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Course Title: Biochemistry and Molecular Biology
Course No. PHR 202
Course Teacher: Shahana Sharmin
Department of pharmacy, BRAC University
Bonds in Protein
• Peptide bond: In protein , the α-
carboxyl group of one amino acid is
joined to the α-amino group of another
amino acid by peptide bond with
removal of water. It is also called an
• Many amino acids joined by peptide
bonds form a polypeptide chain, which
is not branched.
• An amino acid in a polypeptide is
called a residue.
• The sequence of amino acids in a
polypeptide chain is written starting
with the amino terminal residue. Thus
in the tripeptide Ala-Gly-Try(AGW),
alanine is the amino terminal residue
and tryptophan is the carboxy terminal
• A polypeptide chain consists of a
regularly repeating part, called the
main chain and a variable part
comprising the distinctive side chain.
The main chain is sometime called
Peptide bond is rigid and planer
• Pauling and Corey found that peptide unit is
rigid & planner. Resonance stabilization of the
bond between the carbonyl carbon & the
nitrogen atom of the peptide unit confers partial
double bond character and hence rigidity on the
C-N bond. There is no freedom of rotation about
• Consequently all four atoms of the peptide unit
lie in the same plane. The length of the peptide
bond is 1.32A, which is between that of C-N
single bond (1.49A) & C=N double bond (1.27A).
• In contrast, the link between the α-carbon & the
peptide nitrogen & the bond between the α-
carbon & the carbonyl are pure single bond.
Consequently there is a large degree of
rotational freedom about these bonds on either
side of the rigid peptide unit. The rigidity of the
peptide bond enables protein to have well
defined 3D forms. The freedom of rotation on
either side of the peptide unit is equally important
because it allows proteins to fold in many
Bonds in Protein…
• Disulfide bond: In addition to peptide
bonds, covalent disulfide bonds can
form between cysteine residues by
forming a sulfide bridge (-S-S-) formed
from the same or different
polypeptides. The product is a cysteine
• The cross links are formed by the
oxidation of two cystein residues.
• Intercellular proteins usually lack
disulfide bonds, whereas extracellular
proteins often contain several.
• Importance of disulfide bond:
• Without it protein has no
pharmacological or enzymatic
• This bond confer additional stability to
specific confirmations of proteins such
as enzyme(e.g. Ribonuclease) and
structural proteins (e.g.. Keratin).
• It is important for the 3D structure of
proteins for performing the function.
Figure : The disulfide bond between two cysteine residues.
• Amino acids are molecules containing an
amine group, a carboxylic acid group and a side-
chain that varies between different amino acids.
The key elements of an amino acid are carbon,
hydrogen, oxygen, and nitrogen.
• An alpha-amino acid has the generic formula
H2NCHRCOOH, where R is an organic
substituent, the amino group is attached to the
carbon atom immediately adjacent to the
carboxylate group (the α–carbon).
Proteins are built of 20 amino acids
• Amino acids with aromatic side chain:
Phenylalanine (F) Tyrosin(Y) Tryptophan(W)
– All the three have π electron clouds.
– Phenylalanine & Tryptophan are hydrophobic but Tyrosin is hydrophilic.
– Tryptophan contains an indole ring.
• Amino acids containing sulfur atoms:
Cysteine (C) Methionine (M)
– Both amino acids are hydrophobic.
– Cystein contains a sulfhydral group (-SH) & Methionine contains a sulfur atom in threonine linkage
Proteins are built of 20 amino acids
• Amino acids containing aliphatic hydroxyl groups:
Serine(S) Threonine (T)
– Both of them are much more hydrophilic and reactive.
• Amino acids containing basic side chain:
Lysine (K) Arginine (R) Histidine (H)
– All are hydrophilic .
– Lysine and Arginine are positively charged at neutral pH but Histidine can be uncharged or
positively charged depending on its local environment.
– Histidine is often found in the active sited of enzymes, where its imidazole ring canreadily switch
between those states to catalyze the making and breaking bonds.
• Amino acids containing acidic side chains and their amide derivatives:
Aspartate (D) Glutamate (E)
Asparagine (N) Glutamine (Q)
– Uncharged derivatives of Aspartate and Glutamate are Asparagine and Glutamine,
which contain a terminal amide group in place of a carboxylate.
Proteins are built of 20 amino acids
Protein have unique amino acid
• In 1953, Fredrick Sanger determined the amino acid sequence of
Insulin, a protein hormone. This work is a landmark in biochemistry
because it showed for the first time that a protein has a precisely
defined amino acid sequence.
• Sanger’s approach was first to separate the two polypeptide chain A
& B of insulin & then to convert them by specific enzymatic cleavage
into smaller peptides that contained regions of overlapping
sequence. Using 1-fluoro-2,4- dintrobenzine (Sanger’s reagent) he
removed & identified one at a time, the amino terminal residues of
Protein have unique amino acid
• By comparing the sequence of overlapping peptides, he deduced an
unambiguous primary structure of both the A & the B chain.
• Now the complete sequence of more than 1000 proteins are known.
The sequence of nucleotides in RNA, which in turn specifies the
amino acid sequence of a protein. In particular, each of 20 amino
acid is encoded by one or more specific sequences of three
nucleotides. Furthermore, proteins in all organisms are synthesized
from their constituent amino acids by a common mechanism.
Importance of amino acid
• Amino acids sequences are important for several reasons
• Knowledge of the sequence of a protein is very helpful indeed
essential in elucidating its mechanism of action.
• Analysis of relations between amino acid sequence & 3D structure
of proteins are uncovering the rules that govern the folding of
polypeptide chains. Amino acid sequence is the link between the
generic message in RNA & 3D structure that performs a proteins
• Sequence determination is part of molecular pathology. Alterations
in amino acid sequence can produce abnormal function & disease,
such as sickle cell anemia & cystic fibrosis.
• The sequence of a protein reveals much about its evolutionary
history. Protein resemble one another amino acid sequence only if
they have a common ancestor. Consequently molecule events in
evolution can be traced from amino acid sequences.
• The alpha helix is one of the most common secondary structures in
proteins. Amino acid side chains project outwards from the polypeptide
backbone that forms the core of the helix.
• The chain is stabilized in this conformation by hydrogen bonds between the
backbone amino group of one amino acid and the backbone carbonyl group
of another amino acid that is four positions away.
• These interactions do not involve side chains. Thus
many different sequences can adopt an a helical structure.
• Alpha helices are regular cylindrical structures.
• One full turn occurs every 3.6 residues and extends the length of the helix
by approximately 0.5 nm.
• The screw sense of a helix can be right handed (clockwise) or left handed
(counterclockwise), the α-helices found in protein are right handed.
• The α-helical content of proteins ranges widely, from nearly none to almost
100%. For example –
– The digestive enzyme chymotripsin is virtually devoid of α-helix.
– In contrast, about 7.5% of myoglobin and hemoglobin is α-helical.
Coiled-coil structure of Alpha
helix: Super helix
• Two or more α-helices can entwine to form
very stable structures, which can have a
length of 100AO
or more. These structures are
called coiled-coil structure of α-helix. Such α-
helical coiled coils are found in myosin &
tropomyosin in muscle, fibrin in blood clots &
keratin in hair.
• 3-5% amino acids are responsible for coiled-
* Why the elucidation of a structure of a α-helix
is a landmark in molecular biology?
The elucidation of structure of α-helix is a
landmark in molecular biology because it
demonstrated that the confirmation of a
polypeptide chain can be predicted of the
properties of its compounds are rigorously &
Beta pleated sheet
• The beta sheet is another common secondary structure. In contrast
to an alpha helix, it is formed by hydrogen bonds between backbone
atoms on adjacent regions of the peptide backbone, called beta
• These interactions do not involve side chains. Thus, many different
sequences can form a beta sheet.
• A beta sheet is a regular and rigid structure often represented as a
series of flattened arrows. Each arrow points towards the proteins
• In the example shown here the two middle strands run parallel—that
is, in the same direction—whereas the peripheral strands are anti-
• The amino acid side chains from each strand alternately extend
above and below the sheet, thereby allowing each side to have
distinct properties from the other. Beta sheets are usually twisted
and not completely flat."
Animation of Beta pleated sheet
Difference between α-helix &
• A polypeptide chain in a β-pleated sheet called β-
strands, is almost fully extended rather than being tightly
coiled as in the α-helix.
• The axial difference in β-sheet between adjacent amino
acids is 3.5 AO
, in contrast with 1.5AO
for the α-helix.
• Another differences is that the β-pleated sheet is
stabilized by hydrogen bonds between NH & CO groups
in different polypeptide strands, whereas in the α-helix
the hydrogen bonds are between NH & CO group in the
• According to running directions β-sheet is classified as
parallel & anti-parallel & according to screw, sense α-
helix is clockwise & anticlockwise.
Beta-turn or hairpin turn
• Most proteins have compact,
globular shapes which can
change direction frequently. The
predominant type in cytosol
changes direction of β-sheets are
accomplished by the structure
known as β-turn or β-bend that
often connects the ends of two
adjacent strands of anti-parrallel
β-sheet. The essence of this
hairpin turn is that the CO group
of residue n of a polypeptide is
hydrogen bonded to the NH group
of residue n+3. Thus polypeptide
chain can abruptly reverse its
direction. They are also known as
reverse turn or hairpin bend.
Fig : Structure of β-turn. The NH & CO
groups of residue 1 of the tetra-polypeptide
shown here are hydrogen bonded,
respectively, to the CO & NH groups of
residue 4 which results in a hairpin turn)
• According to the architecture protein have four structure levels.
– Primary structure
– Secondary structure
– Tertiary structure
– Quaternary structure
• Primary structure: The linear sequence of amino acids in a whole protein is called primary
structure of proteins.
Understanding the primary structure of protein is necessary to diagnose or study certain disease
which result in proteins with abdominal amino acid sequences.
• Secondary structure: It is the 3D structure of short (3 to 30 residue), adjacent segments of
polypeptide into geometrically ordered units where the amino acids are located near to each
other in the linear sequence. E.g. α-helix, β-sheet.
• Tertiary structure: It is the entire 3D of whole protein where the amino acids are located close
each other or far away from each other in linear sequence.
It indicates the 3D space, how secondary structure features- helices, sheets, bends, turns &
loops assemble to form domains & how three domain relate spatially to one another.
• Quaternary structure: It is the combination of more than one polypeptide or protein that may be
structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called
the quaternary structure of the protein. If there are two subunits the protien is known as dimeric, if
there are three subunits, trimeric & if there are several subunits , multimeric. E.g. haemoglobin is
trimeric which contains 4 polypeptide chains (two α chains & two β chains)
Subunits are held together by non-covalent interactions. (E.g. H-bond, ionic bond, etc. )
Animation of Protein structure
Classification of amino acids according to
their H-bond potentialities..
• According to H-bond potentialities, amino acids can be classified into following 3
• Hydrogen bond donor: The side chains of Tryptophan & Arginine can serve as H-
bond donors only.
• Hydrogen bond donor & acceptor: Like the peptide unit itself, the side chains of
Asparagine, Glutamine, Serine & Threonine can serve as hydrogen bond donors &
H donor group of Tryptophan H donor group of Arginine
H acceptor & donor group of GlutamineH acceptor & donor group of Asparagine
H donor H donor
Classification of amino acids according to
their H-bond potentialities..
• Hydrogen bond donor and/or acceptors: The hydrogen bonding capabilities of
Lysine (the terminal –NH2 group), Aspartic & Glutamic acid (the terminal COOH
group), Tyrosin & Histidine vary with pH. The H-bonding modes of these ionizable
residues are pH dependent.
Protonated form of Aspartic Acid
Ionized form of Glutamic Acid
Protein possess 3D structure
• Protein possess 3D structure. The unique dimensional structure of each polypeptide is
determined by its amino acid sequence. Several types of interactions cooperate in
stabilizing tertiary structures of protein.
• Disulfide bond: A disulfide bond is a covalent linkage from the sulfahydral group (-SH) of
each of two cystine residue to produce a cystine residue. When cystine residues perform
covalent bonding in their side chains, the polypeptide chain (-S-S-) becomes folded.
• Hydrophobic interactions: In an aqueous environment, protein fold is driven by the
strong tendency of hydrophobic residues to be excluded from water. Note that water is
highly cohesive & the hydrophobic groups are thermodynamically more stable when
cluster in the interior of the molecule than when extended into the aqueous surroundings.
The polypeptide chain therefore fold spontaneously so that its hydrophobic side chains are
buried and its polar, charged chains are on the surface. Thus 3D structure formation is
• Presence of amino acid Proline: Proline has a cyclic structure which markedly present in
protein structure. Proline reverse the dimension of the polypeptide chain and thus provides
• Hydrogen bonds: Amino acid side chains containing oxygen or nitrogen bound hydrogen
can form hydrogen bonds with electron-rich atoms. For example – alcohol group of Serine
& Threonine can form hydrogen bond with oxygen of carboxyl groups or carboxy groups of
peptide bonds. Formation of hydrogen bonds enhance the formation and stabilization of
three dimensional structure of protein.
Denaturation of protein
• Denaturation of protein means destruction of 3D structure of native or wild
• A native protein is converted into a denatured protein by cleavage of
disulfide bonds present in it. For e.g. –
• Ribonuclease a single polypeptide chain consisting of 124 amino acid
residues has four disulfide bonds. This four disulfide bonds can be cleaved
reversibly by reducing them with reducing agents. There are two types of
• Physical agent
– Thermal Denaturation.
– Mechanical treatments.
– Hydrostatic Pressure.
• Chemical agent
– Aqueous Organic Compounds.
– Effect of pH.
– Changes in Dielectric constant.
– Ionic strength.
• Thermal denaturation : Rate of denaturation depends on the temperature.
As Temperature is increased
– Affect interactions of tertiary structure
– Increased flexibility → reversible
– H-bonds begin to break → water interaction
– Increased water binding
– Increased viscosity of solution
– Structures different from native protein
– Loss of solubility
– Water content affects heat denaturation
– Splitting of disulfide bonds
– Chemical alterations of amino residues
– Inter- or Intra- crosslinks
• Liquid-air or Liquid-liquid interfaces
• If allowed at interfaces → Unfold
• Depends on
– Rigidity of the 3-D structure
– Number and location of hydrophobic groups
– Accelerated if applied energy to cause shear
• Aqueous Organic Compounds:
• Urea and Guanidine salts
Decrease hydrophobic interactions
• Surface-active agents (SDS)
Disrupt hydrophobic interactions
Increase internal repulsive forces
• Reducing agents
Reduce disulfide crosslinks (β –mercaptoethanol)
• Effect of pH:
• Proteins at physiological pH
When pH is lower than pI --
• Strong acid with high temperature causes deamination followed by Hydrolysis peptide
When pH is higher than pI --
• Formation of fibers causes denaturation
• Chemical modifications lead to denaturation
• Very high pH at elevated temperature results in peptide hydrolysis.
This agents have different mechanism of action. β-marcaptoethanol is
used to reduce the disulfide bond present in a native protein and β-
marcapto forms mixed disulfides with Cystein side chains. In the
presence of excess amount of β-marcaptoethanol the mixed
disulfides also are reduced, so that the final product is a protein in
which the disulfides (Cysteins) are fully converted into sulfhydrals
Mechanism of action of reducing
agent for Denaturation…
• Mechanism of action of reducing agent:
2. When ribonuclease can not be readily reduced by β-marcaptoethanol at
C and pH 7, then the Proline is partly unfolded by agents such as Urea or
Guanidine hydrochloride. These agents disrupt noncovalent interactions.
Thus when ribonuclease is treated with β-marcaptoethanol in 8M urea, the
product is fully reduced, the poly chain becomes devoid of enzymatic
activity. In other words ribonuclease is denatured by this treatment.
Mechanism of action of reducing
agent for Denaturation…
Effects of Denaturation in the body
Protein Denaturation results in the unfolding & disorganization of
the protein structure, which are accompanied by hydrolysis of
peptide bonds & finally protein loses its biological activity.
Denaturation of protein hamper the following functions-
1. Specific protein molecules called enzyme loses their catalytic
power & all chemical reactions in biological systems catalyzed by
those enzymes are hampered.
2. Proteins e.g. – hemoglobin, myoglobin etc. can not transport
molecules or ions.
3. Muscle contraction may be hampered.
4. Highly specific proteins like antibodies can not maintain the
5. Receptor proteins can not generate & transmit nerve impulses.
6. Improved digestibility
7. Increased intrinsic viscosity
8. Inability to crystallize
Renaturation of protein
• During renaturation, all the disulfide, hydrogen, and
hydrophobic bonds that stabilize the native
conformation are re-formed.
• Christian Anfinsen made a critical observation that the
denatured ribonuclease, freed of urea and β-
mercaptoethanol by dialysis, slowly regained enzymatic
• This prove that in suitable oxidizing condition,
denatured reversible produce oxidsed protein.
• For e.g. denatured ribonuclease is converted into native
ribonuclease by the following process-
– At first the denatured protein is freed from urea &
β-mercaptoethanol by dialysis in dialysis bagskept
in buffer. Then it is exposed to air for air oxidation
of the sulfahydral groups in reduced ribonuclease.
When the sulfhydral of the denatured enzyme
become oxidised by air, the enzyme is
sponteneaously refolded into a catalyticallly active
– Detailed study shows that nearly all the original
enzymatic activity is regained if the sufhydral are
oxidized under properties of refolded enzyme will
be virtually identical with those of the native
• A large amount of protein in there native state are not pure. Several thousand protein have
been purified in active from on the basis of some characteristics, such as
– Specific binding affinity
• At each step in purification, the preparation is assayed for a distinctive property of the protein
of interest (e.g. – enzymatic activity) to assess the efficacy of the procedure. There are
various methods of purification of protein. They are -
– Purification according to size :
• Gel filtration chromatography.
– Purification according to solubility:
• Salting out
– Purification based on charge:
• Ion exchange chromatography
– Purification based on affinity:
• Metal-Chelate affinity chromatography
Protein purification according to size
• From a mixture of protein of different sizes, desired
proteins can be separated from small proteins and other
molecules by dialysis. The procedure is –
– The protein mixture is taken in a dialysis bag made
up of semi-permeable membrane, such as cellulose
membrane with pores.
– The bag is then kept in a buffer solution maintaining
the pH of buffer solution to that pH at which the
desired protein is stable.
– Then the molecules having dimensions significantly
greater than the pore diameter are retained inside
the dialysis bag, whereas smaller molecules and
ions traverse the pore of such a membrane and
emerge in dialysate outside the bag.
• We will not get 100% pure protein molecules by this
method because the intermediate size molecules are
also present in the dialysis bag with large molecules.
• To make this process as accurate as possible, we must
know the molecular weight of the desired protein to fix
the molecular weight cut off.
Protein molecules (red) are retained
within the dialysis bag, whereas small
molecules (blue) diffuse into the
• Gel-Filtration Chromatography:
• More discriminating separations on the basis of size can be achieved
by the technique of gel-filtration chromatography .
• The sample is applied to the top of a column consisting of porous beads
made of an insoluble but highly hydrated polymer such as dextran or
agarose (which are carbohydrates) or polyacrylamide. Sephadex,
Sepharose, and Bio-gel are commonly used commercial preparations
of these beads, which are typically 100 μm (0.1 mm) in diameter.
• Small molecules can enter these beads, but large ones cannot. The
result is that small molecules are distributed in the aqueous solution
both inside the beads and between them, whereas large molecules are
located only in the solution between the beads. Large molecules flow
more rapidly through this column and emerge first because a smaller
volume is accessible to them.
• More larger quantities of protein can be separated by gel filtration
chromatography than by gel electrophoresis but at the price of lower
Protein purification according to size
Figure : Gel Filtration Chromatography
A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large
proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones.
Protein purification according to solubility
• Salting out:
• The solubility of most proteins is lowered at high salt concentration. This effect is
called salting out which is very useful.
• The dependence of solubility on salt concentration differs from one protein to
another. Hence salting out can be used to fractionate proteins. E.g. 0.8 M ammonium
sulfate precipitates fibrinogen, a blood clotting protein, where as 2.4 M is needed to
precipitate serum albumin.
• At first protein mixture is added to a certain concentration of (NH4)SO4 salt and some
ppt is formed. Thus protein mixture is added to different concentrations of salt.
Different salt concentrations causes precipitation of different proteins. Precipitation of
different proteins are collected and dissolved in buffer [Phosphate buffer –
combination of KH2PO4 (acidic) and K2HPO4 (basic)]. Then by running them in SDS-
PAGE with marker desired protein is detected. The salt concentration which caused
precipitation of that desired protein is used for further purification of that desired
– It is used for purifying proteins.
– It is used for concentrating dilute solution of protein.
– It is used for fractionating protein.
• Ion-Exchange Chromatography:
• Proteins can be separated on the basis of their
net charge by ion-exchange chromatography. If a
protein has a net positive charge at pH 7, it will
usually bind to a column of beads containing
carboxylate groups, whereas a negatively
charged protein will not .
• A positively charged protein bound to such a
column can then be eluted (released) by
increasing the concentration of sodium chloride
or another salt in the eluting buffer because
sodium ions compete with positively charged
groups on the protein for binding to the column.
• Proteins that have a low density of net positive
charge will tend to emerge first, followed by those
having a higher charge density. Positively
charged proteins (cationic proteins) can be
separated on negatively charged carboxymethyl-
cellulose (CM-cellulose) columns.
• Conversely, negatively charged proteins (anionic
proteins) can be separated by chromatography
on positively charged diethylaminoethyl-cellulose
Protein purification based on charge
• Metal-Chelate Affinity Chromatography:
• It is another powerful and general applicable means of purifying proteins. It is
considered as the best method of protein purification. It is called one stop
• In this procedure, there is a complex between Histidine-Imidazole ring(of protein)
and metal. Under suitable conditions, high concentration of imidazole releases
protein from the complex of metal-protein. This is a very specific method for
purification of protein.
• Different metals are used for the purpose of purification by this process, e.g. –
nickel, cobalt, calcium, manganese, cadmium, zinc, copper etc.
• Insulin is a protein which is purified by this method where about 95% pure insulin
can be obtained. If this method is performed after dialysis, the yield will be 95%.
• Collection of protein solution / mixture:
• At first the desired protein is converted into histidine-tag-protein. For e.g., we will
use histidine-tag-insulin which is obtained by cloing insulin into a plasmid
containing histidine-tag DNA. Then it is inserted in E.coli and plasmid will produce
insulin containing six histidine (the histidine-tag-insulin) Sonication and
centrifugation of such E. coli will produce hundreds of proteins in the solution
Protein purification based on affinity
• Then Ni-sulfate is poured into resin column where resin-Ni complex is
formed. Protein solution is then poured into resin column and there will be a
complex of resin-Ni-histidine tag insulin. No other protein binds with the
complex and simply go through the column.
• As nickel binds with the nitrogen of imidazole ring present in histidine the
resin-Ni-histidine tag insulin forms a strong complex. Now, finally, very high
concentrated imidazole ring is added to the column so that Ni binds with this
imidazole and release the histidine-tag-insulin. Then this released histidine-
tag-insulin is collected to perform dialysis so that excess imidazole becomes
separated. Thus, 95% pure protein is obtained.
• The actual insulin and modified histidine-tag-insulin have some biological
Protein purification based on affinity
• Electrophoresis: A molecule with a net charge will move in an electric field. This
phenomenon is termed as electrophoresis. This offers a powerful means of separating
proteins and other macromolecules, such as DNA and RNA.
• Why the process is carried out in gel?
Electrophoretic separations are nearly always carried out in gels (or on solid support
such as paper) rather than in free solution, for two reasons:
– Firstly, gels suppress connective currents produced by small temperature
gradients, a requirement for effective separation.
– Secondly, gels serve as molecular sieves that enhances separation.
• Mobility of molecules in gel:
– Molecules that are small compared with the pores in the gel readily through the
– Molecules much larger than the pores are almost immobile.
– Intermediate-size molecules move through the gel with various degrees of facility.
• Media of electrophoresis:
• Polyacrylamide gels are choice supporting media for electrophoresis because
– They are chemically inert and are readily formed by the polymerization of
– Morever, their pore sizes can be controlled by choosing various concentrations of
acrylamide and methylene-bis-acrylamide at the time of polymerization.
Formation of polyacrylamide gel
Acrylamide reacts with
form polyacrylamide gel
where Pursulfate (S2O8
converted into Sulfate free
) which act as
Figure: Formation of a
A three-dimensional mesh
is formed by co-
monomer (blue) and cross-
• Proteins can be separated largely on the basis of mass by electrophoresis in a polyacrylamide gel
under denaturating conditions.
• The mixture of protein is first dissolved in a solution of sodium dodecyl sulfate (SDS), an anionic
detergent that disruptd nearly all noncovalent interactions in native proteins.
• Marcaptoethanol or dithiothreitol is also added to reduce disulfide bonds.
• Anion of SDS bind to main chains at a ratio about one SDS for every two amino acid residues,
which gives a complex of SDS with a denatured protein a large net negetive charge that is
roughly proportional to the mass of the protein.
• The negetive charge acquired on binding SDS is usually much greater than the charge on the
• The SDS complexes with the denatured proteins are then electrophorised on a polyacrylamide
gel, typically in the form of a thin vertical slab.
• The directyion of electrophoresis is from top to bottom.
• Finally, the proteins in the gel can be visualized by staining them with silver or a dye such as
Coomassic blue, which reveals a series of bands.
• Radioactive labels can be detected by placing a sheet of X-ray film over the gel, a process
• Small proteins move rapidly through the gel, whereas large ones stay at the top, near the point
of application of the mixture.
• The mobility of most polypeptide chain under these conditions is inversely proportionate to the
logarithm of their mass.
• Thus proteins of different molecular weight are separated and from the bands or SDS-PAGE,
molecular weight of different proteins determined.
Figure : Polyacrylamide Gel Electrophoresis
(A)Gel electrophoresis apparatus. Typically, several samples undergo electrophoresis on one
flat polyacrylamide gel. A microliter pipette is used to place solutions of proteins in the wells of
the slab. A cover is then placed over the gel chamber and voltage is applied. The negatively
charged SDS (sodium dodecyl sulfate)-protein complexes migrate in the direction of the
anode, at the bottom of the gel.
(B)(B) The sieving action of a porous polyacrylamide gel separates proteins according to size,
with the smallest moving most rapidly.
Figure: Staining of Proteins After
Electrophoresis. Proteins subjected to
electrophoresis on an SDS-
polyacrylamide gel can be visualized by
staining with Coomassie blue.
Figure :Electrophoresis Can Determine Mass
The electrophoretic mobility of many proteins in
SDS-polyacrylamide gels is inversely
proportional to the logarithm of their mass.
• Proteins can also be separated electrophoretically on the basis of their
relative contents of acidic and basic residues.
• The isoelectric point (pl) of a protein is the pH at which its net charge is
zero. At this pH, its electrophoretic mobility is zero because z in equation 1
is equal to zero.
• For example, the pI of cytochrome c, a highly basic electron-transport
protein, is 10.6, whereas that of serum albumin, an acidic protein in blood, is
4.8. Suppose that a mixture of proteins undergoes electrophoresis in a pH
gradient in a gel in the absence of SDS.
• Each protein will move until it reaches a position in the gel at which the pH is
equal to the pI of the protein. This method of separating proteins according
to their isoelectric point is called isoelectric focusing.
• The pH gradient in the gel is formed first by subjecting a mixture of
polyampholytes (small multicharged polymers) having many pI values to
• Application: Isoelectric focusing can readily resolve proteins that differ in pI
by as little as 0.01, which means that proteins differing by one net charge
can be separated.
Figure :The Principle of Isoelectric Focusing
A pH gradient is established in a gel before loading the sample.
(A) The sample is loaded and voltage is applied. The proteins will migrate to their
isoelectric pH, the location at which they have no net charge.
(B) The proteins form bands that can be excised and used for further
Two-dimensional method/Combined method
• Isoelectric focusing can be combined with SDS-PAGE to obtain very high
• A single sample is first subjected to isoelectric focusing.
• This single-lane gel is then placed horizontally on top of an SDS-polyacrylamide
• The proteins are thus spread across the top of the polyacrylamide gel according
to how far they migrated during isoelectric focusing.
• They then undergo electrophoresis again in a perpendicular direction (vertically)
to yield a twodimensional pattern of spots.
• In such a gel, proteins have been separated in the horizontal direction on the
basis of isoelectric point and in the vertical direction on the basis of mass.
• By this combined method, we can separate thousnad of proteins.
• We can purify protein molecules
• Two proteins having same isoelectric point (pI) but different mole. Wt. can be
• Two proteins having different isoelectric point (pI) but same mole. Wt. can be
Two-dimensional method/Combined method
Figure :Two-Dimensional Gel Electrophoresis
(A) A protein sample is initially fractionated in one dimension by isoelectric focusing as described .
The isoelectric focusing gel is then attached to an SDS-polyacrylamide gel, and electrophoresis is
performed in the second dimension, perpendicular to the original separation. Proteins with the same
pI are now separated on the basis of mass. (B) Proteins from E. coli were separated by two-
dimensional gel electrophoresis, resolving more than a thousand different proteins. The proteins were
first separated according to their isoelectric pH in the horizontal direction and then by their apparent
mass in the vertical direction.
Synthesis of Peptide
• General consideration:
• Peptides are synthesized by linking an amino group to a carboxyl group that has been
activated by reacting it with a reagent such as dicyclohexylcarbadiimide (DCC).
• The t-BOC amino acid is activated by DCC.
• Then the amino acid reacts with another amino acid attached to resin. Here the attack of
a free amino group on the activated carboxyl leads to formation of a peptide bond an the
release of dicyclohexyl urea.
• After formation of peptide, this t-BOC protecting group can bind subsequently removed by
exposing the peptide to dilute acid which leave peptide bond intact.
• Note: Here the t-BOC amino acid is used because if a single free amino group and a
single free carboxyl group of the same amino acid can bind in presence of DCC which
inhibit the formation of peptide.
Protein synthesis by solid phase method
• Peptide can be synthesized by a solid phase method derived by R. Bruce Merrifield. Amino
acids are added stepwise to a growing peptide chain that linked to an insoluble matrix, such as
• All reactions are carried out in a single vessel, which eliminate losses due to repeated transfers
• Anchoring: The carboxy terminal amino acid of the desired peptide sequence is first anchored
to polystyrene beads.
• Deprotection: The t-BOC protecting group of this amino acid is removed by treating with F3-
• Coupling: The next amino acid (in the protected t-BOC form) and dicyclohexylcarbodiimide (the
coupling agent) are added together.
• After formation of peptide bonds, excess reagents and dicyclohexylurea are washed away,
which leaves the beads with the desired dipeptide product.
• Repitition: Additional amino acids are linked by repeating 2nd
• Release or cleavage: At the end of the synthesis, the peptide is released from the beads by
adding hydrofluoric acid (HF), which cleaves the carboxyl ester anchor without disrupting
• Protective groups on potentially reactive side chains are also removed at this time.
• The purification of the synthesized peptide is not required because the desired product at each
stage is bound to beads that can be rapidly filtered and washed.
by solid phase
Fig: Sequence of
steps in solid
(1) Anchoring of
(3)Coupling of the
next residue, step
(2) & (3) are
repeated for each
added amino acid,
(4) The complete
peptide is released
from the resin.
Importance of synthesizing peptides of
1. Synthetic peptides can serve as antigens to
stimulate the formation of specific antibodies. For
instance, as discussed earlier, it is often more efficient
to obtain a protein sequence from a nucleic acid
sequence than by sequencing the protein itself. Peptides
can be synthesized on the basis of the nucleic acid
sequence, and antibodies can be raised that target
these peptides. These antibodies can then be used to
isolate the intact protein from the cell.
2. Synthetic peptides can be used to isolate receptors
for many hormones and other signal molecules. For
example, white blood cells are attracted to bacteria by
formylmethionyl (fMet) peptides released in the
breakdown of bacterial proteins. Synthetic
formylmethionyl peptides have been useful in identifying
the cell-surface receptor for this class of peptide.
Moreover, synthetic peptides can be attached to
agarose beads to prepare affinity chromatography
columns for the purification of receptor proteins that
specifically recognize the peptides.
3. Synthetic peptides can serve as
drugs. Vasopressin is a peptide
hormone that stimulates the
reabsorption of water in the distal
tubules of the kidney, leading to the
formation of more concentrated urine.
Patients with diabetes insipidus are
deficient in vasopressin (also called
antidiuretic hormone), and so they
excrete large volumes of urine (more
than 5 liters per day) and are
continually thirsty. This defect can be
treated by administering 1-desamino-
8-d-arginine vasopressin, a synthetic
analog of the missing hormone. This
synthetic peptide is degraded in vivo
much more slowly than vasopressin
and, additionally, does not increase
the blood pressure.
4. Finally, studying synthetic peptides
can help define the rules governing
the three-dimensional structure of
Importance of synthesizing peptides of
• Edman deviced a mothod for labeling the amino-terminal residue and
cleaving it from the peptide without disrupting the peptide bonds
between the other amino acid residues.
• Principle: The Edman degradation sequentially removes one residue
at a time from the amino end of a peptide.
• Mechanism: Phenylisothiocyanate reacts with the uncharged terminal
amino group of the peptide to form a phenylthiocarbamoyl derivative.
• Then under mildly acidic condition, a cyclic derivatives of the terminal
amino acid is liberated, which leaves an intact peptide shortened by
one amino acid.
• The cylcic compound is a phenylthiohydantoin (PTH) – amino acid,
which can be identified by chromatographic procedures. [Furthermore,
the amino acid composition of the shortened peptide (Arg, Asp, Gly,
Phe) can be compared with that of the original peptide. (Ala, Arg, Asp,
• The difference between these analysis is one Alanine residue, which
shows that Alanine is the amino-terminal residue of the original peptide.
Edman Degradation (Protein
The Edman Degradation. The labeled amino-terminal residue (PTH-alanine in the first
round) can be released without hydrolyzing the rest of the peptide. Hence, the amino-
terminal residue of the shortened peptide (Gly-Asp-Phe-Arg-Gly) can be determined
Figure :Separation of PTH-Amino Acids
PTH-amino acids can be rapidly separated by high-pressure liquid chromatography (HPLC). In this
HPLC profile, a mixture of PTH-amino acids is clearly resolved into its components. An unknown
amino acid can be identified by its elution position relative to the known ones.
• Edman degradation is carried out on a machine called a
sequenator. In a liquid-phase sequenator, a thin film of
protein in a spinning cylindrical cup is subjected to the
• The reagents and extracting solvents are passed over the
immobilized film of protein, and the released PTH-amino
acid is identified by HPLC. One cycle of the Edman
degrdation is carried out in less than two hours.
• Hundreds of proteins have been sequenced by Edman
degradation of peptides derived from specific cleavages.
• It is a demanding and time consuming process.
Amino Acid sequences are sources of many
kinds of Insight
• A protein's amino acid sequence, once determined, is a valuable source of
insight into the protein's function, structure, and history.
1.The sequence of a protein of interest can be compared with all other known
sequences to ascertain whether significant similarities exist.. If the newly isolated
protein is a member of one of the established classes of protein, we can begin to
infer information about the protein's function. For instance, chymotrypsin and
trypsin are members of the serine protease family, a clan of proteolytic enzymes
that have a common catalytic mechanism based on a reactive serine residue. If
the sequence of the newly isolated protein shows sequence similarity with trypsin
or chymotrypsin, the result suggests that it may be a serine protease.
2.Comparison of sequences of the same protein in different species yields a
wealth of information about evolutionary pathways. For example, a comparison
of serum albumins found in primates indicates that human beings and African
apes diverged 5 million years ago, not 30 million years ago as was once thought.
3.Amino acid sequences can be searched for the presence of internal repeats.
Such internal repeats can reveal information about the history of an individual
4.Many proteins contain amino acid sequences that serve as signals
designating their destinations or controlling their processing. A protein
destined for export from a cell or for location in a membrane, for example,
contains a signal sequence, a stretch of about 20 hydrophobic residues near
the amino terminus that directs the protein to the appropriate membrane.
Another protein may contain a stretch of amino acids that functions as a
nuclear localization signal, directing the protein to the nucleus.
5.Sequence data provide a basis for preparing antibodies specific for a protein
of interest. Peptides with these sequences can be synthesized and used to
generate antibodies to the protein. These specific antibodies can be very
useful in determining the amount of a protein present in solution or in the
blood, ascertaining its distribution within a cell, or cloning its gene .
6.Amino acid sequences are valuable for making DNA probes that are specific
for the genes encoding the corresponding proteins. Knowledge of a protein's
primary structure permits the use of reverse genetics. DNA probes that
correspond to a part of the amino acid sequence can be constructed on the
basis of the genetic code.
Amino Acid sequences are sources of many
kinds of Insight
Recombinant DNA Technology Has Revolutionized
• Hundreds of proteins have been sequenced by Edman degradation of peptides derived
from specific cleavages. A huge effort is required to elucidate the sequence of large
proteins, those with more than 1000 residues. For sequencing such proteins, a
complementary experimental approach based on recombinant DNA technology is often
• Long stretches of DNA can be cloned and sequenced, and the nucleotide sequence
directly reveals the amino acid sequence of the protein encoded by the gene.
Recombinant DNA technology is producing a wealth of amino acid sequence information
at a remarkable rate.
• With the use of the DNA base sequence to determine primary structure, there is still a
need to work with isolated proteins. The amino acid sequence deduced by reading the
DNA sequence is that of the nascent protein, the direct product of the translational
• Cysteine residues in some proteins are oxidized to form disulfide links, connecting either
parts within a chain or separate polypeptide chains.
• Chemical analyses of proteins in their final form are needed to delineate the nature of
these changes, which are critical for the biological activities of most proteins.
1. DNA unwinds
2. mRNA copy is made of one of the DNA strands.
3. mRNA copy moves out of nucleus into cytoplasm.
4. tRNA molecules are activated as their complementary amino acids
are attached to them.
5. mRNA copy attaches to the small subunit of the ribosomes in
cytoplasm. 6 of the bases in the mRNA are exposed in the
6. A tRNA bonds complementarily with the mRNA via its anticodon.
7. A second tRNA bonds with the next three bases of the mRNA, the
amino acid joins onto the amino acid of the first tRNA via a peptide
8. The ribosome moves along. The first tRNA leaves the ribosome.
9. A third tRNA brings a third amino acid
10.Eventually a stop codon is reached on the mRNA. The newly
synthesised polypeptide leaves the ribosome.
•DNA is a very long molecule that looks like a twisted ladder.
•It is made up of 4 different subunits called nucleotides which can be
arranged in any order
•DNA has two strands.
•The strands are stuck together by the complementary bases.
•Adenine to Thymine A-T
•Cytosine to Guanine C-G
It is the Sequence of bases that act like a code
The sequence (order) of bases tells the cell what
proteins to make.
The sequence of bases dictates the sequence of
amino acids, which determines the shape of a
If the protein is the wrong shape it will not work
properly (it may work differently)
So if the sequence in the DNA is wrong it may result
in a genetic disease
(making a mRNA copy of DNA)
•The part of the DNA molecule (the gene) that the cell
wants the information from to make a protein unwinds to
expose the bases.
•Free mRNA nucleotides in the nucleus base pair with
one strand of the unwound DNA molecule.
•The mRNA copy is made with the help of RNA polymerase. This enzyme joins
up the mRNA nucleotides to make a mRNA strand.
•This mRNA strand is a complementary copy of the DNA (gene)
•The mRNA molecule leaves the nucleus via a nuclear pore into the cytoplasm
pick up their specific amino acids from the
mRNA used to make polypeptide chain
•First the mRNA attaches itself to a ribosome (to the small subunit).
•Six bases of the mRNA are exposed.
•A complementary tRNA molecule with its attached amino acid (methionine) base
pairs via its anticodon UAC with the AUG on the mRNA in the first position P.
•Another tRNA base pairs with the other three mRNA bases in the ribosome at
•The enzyme peptidyl transferase forms a peptide bond between the two amino
•The first tRNA (without its amino acid) leaves the ribosome.
The ribosome moves along the mRNA to the next codon (three bases).
The second tRNA molecule moves into position P.
Another tRNA molecule pairs with the mRNA in position A bringing its amino acid.
A growing polypeptide is formed in this way until a stop codon is reached.
End of Translation
A stop codon on the mRNA is reached and this signals the ribosome
to leave the mRNA. A newly synthesised protein is now complete!
Animation of protein synthesis
Protein Synthesis Animation Video.mp4
• Short note on:
– Iso-electric point
– Native protein
– Denatured protein
– Zwitter ion
• How α-amino acids are optically active?
• What is the difference of proline from other amino
• Why protein possess three dimensional structure?
• Biochemistry, 5th ed., J.M. Berg, J.L. Tymoczko
and L. Stryer, Freeman (1995) (or an earlier
• Principles of Biochemistry – Leninger.