• To know the chemical structure and and be able to identify amino
acids of biochemical importance.
•To study the physicochemical properties of amino acids
•To appreciate the relationship between amino acids and
•To identify specific peptides as active endogenous biomolecules
Structure of amino acids
•Amino acids are the basic structural units of peptides and proteins.
They consist of:
• amino group
• a carboxyl group
• a hydrogen atom
• a distinctive R group bonded to a C atom known as the α-carbon
• The R group is referred to as the side chain.
Physicochemical properties of amino acids
• Amino acids in solution at neutral pH are predominantly dipolar ions
(Zwitterions) rather than unionised molecules.
• In the dipolar state the amino group is protonated (NH3+) and the
carbonyl group is dissociated (-coo- ).
• In addition to the ionisable α-substituents several of the amino acids have
Other ionisable groups. The pH at which the net ionic charges are
balanced is the isoelectric point (IEP).
Amino acids are optically active
tetrahedral array of 4-different groups about the α-carbon confers
optical activity on amino acids
• The α-carbon of all amino acids are asymetric so there are two
stereoisomers of each except glycine.
• The 2 mirror image forms are the:
• By convention if the α-NH
is projected to the left, the a.acid has an
absolute L-configuration. Its optical enantiomer with α-NH3+ projected
towards the right has a D-configuration.
Occurrence of L and D-amino acids in proteins
• All amino acids of animal proteins are L-isomers and are
sometimes referred to as natural amino acids.
•Some D-a.acids occur in plants and bacteria eg. D-alanine
and D-glutamic acid occur in bacteria cell wall.
• Some antibiotics contain D-amino acids
Eg. D-alanine, D-glutamic acid and D-ornithine are
present in bacitracin-an active agent against G-ve bacteria.
•Polymyxins contain D-phenylalanine
The common amino acids in proteins
• 20 kinds of side chains varying in:
•Hydrogen bonding capacity
are commonly found in proteins
• All proteins in all species of bacteria & man are constructed from the
same set of 20 amino acids.
•The remarkable range of functions exhibited by proteins result from
the diversity and versatility of the 20 kinds of building blocks-amino
The 20 amino acids and their abbreviations
Aspartic acid & Asparagine
Glutamine or Glutamic acid
• simplest amino acid with H as side chain, not optically active.
•Residues found in almost all proteins particularly abundant in
•A neurotransmitter in the CNS
•Sometimes combined with antacids in the treatment of gastric hyperacidity
to reduce gastric irritation due to buffering capacity of the amino group.
•Been used in the treatment of myasthenia gravis, benefit is doubtful.
•Has methyl group as its side chain
•Occurs in almost all proteins
•Its structural isomer β-alanine occurs in tissues in the free form and as a
component of coenzyme A and the peptide carnosine & anserine, β-alanine
not a component of protein.
Valine, leucine, isoleucine & proline (amino acids with hydrocarbon
Proline differs from other amino acids in containing a 20 rather than a
10 amino group.
The side chain of proline is bonded to:
• both the amino group and the α-carbon to form a cyclic structure.
•The cyclic structure results in the distortion of the linear symmetry
of the polypepetide chain into which proline is incorporated
•Further, when the >NH group is engaged in an amide link, there is no H
atom remaining to participate in H-bonding
•Therefore, proline is an important determinant of the 3-dimensional shape
of peptide chains.
•Some hydroxy derivatives of proline are found only in collagen & gelatin
•Valine leucine and isoleucine are essential amino acids, thus has to be
taken in the diet to support life and growth possibly bcos the body lacks the
capacity to synthesize branched aliphatic chains, hydrophobic?
Serine & Threonine:
•The –OH group in the side chain of serine can engage in H-bonding
and ester formation.
•Serine is therefore an important reactive component of proteins.
•An antibiotic, a diazoacetyl`ester (azaserine) has anti-tumour and
Aromatic amino acids, phenylalanine, tyrosine &
•Phenylalanine & tyrosine are important constituents of proteins
•Are precursors for the synthesis of important regulatory proteins as
neurotransmitters and hormones.
• Through the formation of another amino acid
dihydroxyphenylalanine (DOPA), the catecholamines eg noradrenaline are
Synthesis of catecholamines from phenylalanine and tyrosine
•Starting point is the essential aa Phe & Tyr derived from protein in the diet
Generally, dietary intake of tyrosine is more than adequate
for catecholamine synthesis
•Phenylalanine can be converted to tyrosine by the enzyme phenylalanine
Hydroxylase. Enzyme absent in the congenital disease phenylketonuria.
•L-DOPA used in the treatment of Parkinsons disease.
•Note: Rational for L-Dopa/carbidopa combination in Parkinson’s
•Methyldopa used to treat high B.P
•DOPA involved in melanin formation
•Tyrosine gives rise to the thyroid hormones
•Tryptophan is the precursor of 5-hydroxytryptamine or serotonin
which has several actions on the CVS, GIT, genital tract and
the respiratory system.
•Side chains of the aas described are uncharged at physiological PH
•The ff aas have charged side chains:
•Aspartate & glutamate
•Arginine and lysine have basic side chains
•Aspartate and glutamate have negatively charged side chains (acidic)
•Histidine may have +vely charged or neutral depending on its local
•Histidine is an important reactive aa in proteins because the pka of
the nitrogen in the imidazole ring of histidyl residues in proteins is
in the range of pH of the tissues.
•Histidine is also an important precursor of the pharmacologically active
•Lysine is an important component of proteins due to reactivity
of the amino group on the side chain.
•Has been used as an adjunct to mercurial diuretics in the treatment of
•Hydroxylysine, a derivative occurs in collagen.
•Arginine involved in the synthesis of urea
•Has been given in the treatment of hepatic failure where there is
Coma due to high levels of ammonia.
•Acts predominantly by stimulating the synthesis of urea.
•Large doses stimulate the secretion of insulin & G-hormone
•Aspartate & glutamic acid have carboxyl groups on their side chains
which are completely ionised at the pH of the tissues
•The acidic groups are masked by being converted to the
corresponding amides, asparagine and glutamine
Asparagine and glutamine (Amide derivatives of aspartate & glutamate
•Methionine & cysteine have S in their side chains
•Methionine is a component of most proteins acting as a methyl
donor in biochemical processes.
•The demethylated derivative homocysteine occcurs in tissues
•It is a dietary component and may be replaced by D-methionine
which is deaminated and then reaminated to the L-form.
•Methionine has a lipotropic action and prevents fatty infiltration
of the liver. Given as a supplement to patients with liver diseases
Unable to take adequate diet.
•Cys plays a role in determining the tertiary structure of proteins
•2 mols in adjacent polypeptide chains combine through their
thiol groups to form a cystine residue
•Cysteine and methylcysteine have been reported to increase the rate
of wound healing
•Acetylcysteine has been used as a mucolytic
•Methionine and acetylcysteine have been used in the
treatment of paracetamol overdosage
•Acetylcysteine increase GSH formation in the liver
•Methionine increases conjugation rxn in the liver
•GSH conjugates N-acetyl-p-benzoquinone imine.
•Note: Some proteins contain special aas that
supplement the 20 basic aas eg. Collagen contains hydroxyproline
Such proteins are formed by modification of a common aa
after it has been incorporated into a polypeptide chain.
The peptide bond
•Amino acids are the building blocks of
peptides and proteins.
• The peptide bond is formed by the interaction of
two amino acids with the elimination of a
molecule of water between the -NH2 and –COOH
•Properties & occurrence of peptide bonds in
peptides & proteins
•The sharing of electrons b/n the –C=O and –C-NH confers
rigidity on the peptide bond.
•In proteins and polypeptides aas are linked by peptide bonds
between the α-amino group of one aa and the carboxyl group of
•Peptide bond is therefore an amide linkage
•A single polypeptide chain may contain up to several hundreds
of aas joined together linearly.
•The carbonyl oxygen and the amide hydrogen of the peptide
bonds participate in H-bonding interactions leading to the
Structure of Peptide
• The peptide bond is an amide bond.
• Amides are very stable and neutral.
Oligopeptides, polypeptides and proteins
•Amino acids may be linked by peptide bonds to form:
•Oligopeptides (several aa residues)
•Polypeptides (many residues)
•Proteins consist of one to several peptides
•N-terminal or the amino terminal is the terminal bearing
the free amino group.
•C-terminal or the carboxyl terminal is the one bearing the
free carboxyl group.
•The amino acid residues are numbered starting at the Nterminal, the direction in which aas are incorporated
during protein synthesis.
•Small peptides may break the rule governing peptides
•They may contain D-amino acids (some antibiotics)
•The amino acids may be linked by bonds other
than the standard peptide bond (the glutamyl residues
•2 dipeptides carnosine & anserine
occur particularly in muscle.
•Carnosine in man and other animals and
anserine in birds
•Glutathione is a tripeptide of considerable
•Amino acid residues in glutathione are:
•GSH acts as a coenzyme for several enzymatic reactions
•2 mols may be linked through their –SH group under
Oxidising conditions to form oxidised glutathione GSSH
•Several members of biochemical & Pharmacological
•Include the following nonapeptides of the Post Pituitary
•Antidiuretic hormone (ADH)
•Longer chain polypeptide hormones of the pancreas
•Hormones of the anterior Pituitary
•ACTH OR Corticotrophin 39
•Substances having intense activity on smooth muscle:
•Substances regulating secretions in the GIT:
•A substance regulating circulation
Role of Angiotensin in
Have we achieved our objectives?
To know the chemical structure and be able to identify amino acids of
To study the physicochemical properties of amino acids
To appreciate the relationship between amino acids and
To identify specific peptides as active endogenous biomolecules
CLASSIFICATION OF PROTEINS
•May be classified as follows:
•Simple (contain only amino acids
•Conjugated (contain additional substances)
•High (50,000-several millions)
•Fibrous (long in proportion to their diameter)
•Globular (less asymmetric)
Generally however, proteins are classified as:
Water soluble, and soluble in dilute salt solutions
Precipitated with full saturation in ammonium sulphate
Eg. Plasma albumin
Soluble in dilute salt solutions
Insoluble in water and strong salt solutions
Insoluble in aqueous solutions eg. Keratin, collagen, fibrin
Contain High proportion of arginine
Of low MW, not coagulated by heat, soluble in water to give
appreciably alkali solution
•Soluble in water to give weakly alkali solution, conjugated
•Glycoproteins or mucoproteins
carbohydrate derivatives-blood group substances
•Lipoproteins-plasma lipoproteins, components of cell
Membranes and subcellular organells
•Phosphoproteins-phosphoesters with serine or
threonine residues eg. casein
•Flavoproteins-flavine-adenine dinucleotide, various
reduction and oxidative enzymes
•Haemoproteins-Iron-porphyrin (haem) groups eg. Hb,
•Metalloproteins-containing metal groups eg carbonic
•Greek – proteios – of 1° importance
•Polymers of amino acids linked by peptide
•Proteins are the most important of all
•A copolymer is a polymer made from more
than one type of monomer molecule.
•Twenty different α-amino acids can link to
•Prefer to use fat, CHO for energy
•CHO and fat are protein sparing
–During prolonged strenuous exercise,
about 15% of the muscles need met with
protein (break down own tissue)
–If protein intake is inadequate, body
protein → energy e.g. starvation
•Proteins - Properties
•Proteins are extremely large natural polymers
with molecular weights reaching several
•Compare a typical organic molecule -benzoic
acid (C6H5COOH MW = 132).
•The small protein haemoglobin has the
•Its molecular weight = 65,000.
•Proteins are too large to pass through cell
membranes and remain trapped in the cells
where they are made.
•In disease or trauma, cells are damaged
and proteins can escape.
•Detection of proteins in urine indicates
kidney damage. Heart attack releases
specific heart cell proteins into the blood.
of Some Important Proteins
No. of aa residues
•Proteins are linear polymers built of monomer
units called amino acids
•Proteins contain a wide range of functional
•Proteins can interact with one another and with
other biological macromolecules to form complex
•Some proteins are quite rigid, whereas others
display limited flexibility
•Function of a protein is directly
dependent on its three-dimensional
•Proteins spontaneously fold up into
three-dimensional structures that are
determined by the sequence of amino
acids in the protein polymer.
•alcohols, thiols, thioethers, carboxylic
acids, carboxamides, and a variety of
•combined in various sequences, this
array of functional groups accounts for
the broad spectrum of protein function.
–macro-molecular machines that carry out
the accurate replication of DNA,
– the transmission of signals within cells,
–many other essential processes.
•Rigid units can function as structural
elements in the cytoskeleton (the internal
scaffolding within cells) or in connective
•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
•Other properties of proteins
-a protein containing solution centrifuged
at sufficiently high speed will have its molecules
settled at a constant rate when the centrifugal
force exceeds the dispersant forces on the molecules
-The pH determines the properties of the protein such
as solubility, viscousity and enzymatic activity.
-the precise location of an antigenic substance can be
determinedby an antibody that reacts specifically with it.
-At any pH other than the IEP, a protein will migrate in an electric
field. The differential rates of migration can be used to separate
Hydrolysis (enzymatic or heat) of the
amides regenerates the amino acids:
•The amide linkage is split as indicated.
•Regeneration of component amino acids
The very large protein is broken down into
smaller, water soluble components:
•These small molecules may move through the
organism to be reassembled elsewhere.
•General functions of proteins
•Most versatile macromolecules in living
•serve crucial functions in essentially all
–transport and store other molecules such as
– provide mechanical support and immune
– transmit nerve impulses, and
– control growth and differentiation
Roles of Proteins
Transport, metabolic control, contraction,
and catalysis of chemical transformations.
provide the matrix for bone and connective
give structure and form to the human
•Enzymes-dynamic proteins. almost all biological
reactions are enzyme catalyzed . Allows the
reaction to occur at a rate compatible with life.
•Haemoglobin and myoglobin
•transport oxygen in blood and in muscle
•transports iron in blood.
•many drugs and xenobiotics compounds are
transported bound to albumin.
•Others transport hormones in blood from their site of
synthesis to their site of action
–Immunoglobulins and interferons
act against bacterial or viral infection.
formed where required to stop the loss of
blood on injury to the vascular system.
–Many hormones are proteins.
–Protein hormones include insulin, thyrotropin,
somatotropin (growth hormone), luteinizing
hormone, and follicle stimulating hormone.
Important peptide hormones include
adrenocorticotropin, antidiuretic hormone,
glucagon, and calcitonin.
–Myosin and actin
function in muscle contraction.
•Control And Regulation Of Gene Transcription And
–histone proteins closely associated with DNA, the
repressor and enhancer proteins that control gene
expression, and the proteins that form a part of the
–collagen and elastin,
form the matrix for bone, ligaments, connective
tissue and skin
Provide structural strength and elasticity to
Keratin is the 1º component of human hair, nails,
skin, and tooth enamel – fibrous sulfur-containing
•Levels of protein structure
–The amino acid sequence in a polypeptide chain
–Consists of local regions of polypeptide chains
formed into structures that are usually stabilized
by hydrogen bonds
–Involves folding of the secondary elements into
an overall three-dimensional conformation
–Combination of 2 or more subunits each
composed of a polypeptide chain
levels of organization
structure = specification of the sequence
of amino acids i.e. the order in which amino acid
residues are linked together in a protein.
since every polypeptide begins with free
amino group, this is called the N-terminus. The
opposite end of the polypeptide has a free carboxyl
group, called the C-terminus.
N and C t er m inal of polypept ides
•Amino Acid Sequences Have Direction
•Leu-enkephalin - an opioid peptide, modulates the perception of pain.
• reverse pentapeptide, Leu-Phe-Gly-Gly-Tyr (LFGGY), is a different molecule and
shows no such effects
of a regularly repeating part,
called the main chain or backbone
and a variable part, comprising the
distinctive side chains
•Why know the sequence of amino acids
in a polypeptide chain?
• Elucidating its mechanism of action (e.g., the
catalytic mechanism of an enzyme)
– proteins with novel properties can be generated
by varying the sequence of known proteins.
• Second, amino acid sequences determine the
three-dimensional structures of proteins.
– sequence is the link between the genetic
message in DNA and the three-dimensional
structure that performs a protein's biological
•Oxytocin & Vasopressin
•ADH and oxytocin each have nine (9) amino acids.
•Each has cysteine residues at amino acid positions 1 and 6. These
cysteine residues form a disulfide bond with one another to create a
cyclic six amino acid ring with 3 amino acid residues hanging off.
•ADH and oxytocin share 7 amino acids in common and differ only at
amino acid positions 3 and 8.
•Oxytoxin is Isoleucine-3, Leucine-8 while ADH is Phenylalanine-3,
•Functions of Oxytocin & ADH
stimulates contraction of uterine smooth
muscle. It is secreted during labor to effect delivery
of the fetus.
Oxytocin also stimulates contraction of smooth
muscle in the mammary glands (myoepithelial
in low doses controls the resorption of water
by the distal tubules of the kidneys and regulates
the osmotic content of blood.
At high doses, ADH causes contraction of
arterioles and capillaries, especially those of the
coronary vessels, to produce localized increases in
Receptors, V1 – bood vessels, V2- kidney
1 0 structure of Insulins Used in the Treatment of DM
*Positions 1 and 2 of B chain are both Ala in chicken and duck; whereas
in the other species in the table, position 1 is Phe and position 2 is Val in
by various kinds of bacteria
restriction enzymes recognize specific
sequences of DNA and cut the double strand
where the sequence occurs.
Treating the DNA of two different organisms
with the same restriction enzyme produces
complementary fragments, or fragments with
ends that fit together
These can be combined in a hybrid DNA
molecule that, if part of a living cell,
expresses traits of both parents.
•Recombinant DNA Technology
In genetic engineering, scientists use restriction
enzymes to isolate a segment of DNA that
contains a gene of interest—for example, the
gene regulating insulin production.
2. A plasmid extracted from its bacteria and
treated with the same restriction enzyme can
hybridize with this fragment’s “sticky” ends of
3. The hybrid plasmid is reincorporated into the
bacterial cell, where it replicates as part of the
4. A large number of daughter cells can be
cultured and their gene products extracted for
•Short-acting insulin analogs are designed to
overcome the limitations of conventional
regular human insulin.
•Insulin lispro (Humalog), formerly called
LYSPRO from the chemical nomenclature
•faster subcutaneous absorption,
•an earlier and greater insulin peak, and
•a shorter duration of action
insulin lispro, reversal of the proline at B-28 and
the lysine at B-29 results in more rapid dissolution of
this insulin to a dimer and then to a monomer that is
absorbed more rapidly after subcutaneous injection
fold in a series of stages. The first
level of folding is called the secondary (2˚)
of the most common 2˚ folding patterns is
called the alpha-helix , discovered by Pauling and
–Alpha helix: Hydrogen bonds can form readily
between C=O groups in the backbone and N-H
groups four amino acid residues further along
–This regular pairing pulls the polypeptide into a
helical shape that resembles a coiled ribbon.
20 structure contd
•Another common folding pattern is called
beta pleated sheet .
•Some protein regions remain in random
coil, no regular pattern of secondary
•Different proteins have different degrees
of alpha helix, beta sheet, and random
•Silk is a protein stabilized entirely by
pleated sheet; keratin (in hair) is
stabilized entirely by alpha helix. Most
proteins have some of both.
•Hydrogen-Bonding Scheme For an α helix
CO group of residue n forms a hydrogen
bond with the NH group of residue n+ 4.
Structure of an α-helix
backbone is folded into a
spiral that is held in
place by hydrogen bonds
(black dots) between
backbone oxygen atoms
and hydrogen atoms.
Note that all the
hydrogen bonds have
the same polarity. The
outer surface of the helix
is covered by the sidechain R groups.
•A simple two-stranded β sheet with antiparallel β strands.
• A sheet is stabilized by hydrogen bonds (black dots)
between the β strands.
•The planarity of the peptide bond forces a β sheet to be
pleated; hence, this structure is also called a β pleated
sheet, or simply a pleated sheet.
view of a β sheet showing how the
R groups protrude above and below the
plane of the sheet.
Secondary structure is not just hydrogen bonds.
Helix: Right-handed helix with 3.6 amino acid residues per
turn. Hydrogen bonds are formed parallel to the helix axis.
Sheet: A parallel or antiparallel arrangement of the polypeptide
chain. Hydrogen bonds are formed between the two (or more)
Turn: A structure in which the polypeptide backbone folds
back on itself. Turns are useful for connecting helices and
•Secondary structure exists to provide a way to form hydrogen bonds in the
interior of a protein.
•These structures (helix, sheet, turn) provide ways to form regular hydrogen
•These hydrogen bonds are just replacing those originally made with water.
• As a protein folds, many hydrogen bonds to water must be broken.
•If these broken hydrogen bonds are replaced by hydrogen bonds within
the protein, there is no net change in the number of hydrogen bonds
•Hydrogen bonding does contribute somewhat to the overall stability
of a protein; however, the hydrophobic interaction usually dominates
the overall stability
Highly elongated protein molecules whose shapes
are dominated by a single type of secondary
1. Coiled Coil
durable, insoluble, unreactive
2. β Sheet
3. Triple Helix
strong, high tensile strength
• principal component of hair, nails, wool, horns,
hooves, scales, feathers, shells
∀ α keratin - in mammals
∀ β keratin - in birds and reptiles
The α-keratin chain is an α-helix. Pairs of α-helix chains are
interwound to form a two-chain coiled coil. The chains wind in a
Each α-keratin chain consists of ~310 residues having a 7-residue
where residues a and d are nonpolar
Silk - a β sheet
• consists of
antiparallel β sheets
• 6-residue repeat
• The β sheets stack
to form a
Collagen - a triple helix
• Single collagen molecule
contains 3 polypeptide chains.
• Each chain is a left-handed
helix (3 residues/turn).
• 3 helical chains are twisted
together in a right-handed
manner to form a superhelical
• Many varieties - eg., Type I has
two α 1 and one α 2 chains
Collagen - distinctive amino acid
30% Gly and 15-30% Pro or Hyp (hydroxyproline)
(-Gly-X-Pro-) repeats or (-Gly-X-Hyp-) repeats
• Scurvy (vitamin C deficiency) - improper fibers,
skin lesions, fragile blood vessels, poor wound
healing, due to decreased Hyp formation
• Osteogenesis imperfecta (brittle bone disease)
(OI) a group of heritable disorders with an incidence
of about 1 in 10, 000- abnormal bone formation in
infants, varies from mild to lethal.
• Defect due to mutation in the genes for procollagen
Type I, single base change in the codon for glycine
resulting in the disruption of the triple helical
• Ehler-Danlos syndrome - hyperextensibility of joints
and skin (“loose” skin), mutations: Gly replaced with
Ser or Cys
Schematic Views of α-Helices
• A ball-and-stick model.
• A ribbon depiction.
• A cylindrical depiction.
• Ferritin, an iron-storage protein, is built
from a bundle of a helices.
Major Histocompatibility Complex
Model of binding site in class I MHC (major
histocompatibility complex) molecules, which are involved in
A sheet comprising eight antiparallel β strands (green)
forms the bottom of the binding cleft, which is lined by a pair
of α helices (blue).
A disulfide bond is shown as two connected yellow spheres.
The MHC binding cleft is large enough to bind a peptide 8
10 residues long.
continue folding beyond the
formation of secondary structure.
is only with the complete, compact folding
into tertiary (3°) structure that they attain
their "native conformation" and become active
proteins (as a result of the creation of active
that contribute to tertiary folding
–sulfhydryl bonds (-S-S- bonds). These are
especially important, because they are
covalent bonds and quite strong compared to
synthesis generates a linear
sequence that has to be folded with
hydrophilic groups on the outside and
hydrophobic groups buried (if it is water
primary structure determines the
the number of possible structures
it is not possible that the protein tests
every one of them to find the lowest
is thought that secondary structures, called
‘molten globules’, facilitate the folding process.
problem is that as proteins are
synthesised hydrophobic regions must not be
exposed to an aqueous environment or they
will associate to form aggregates.
is achieved by chaperones that bind to
hydrophobic regions and subsequently detach
to allow correct folding.
Protein Folding contd
process allows the correct folding of even
large proteins since these fold sequentially as
they are synthesised.
proteins require chaperonins that
enclose the protein to be folded in a cavity
away from the rest of the cell.
and chaperonins do not direct
protein folding but simply provide conditions
where it can occur properly.
cells exposed to a near lethal temperature
rise heat shock proteins are synthesised.
These allow existing proteins to refold correctly.
Examples include Hsp 70 and Hsp 60
Prion diseases and protein folding
pathogens composed entirely of proteins
A number of neurological degenerative diseases are known
to be caused by prions
–These include Creutzfeldt–Jacob disease (CJD) and kuru
in humans and scrapie (Bovine spongiform
encephalopathy,BSE) in sheep.
–Mad cow disease is also caused by a prion.
they are infectious no nucleic acid has been
identified and it is now thought that a protein infectious agent
or prion is responsible.
scrapie there is a normal brain protein (PrPc) which
becomes converted to the scrapie form (PrPsc).
have the same primary structure but different
secondary and tertiary structures.
is suggested that the prion form converts the
normal form to the prion form, i.e. the process is
are two possible mechanisms for this
–The association of the normal form with the prion form
may be sufficient to cause the change
–There may be an involvement of a chaperone and ATP
in the unfolding and refolding
in the normal gene for PrP may make
the formation of PrPsc more likely.
long protein sequence frequently folds into a
series of compact, semi-independent regions called
domain has a hydrophobic core and a
hydrophilic exterior and generally are 100-150
amino acids in length.
of a single protein are usually connected
of polypeptide chain lacking a usual
secondary structure (random coil) or
a cleft or less dense region of tertiary structure.
Sometimes a binding site is found in a cleft between
are frequently associated with a
specific function of the protein.
example: binding sites for two different
substrates or a substrate and effector could be in
two different domains.
dehydrogenase..one domain binds NAD+ and the
second domain binds glyceraldehyde-3phosphate.
The cell-surface protein, CD4
•Cell surface protein found on some cells of the
•Has an extracellular and cytoplasmic portions.
•(HIV) attaches itself to the extracellular portion,
which comprises of four similar domains of
approximately 100 amino acids each
proteins are made of multiple
polypeptide subunits, which must be
assembled together after each individual
polypeptide has reached its 3° structure.
–Hemoglobin (blood protein involved in oxygen
transport) has four subunits .
–Pyruvate dehydrogenase (mitochondrial
protein involved in energy metabolism) has 72
of 2 heavy and 2 light chains.
A disulfide bond joins a L chain to a H
chain and the two L-H chain pairs are
bound together by two disulfide bonds
between the H chains.
The variable regions of an L and H chain
come together to form the antigen binding
site of the immunoglobulin.
Structure of antibodies
heavy and light chains come together
to form Fab domains, which have the
antigen-binding sites at the ends.
two heavy chains form the Fc domain.
The Fab domains are linked to the Fc domain
by flexible linkers
Myoglobin and Hemoglobin
proteins are involved in oxygen transport.
= intracellular protein in muscle
= intracellular protein in red blood cells
Why study them?
proteins in human health
model in studying protein structure, binding, function
a heme group
–porphyrin ring system
N atoms in porphyrin
Fe(II) binds O2
–with O2 = scarlet
–no O2 = dark purple
oxidized to Fe(III) dark brown, does not
physiological role is to facilitate oxygen
transport in muscle.
increases oxygen solubility in aqueous
aquatic mammals, myoglobin also functions to
store oxygen (10-fold more in seals and whales)
binding of O2 to myoglobin (Mb)
Mb + O2
protein in red blood cells
function is to transport oxygen
oxygen in lungs and releases oxygen
–two α-subunits and two β subunits - α2β2
–each subunit contains a heme group
–Fe(II) binds O2
O2 = scarlet
O2 = dark purple
•Over 300 variations of amino acid sequences of normal
adult haemoglobin (HbA) have been reported.
-insertion of incorrect amino acid into either β or α-chain
during protein synthesis
•Haemoglobin variants may function normally or abnormally
depending on the nature and position of the substitution
Phe CD1(42)β → Ser
Val E11(67) β → Asp
Leu H19(136) α → Pro
Gly B6(24) β → Val
Tyr C1(35) α → Phe
His E7(58) α → Tyr
Val E11(67) β → Glu
His F8(87) α → Tyr
Asp G1(99) β → His
Asn G4(102) β → Thr
Weakens heme binding
Weakens heme binding
Disrupts the H helix
Disrupts the B-E helix interface
Disrupts hydrogen bonding at the α1-β1
Promotes methemoglobin formation
Promotes methemoglobin formation
Promotes methemoglobin formation
Disrupts a hydrogen bond that stabilizes
the T conformation
Disrupts a hydrogen bond that stabilizes
the R conformation
Glu A6(6) β → Val Deoxyhemoglobin S forms insoluble
filaments that deform erythrocytes.
Mutant Val on one β subunit interacts
in hydrophobic pocket of another β
subunit , forming linear polymers.
Sickle Cell Disease
common hereditary blood disorder
common of the conditions is sickle cell anaemia (SCA)
affecting mainly the black population.
SCA, the Haemoglobin called HbS contains normal α-chains
but its β-chain contain valine instead of glutamate at
residue 6, ie, a hydrophobic amino acid replaces an acidic
hydrophobic valine is able to interact with the β85-Phe and
β88-leu of an adjacent deoxy HbS.
Consequences of the alteration:
•Modification of the Hb conformation, stacking of 280
million Hb molecules within each erythrocyte altered by
the production of fibrous aggregates.
in shape of erythrocytes from a biconcave disc to
a crescent or sickle shape on deoxygenation
In homozygotes the erythrocytes interact to form
clumps, occlusion of capilaries and consequent reduction
in blood flow. Organ damage!
is characterized by episodes of pain, chronic
hemolytic anemia and severe infections, usually beginning
in early childhood
Sickle cell anaemia
conditions such as low
O2 levels, RBCs with HbS
distort into sickle cells
sickled cells can
block small vessels
occlusions which may
cause necrosis of the
Sickle Cell Anaemia
–gel electrophoresis. Because sickle
hemoglobin lacks a glutamate, it is less acidic
than HbA. Hemoglobin HbS, therefore, does
not migrate as rapidly towards the anode as
–It is also possible to diagnose sickle-cell
anemia by recombinant DNA techniques.
SCA – Management
a combination of fluids, analgesics,
antibiotics and transfusions are used to
symptoms and complications.
–Hydroxyurea, an antitumor drug, has been
shown to be effective in preventing painful
–Hydroxyurea induces the formation of fetal
Hb (HbF) - a Hb normally found in the fetus
or newborn - which, when present in
individuals with SCA, prevents sickling.
Degradation of Proteins
have both extracellular and intracellular pathways for
The major extracellular pathway is the system of digestive
proteases, which break down ingested proteins to
polypeptides in the intestinal tract.
endoproteases such as trypsin and chymotrypsin, which
cleave the protein backbone adjacent to basic and aromatic
exopeptidases, which sequentially remove residues from
the N-terminus (aminopeptidases) or C-terminus
(carboxypeptidases) of proteins; and
peptidases, which split oligopeptides into di- and
tripeptides and individual amino acids.
These small molecules then are transported across the
intestinal lining into the bloodstream
life span of intracellular proteins varies from
as short as a few minutes for mitotic cyclins, which
help regulate passage through mitosis, to as long as
the age of an organism for proteins in the lens of
have several intracellular proteolytic
pathways for degrading misfolded or denatured
proteins, normal proteins whose concentration must
be decreased, and foreign proteins taken up by the
major intracellular pathway involves
degradation by enzymes within lysosomes,
membrane-limited organelles whose interior is
from the lysosomal pathway are
cytosolic mechanisms for degrading proteins.
The best-understood pathway, the ubiquitinmediated pathway, involves two steps: •addition
of a chain of ubiquitin molecules to
an internal lysine side chain of a target protein
of the ubiquitinated protein by a
proteasome, a large, cylindrical multisubunit
numerous proteasomes present in the cell
cytosol proteolytically cleave ubiquitin-tagged
proteins in an ATP-dependent process that yields
peptides and intact ubiquitin molecules
The Ubiquitin-mediated Pathway
be targeted for degradation by the ubiquitinmediated pathway, a protein must contain a
structure that is recognized by a ubiquitinating
conjugating enzymes recognize different
degradation signals in target proteins.
– For example, the internal sequence Arg-X-X-Leu-Gly-X-Ile-
Gly-Asx in mitotic cyclin is recognized by the ubiquitinconjugating enzyme E1.
–Internal sequences enriched in proline, glutamic acid,
serine, and threonine are recognized by other enzymes.
The Ubiquitin-mediated Pathway contd
life span of many cytosolic proteins is
correlated with the identity of the N-terminal
residue, suggesting that certain residues at the Nterminus favor rapid ubiquitination.
–For example, short-lived proteins that are degraded
within 3 minutes in vivo commonly have Arg, Lys, Phe,
Leu, or Trp at their N-terminus.
–In contrast, a stabilizing amino acid such as Cys, Ala,
Ser, Thr, Gly, Val, or Met is present at the N-terminus in
long-lived proteins that resist proteolytic attack for more
than 30 hours.
The Ubiquitin-mediated Pathway
newly synthesized proteins have
methionine, a stabilizing amino acid, at
enzymatic alteration that
generates one of the destabilizing amino
acids at the N-terminus is necessary to
target a protein for degradation
is the breaking of the
noncovalent bonds which determine the
structure of a protein.
disruption of tertiary structure is
achieved by reduction of the disulfide bonds
in a protein.
Generally, the denatured protein will lose
its activity, antigenicity, and become
Denaturation occurs when:
–hydrogen bonds are disrupted
–disulfides are reduced
–soaps separate the hydrophobic sections
–acids or bases neutralise the salt bridges
–metals complex with functional groups to
form insoluble salts.
Any chemical or physical agent that destroys
and changes protein conformations causes
Heavy metals UV
Mechanisms of Denaturation
Disrupts low energy van der Waals forces in
of pH: Lead to changes in the charge of
the protein’s amino acid side chains and results in
the disruption of electrostatic and hydrogen bonds.
like Triton X-100 (nonionic, uncharged)
and sodium dodecyl sulfate (SDS, anionic, charged)
disrupt the hydrophobic forces which fold proteins.
Charged detergents like SDS also disrupt
Mechanisms of denaturation contd
and guanidine hydrochloride disrupt
hydrogen bonding by forming hydrogen
bonds with the protein’s amino acid side
chains that are stronger than those in the
– In addition, these two reagents can disrupt
hydrophobic effects much like detergents.
(βME) and dithiothreitol
(DTT) reduce disulfide bonds.
In vitro denaturation and renaturation of proteins
with an 8 M urea
solution containing mercaptoethanol
(HSCH2CH2OH) completely denatures
The urea breaks intramolecular
hydrogen and hydrophobic bonds,
and the mercaptoethanol reduces
each disulfide bridge (-S-S-) to two
sulfhydryl (-SH) groups.
When these chemicals are
removed by dialysis, the SH groups
on the unfolded chain oxidize
spontaneously to re-form disulfide
bridges, and the polypeptide chain
simultaneously refolds into its native
Mechanisms of denaturation contd
such as urea or guanidinium
chloride effectively disrupt the noncovalent
β-mercaptoethanol - In the presence of a
large excess of β-mercaptoethanol, a protein
is produced in which the disulfides (cystines)
are fully converted into sulfhydryls
Role of β -Mercaptoethanol in Reducing Disulfide Bonds
Disulfides are reduced, and the βmercaptoethanol is oxidized and forms
Denaturation of Ribonuclease
polypeptide chains devoid of cross-links assume a
random-coil conformation in 8 M urea or 6 M guanidinium
When ribonuclease was treated with β-mercaptoethanol in 8 M
urea, the product was a fully reduced, randomly coiled
polypeptide chain devoid of enzymatic activity.
Reestablishing Correct Disulfide Pairing
ribonuclease can be
reformed from scrambled
ribonuclease in the presence of a
trace of b-mercaptoethanol