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Proteomics
Preamble to Proteomics
Preamble to Proteomics
The term “proteome” describes the protein complement
expressed by a genome. The study of the full set of proteins
encoded by a genome is known as proteomics. This large scale
study of protein structure and function, first requires a
thorough understanding of protein composition and their
various structural levels.
Learning Objective
After interacting with this Learning Object, the learner will
be able to,
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Recall Amino acids structures & classification.
Describe acid-base properties of Amino-acids.
Define Peptide bond formation, and,
Recall protein structural levels.
Proteomics
Preamble to Proteomics
Amino acid structures & classification
Amino acids are the building blocks or
monomers that make up proteins.
They consist of a central alpha carbon
atom bonded covalently to an amino
group, a carboxyl group, a hydrogen
atom and a variable side chain, also
called the R group. While most amino
acids have a primary amino group,
proline consists of a secondary amine
group and is therefore an imino acid.
Proteomics
Preamble to Proteomics
Amino acid structures & classification
All amino acids except glycine have a
non-superimposable mirror image due
to the spatial arrangement of four
different groups about the chiral
carbon atom. Rotation of either
isomer about its central axis will never
give rise to the other isomeric
structure.
Proteomics
Preamble to Proteomics
Amino acid structures & classification
Amino acids are classified based on
the properties of their side chains or R
groups which vary in size, structure
and charge. The polarity of the side
chains is one of the main basis for
classification.
Proteomics
Preamble to Proteomics
Acid- base properties of amino acids
All amino acids exhibit a characteristic
titration curve with distinct pK values.
Amino acid taken in an acidic medium
is titrated against 0.1N NaOH in a
burette. The cationic form of the
amino acid is gradually converted into
its neutral or zwitterionic form by loss
of a proton from its COOH group.
Proteomics
Preamble to Proteomics
Acid- base properties of amino acids
Number of equivalents of alkali being
consumed is plotted against the pH of
the amino acid solution to obtain the
titration curve. pK1 of glycine is found
to be 2.34 i.e. it starts to lose its
carboxyl group proton at this pH.
Proteomics
Preamble to Proteomics
Acid- base properties of amino acids
As the titration proceeds, a point of
inflection is seen at which the removal
of proton is believed to be essentially
complete and the amino acid is largely
in its zwitterionic form. For glycine,
this point occurs at pH 5.97.
Proteomics
Preamble to Proteomics
Acid- base properties of amino acids
Removal of the proton from the amino
group constitutes the second stage of
the titration process. The zwitterionic
form is gradually converted into the
anionic form until the pH is
sufficiently alkaline to contain amino
acid only in the alkaline form.
Proteomics
Preamble to Proteomics
Acid- base properties of amino acids
The pK2 of an amino acid is obtained
in the second stage of the titration.
pK2 of glycine is found to be 9.6. Some
amino acids having positively or
negatively charged side chains will
have pK1, pK2 and pKR, which
corresponds to ionization of the side
chain. These amino acids have good
buffering capacity around 1 pH unit on
either side of their pK values.
Proteomics
Preamble to Proteomics
Peptide bond formation
Amino acids are the building blocks or
monomers that make up proteins.
Proteomics
Preamble to Proteomics
Peptide bond formation
Amino acids are oriented in a head-totail fashion and linked together such
that the carboxyl group of one amino
acid combines with the amino group
of another. Two amino acids joined
together by means of such a
condensation reaction with the loss of
a water molecule forms a dipeptide.
Many such amino acids linked together
form a polypeptide.
Proteomics
Preamble to Proteomics
Peptide bond formation
The peptide bond is rigid due to its
partial
double
bond
character.
However, the bonds between the αcarbon and amino and carboxyl groups
are pure single bonds that are free to
rotate.
Proteomics
Preamble to Proteomics
Protein structural levels
Amino acids are joined together in a
head-to-tail arrangement by means of
peptide bonds with the release of
water molecules.
This linear
sequence of amino acids constitutes
the primary structure.
Proteomics
Preamble to Proteomics
Protein structural levels
The folding of the primary structure
into the secondary is governed by the
permissible rotations about the  and
ψ angles. Not all values of these
angles lead to sterically favorable
conformations. The Ramachandran’s
plot
defines
the
regions
of
favorability.
Proteomics
Preamble to Proteomics
Protein structural levels
Amino acids along the polypeptide
backbone interact through hydrogen
bonds leading to secondary structures.
The -helix has intra-chain hydrogen
bonds between the ‘H’ of NH and ‘O’
of CO in every 4th residue. In 
sheets, the backbone is made to
zigzag such that chains are arranged
side by side for hydrogen bonding.
Proteomics
Preamble to Proteomics
Protein structural levels
Amino acids located far apart on the
polypeptide chain interact with each
other by means of hydrogen bonds,
electrostatic interactions, disulphide
bridges etc., allowing the protein to
fold three dimensionally in space,
giving rise to the tertiary structure.
Folding takes place such that the
hydrophobic residues are buried inside
the structure while the polar residues
remain
in
contact
with
the
surroundings.
Proteomics
Preamble to Proteomics
Protein structural levels
Different subunits or polypeptide
chains interact with one another and
are held together by means of ionic,
electrostatic, van der Waals etc
interactions.
Such
multisubunit
proteins are said to have a quaternary
structure.
Proteomics
Preamble to Proteomics
Structure & Classification
1. Amino acid: The basic monomeric unit of
polypeptides and proteins. There are twenty
standard amino acids with different structures
and properties that can be combined in
multiple ways to make up the wide range of
proteins known to us. Each amino acid is also
specified by a three-letter and single letter
code.
2. α-carbon atom: The central carbon atom of
an amino acid which is covalently bonded to an
amino group (NH2), a carboxyl group (COOH), a
hydrogen atom (H) and a variable R group. The
groups are tetrahedrally arranged around the
carbon atom. This carbon atom is an
asymmetric or chiral centre that gives rise to
the phenomenon of optical isomerism thereby
conferring a non-super imposable mirror image
on each of the amino acids except glycine.
3. Side chain: The side chain or R group is
distinct for each amino acid, giving them their
unique properties. It is on the basis of this side
chain that the amino acids are classified into
various groups.
4. Amino group: This consists of an NH2 group
covalently bonded to the central carbon atom.
Depending upon the pH of the surrounding
medium, it either exists as NH2 or NH3+ . Except
for proline, which has a secondary amino group,
all amino acids have only primary amino groups.
5. Carboxyl group: A COOH group covalently
bound to the central alpha carbon atom, which
exists as either COOH or COO- depending on the
pH of the surrounding medium.
Proteomics
Preamble to Proteomics
Acid-base properties
1. Cationic form: All amino acids exist in the
completely protonated form in acidic medium,
known as the cationic form. Both amino and
carboxyl groups are protonated here.
5. Amino acid in acidic medium: To obtain the
titration curve of an amino acid, it is first taken
in a highly acidic medium such that it exists
entirely in the cationic form.
2. Zwitterion: The state in which the amino
acid has no net charge is known as the
zwitterion. It is neutral due to the presence of
equal number of NH3+ and COO- groups.
6. Burette: A graduated, long glass tube fitted
with a stopcock at the end to control the flow of
liquid. This contains the solution against which
titration is to be performed. In this case, the
amino acid is titrated against 0.1N sodium
hydroxide (NaOH).
3. Anionic form: In a highly alkaline medium,
all amino acids exist in their anionic form due
to the presence of COO- group.
4. Titration flask: A conical flask in which the
solution to be titrated is taken along with a
suitable pH indicator.
7. Stand: This has a clamp which can hold the
burette steady while the experiment is being
performed.
Proteomics
Preamble to Proteomics
Acid-base properties
8. Titration curve: The number of equivalents
of alkali being consumed during the titration
process is plotted against pH of the solution in
the flask to yield a unique titration curve for
each amino acid. The titration curve depicted
corresponds to that of glycine.
9. pK: Negative log of the pH at which the
catonic and neutral forms inter-convert (pK1)
and neutral and anionic forms inter-convert
(pK2).
Proteomics
Preamble to Proteomics
Peptide bond
1. Peptide bond: The bond formed during the
process of linking together two amino acids
with the carboxyl group of one amino acid
being linked to the amino group of another
with the concurrent loss of a water molecule.
These bonds are planar in geometry and exhibit
partial double bond character.
2. Dipeptide: Two amino acids bonded through
a peptide bond. Many such amino acids linked
together constitute a polypeptide.
3.ψ (psi) and φ (phi): Angle of rotation about
the bond between the α-carbon atom and
carboxyl and amino groups respectively. These
angles determine which protein conformations
will be favourable.
Proteomics
Preamble to Proteomics
Structural levels
1. Primary structure: The sequence of amino
acids joined together by peptide bonds to
form a linear polymer constitutes the primary
structure of the protein. Linear polypeptide
chains are often cross-linked, most commonly
by two cysteine residues linked together to
form a cystine unit.
2. Secondary structure: The folding of a
polypeptide backbone by means of internal
hydrogen bonds between nearby amino acid
residues giving rise to a regular arrangement
defines the secondary structure of the protein.
α-helices and β-sheets are the most commonly
observed secondary structures of proteins due
to their highly favourably ψ and φ angles as
described by the Ramachandran’s plot. The
amino acid proline tends to disrupt the helix
and is often found at a bend in the structure
known as reverse turns or β bends.
3. Tertiary structure: Interactions (hydrophobic,
electrostatic, hydrogen bonds etc.) between
amino acid side chains located far apart in the
polypeptide sequence cause the protein to fold
resulting in a three-dimensional arrangement of
atoms known as the tertiary structure. The
folding takes place in such a way that the
hydrophobic residues get buried to form the
core while the hydrophilic amino acids remain
on the surface in contact with the polar
surroundings.
4. Quaternary structure: Many proteins have
more than one polypeptide chain, also called a
subunit, that are assembled together by various
interactions like electrostatic, van der Waals,
disulphide bonds etc. giving rise to the
quaternary structure.
Proteomics
Preamble to Proteomics
Structural levels
5. Bonding interactions: Several types of bonds
are
responsible
for
stabilizing
protein
structures. Some of them are:
i) Hydrogen bonds: These are formed between
an electronegative atom (like O or N) and a
highly electropositive atom (like H). They can
be formed within a polypeptide chain
(intrachain), as in the case of secondary
structures, or between different polypeptide
chains (interchain).
ii) Electrostatic interactions: Attractive forces
existing
between
oppositely
charged
groups/atoms, which can stabilize the protein
structure.
iii) Hydrophobic interactions: These are
largely non-specific interactions between nonpolar amino acid side chains, which act to bury
these hydrophobic residues away from a polar
environment.
iv) Van der Waals forces: These are attractive or
repulsive forces caused due to fluctuating
polarization and therefore temporary dipole
formation between nearby particles.
v) Disulphide bridges: Specific interaction and
oxidation of thiol groups of cysteine residues in
different regions of the polypeptide chain(s)
leads to formation of disulphide (S-S) bonds.
Proteomics
Preamble to Proteomics
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