarity. The amino acid residues are
consecutively numbered, starting from the N-terminus, and the eight helices are
likewise designated A through H.

Sickle cell hemoglobin (HbS) Glu (β-chain) replaced by Val
DeoxyHbS undergoes self association and forms a liquid
crystalline phase, which distorts the erythrocytes into a sickle
shape. Sickling: leads to their aggregation and to a decrease in
blood circulation (anemia, tissue infarction and chronic failure of
organ function)
HEMOGLOBIN: colored component of the blood 34% solution
in erythrocytes
Mr 64500 - tetramer composed of 2 polypeptide chain pairs
With a Fe2+ content of 0.334%, the total of 950g Hb in a human
represent 3.5 g or 80% of the total body iron
Several forms of human Hb (α, β, γ, ε, δ)
Presently known 153 abnormal Hb (87 are variations of β-chain)
β-Thalassemia – no β-chain – patients contains HbF instead HbA1

Quaternary structure of hemoglobin: The hemoglobin tetramer
can be envisioned as being composed of two identical dimers. The two
polypeptide chains within each dimer are held tightly together, primarily
by hydrophobic interactions. Ionic and hydrogen bonds also occur
between the members of the dimer.
• T form: The deoxy form of hemoglobin is called the “T” (tense)
form. In the T form, the two αβ dimers interact through a network
of ionic bonds and hydrogen bonds that constrain the movement of
the polypeptide chains. The T form is the low-oxygen-affinity form
of hemoglobin.
• R form: The binding of oxygen to hemoglobin causes the rupture
of some of the ionic bonds and hydrogen bonds between the αβ
dimers. This leads to a structure called the “R”, or relaxed form, in
which the polypeptide chains have more freedom of movement. The
R form is the high-oxygen-affinity form of hemoglobin.

Aggregation or association of two or more identical or
different polypeptide chains by noncovalent interaction
leading to stable oligomeric (or multimeric) structure.
650 proteins (including 500 enzymes) with subunit
structure had been described by 1974. Most of the known
multimeric proteins contain either 2 or 4 similar sized
subunits.
Far less common are proteins with uneven numbers of
subunits (by 1973, 28 trimeric proteins, including 23
enzymes; 5 pentameric, 2 heptameric and 1 nonameric).
Possession of quarternary structure appear to confer a
flexibility of shape and activity, which is necessary for the
physiological role of the proteins. Monomers derived from
multimeric enzymes are usually inactive.
The subunit composition can be determined by
dissociation of aggregate and investigation of the separate
subunits by:ultracentrifugation, polyaerylamide disc
electrophoresis, gel-filtration, ion-exchange chromatography
The structure of the intact aggregate can be studied by:
electron microscopy, low angle X-ray or neutron diffraction.
QUARTERNARY STRUCTURE OF PROTEINS

The binding of a hypothetical pair of proteins by two ionic
bonds, one hydrogen bond, and one large combination of
hydrophobic and van der Waals interactions. The structural
complementarity of the surfaces of surfaces of the two molecules
gives rise to this particular combination of weak bonds and hence to
the specificity of binding between the molecules.

Binding of oxygen to myoglobin and
hemoglobin
Myoglobin can bind only one molecule of
oxygen. Hemoglobin can bind four oxygen molecules
– one at each of its four heme groups.
Oxygen dissociation curve: A plot of Y
measured at different partial pressures of oxygen
(pO2) is called the oxygen dissociation curve. This
graph illustrates that myoglobin has a higher oxygen
affinity at all pO2 values than does hemoglobin.
• Myoglobin (Mb): The oxygen dissociation
curve for myoglobin has a hyperbolic shape. The
equilibrium is shifted to the right or to the left as
oxygen is added to or removed from the system.
Hemoglobin (Hb): The oxygen dissociation
curve for hemoglobin is sigmoidal in shape,
indicating that the subunits cooperate in binding
oxygen. Cooperative binding of oxygen by the
four subunits of hemoglobin means that the
binding of an oxygen molecule at one heme
group increases the oxygen affinity of the
remaining heme groups in the same hemoglobin
molecule. Allosteric effects
The ability of hemoglobin to reversibly
bind oxygen is affected by the pO2, the pH of
the environment, the partial pressure of
carbon dioxide, pCO2, and the availability of
2,3-bisphosphoglycerate. These are
collectively called allosteric (“other site”)
effectors.

Heme-heme interactions
The net effect is that the affinity of
hemoglobin for the last oxygen bound is
approximately 300 times greater than its affinity
for the first oxygen bound.
Loading and unloading oxygen
For example, in the lung, the concentration of
oxygen is high and hemoglobin becomes virtually
saturated (or “loaded”) with oxygen. In contrast,
in the peripheral tissues, oxyhemoglobin releases
(or “unloads”) much of its oxygen for use in the
oxidative metabolism of the tissues.
The concentration of both CO2 and H+
in the
capillaries of metabolically active tissues is higher
than that observed in alveolar capillaries of the
lungs. In the tissues, CO2 is converted by carbonic
anhydrase to carbonic acid:
CO2 + H2O H2CO3
H2CO3 HCO3
–
+ H+
which spontaneously loses a proton, becoming
bicarbonate:
The H+
contributes to the lowering of pH.
This differential pH gradient (lungs having a
higher pH, tissues a lower pH) favors the
unloading of oxygen in the peripheral tissues, and
the loading of oxygen in the lung. Thus, the oxygen
affinity of the hemoglobin molecule responds to
small shifts in pH between the lungs and oxygen-
consuming tissues, making hemoglobin a more
efficient transporter of oxygen.
Source of the protons that lower the pH

Effect of 2,3-bisphosphoglycerate on oxygen affinity
It is the most abundant organic phosphate in the
RBC, where its concentration is approximately that of
hemoglobin. 2,3-BPG is synthesized from an intermediate
of the glycolytic pathway.
• Binding of 2,3-BPG to deoxyhemoglobin: 2,3-BPG
decreases the oxygen affinity of hemoglobin by binding to
deoxyhemoglobin. This binding stabilizes the taut
conformation of deoxyhemoglobin.
HbO2 + 2,3-BPG Hb–2,3-BPG + O
oxyhemoglobin deoxyhemoglobin
• One molecule of 2,3-BPG binds to a pocket, formed by
the two β-globin chains, in the center of the
deoxyhemoglobin tetramer. This pocket contains several
positively charged amino acids that form ionic bonds with
the negatively charged phosphate groups of 2,3-BPG.
• Shift of the oxygen dissociation curve: The presence of
2,3-BPG significantly reduces the affinity of hemoglobin
for oxygen, shifting the oxygen dissociation curve to the
right. This reduced affinity enables hemoglobin to release
oxygen efficiently found in the tissues.
• Response of 2,3-BPG levels to chronic hypoxia or
anemia: The concentration of 2,3-BPG in the RBC
increases in response to chronic hypoxia, such as that
observed in chronic obstructive pulmonary disease
(COPD) like emphysema, or at high altitudes, where
circulating hemoglobin may have difficulty receiving
sufficient oxygen.

1. H+
decreases affinity to O2
2. Blood to tissues → acidification
3. Binding of H+
to Hb conformation
4. Lung O2 to Hb, H+
release
5. Enhancement of pH
6. Blood – O2 binding, tissue – O2
release
BOHR'S EFFECT
Effect of H+
on oxygen binding by hemoglobin (Hb). A. In the tissues,
CO2 is released. In the red blood cell, this CO2 forms carbonic acid, which
releases protons. The protons bind to Hb, causing it to release O2 to the
tissues. B. In the lungs, the reactions are reversed. O2 binds to protonated
Hb, causing the release of protons. They bind to bicarbonate (HCO3
-
),
forming carbonic acid which is cleaved to H2O and CO2, which is exhaled.

1. Selection of protein source (tissues, cells, E.coli, yeasts, cloning)
2. Solubilization
a. cytosol - osmotic lysis of cells by hypotonic solution + centrifugation
b. membrane - homogenization + centrifugation
3. Stabilization - isolation in buffers, temperature, inhibitors of proteases
PROTEIN ISOLATION
PROTEIN PURIFICATION
CHARACTERISTIC PROCEDURE
1. CHARGE 1.Ion-exchange chromatography
2. Electrophoresis
3. Isoelectric focusing
2. POLARITY 1. Adsorption chromatography
2. Paper chromatography
3.Reverse-phase chromatography
4.Hydrophobic chromatography
3. SIZE 1. Dialysis and ultrafiltration
2. Gel electrophoresis
3. Gel filtration chromatography
4. Ultracentrifugation
4. SPECIFICITY 1. Affinity chromatography

In the process of ion exchange, ions that electrostatically bound to an insoluble and chemically inert
matrix are reversibly replaced by ions in solution:
R + A-
+ = R + B-
+ A-
A schematic diagram illustrating the separation of several
proteins by ion exchange chromatography using stepwise elution.
A device for generating a
linear concentration gradient. Two
connected open chambers, which
have identical cross-sectional
areas, are initially filled with
equal volumes of solutions of
different concentrations.
As the column is washed, a process known as elution, those proteins with
relatively low affinities to ion exchanger move through the column faster than the
proteins that bind to the ion exchanger with higher affinities.
ION-EXCHANGE CHROMATOGRAPHY

The molecules are separated according to their size and shape
Molecules with molecular masses ranging below the exclusion limit
(the molecular mass of the smallest molecule unable to penetrate the
pores) of a gel will elute from the gel in the order of their molecular
masses, with the largest eluting first.
Dialysis is a process that
separates molecules
according to size through
the use of semipermeable
membranes containing
pores of less then
macromolecular
dimensions
GEL FILTRATION CHROMATOGRAPHY
At start of
dialysis
(a) (b) At equilibrium
Dialysis
bad
Buffer
Concentrated
solution

Molecule known as a ligand, specifically binds to the protein of interest is
covalently attached to an inert and porous matrix
The separation of
macromolecules by affinity
chromatography.
The formation of cyanogen bromide-
activated agarose (top) and its reaction
with a primary amine to form a covalently
attached ligand for affinity
-chromatography (bottom).
Examples of the various types of nucleophilic groups that can be covalently
attached to epoxy-activated agarose via reation with its epoxide groups.
AFFINITY CHROMATOGRAPHY

ELECTROPHORESIS
Migration of ions in an electric field
Polyacrylamide and agarose
Paper electrophoresis, (a) A schematic diagram of
the apparatus used. The sample is applied to a point on
the buffer-moistened paper. The ends of the paper are
dipped into reservoirs of buffer in which the electrodes
are immersed and an electric field is applied. Uncharged
molecules remain the point of sample application.
Visualization with amido black, Coomassie brilliant blue, silver stain, radioactivity
–
+
–
+
Cathode Cathode
Plastic
frame Plastic
frame
Anode
Anode
Buffer
Buffer
Sample
wells
Sample
wells
Sample
Stacking
gel
Running
gel

[CH3-(CH2)10-CH2-O-SO3
-
]Na+
– sodium dodecyl sulfate SDS-treated
protein stend to have identical charge-to-mass ratios and similar shapes.
SDS-PAG ELECTROPHORESIS
The SDS – polyacrylamide
electrophoresis pattern of the
supernatant (left) and membrane
fractions (right) of various strains of
the Salmonella typhimurium. Samples
of 200-µg of protein each were run in
parallel lanes on a 35-cm - long x 0.8-
mm-thick slab gel containing 10%
polyacrylamide. The lane marked
MW contains molecular weight
standards.
A logarithmic plot of the
molecular masses of 37 different
polypeptide chains ranging from
11 to 70 kD versus their relative
electrophoretic mobilities on an
SDS-polyacrylamide gel.
Many proteins contain more than one polypeptide chain → SDS
treatment disrupt the noncovalent interactions between these subunits

If a mixture of proteins is electrophoresed through a
solution having stable pH gradient in which the pH smoothly
increases from anode to catode, each protein will migrate to
the position in the pH gradient corresponding to its isoelectric
point.
IMMUNOBLOTTING
ISOELECTRIC FOCUSING
The detection of proteins by immunoblotting

Proteins and their biological structures

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