Protein Secondary Structure, Ramachandran Plot, Molecular Docking, Pharmacophore, Heat Map

Pradipta Banerjee, Ph.D.
Dept. of Biochemistry & Plant Physiology,
CUTM, Odisha, India
pradipta.banerjee@cutm.ac.in

Nomenclature of C-atoms

Four Levels of Organization in the
Structure of a Protein
 The amino acid sequence is known as
primary structure of protein.
 Stretches of polypeptide chain that form α
helices and β sheets constitute
protein’s secondary structure.
 Full three-dimensional organization of a
polypeptide chain is referred as the
protein’s tertiary structure,
 and if a particular protein molecule is
formed as a complex of more than one
polypeptide chain, the complete structure
is designated as the quaternary structure.

Characteristic Features
1. Dipeptide bond links 2 amino acids
2. Strong bond, Rigid, planar structure
3. Trans configuration
4. -NH and –CO are polar and can
form H-bonds
5. Have 40% partial double bond
character, due to resonance.
Peptide Bonds

Cis & Trans Peptide Bonds
Cis: Cα groups on
same side of peptide
bond (very rare)
Trans: Cα groups on
same side of peptide
bond

 Peptide groups, with few exceptions, assume the trans conformation: that in which successive
Cα atoms are on opposite sides of the peptide bond joining them.
 This is partly a result of steric interference, which causes the cis conformation to be about 8
kJ.mol-1 less stable than the trans conformation (this energy difference is somewhat less in peptide
bonds followed by a Pro residue and, in fact, 10% of the Pro residues in proteins follow a cis
peptide bond, whereas cis peptides are otherwise extremely rare).
…Cis & Trans Peptide Bonds

Proline, a cyclic secondary amino acid, has
conformational constraints imposed by the cyclic
nature of its pyrrolidine side chain, which is unique
among the “standard” 20 amino acids.

Proteins are molecules that consist of one or more polypeptide
chains. These polypeptides range in length from ~40 to ~34,000
amino acid residues (although few have more than 1500
residues) and, since the average mass of an amino acid residue is
about 110 D, have molecular masses that range from ~40 to
over ~3700 kD.
Polypeptide Chains

Polypeptide Backbone Conformations (of a Protein) May Be Described
by their Torsion Angles
 Torsion angles or rotation angles or dihedral angles are
formed between the Cα-N bond (Φ) and the Cα-C bond (Ψ)
of each of its amino acid residues.
 These angles, Φ and Ψ are both defined as 180° when the
polypeptide chain is in its planar, fully extended (all-trans)
conformation and increase for a clockwise rotation when
viewed from Cα.

α-Helix
The right-handed α-helix: Hydrogen bonds between the
N-H groups and the groups that are four residues back
along the polypeptide chain are indicated by dashed
lines**.
The left-handed α-helix has never been observed (its
helical parameters are mildly forbidden) is that its side
chains contact its polypeptide backbone too closely.
NB: 1-2% of the individual non-Gly residues in proteins
assume this conformation.
The α-helix is a common secondary structural element of
both fibrous and globular proteins.
**Hydrogen bonds of an helix are arranged such that the
peptide N-H bond of the 10th residue points along the
helix toward the peptide group of the 6th residue.

 If a polypeptide chain has a long block of Glu residues, this segment of the chain will not form an helix
at pH 7.0. The negatively charged carboxyl groups of adjacent Glu residues repel each other so strongly
that they prevent formation of helix.
 For the same reason, if there are many adjacent Lys and/or Arg residues, which have positively charged
R groups at pH 7.0, they will also repel each other and prevent formation of the helix.
 Bulk and shape of Asn, Ser, Thr, and Cys residues can also destabilize an helix if they are close together
in chain.
 A Pro residue introduces a destabilizing kink in an helix. Proline is only rarely found within an helix.
 Glycine occurs infrequently in helices for a different reason: it has more conformational flexibility than
other amino acid residues. Polymers of glycine tend to take up coiled structures quite different from an
helix.
Points to Remember: α-Helix

 Βeta pleated sheet’s conformation has repeating Φ and Ψ angles that fall in the allowed region of the
Ramachandran diagram and utilizes the full hydrogen bonding capacity of the polypeptide backbone.
 In pleated sheets, however, hydrogen bonding occurs between neighboring polypeptide chains rather
than within one as in helices.
 Pleated sheets come in two varieties:
1. The antiparallel pleated sheet, in which neighboring hydrogen bonded polypeptide chains run in
opposite directions.
2. The parallel pleated sheet, in which the hydrogen bonded chains extend in the same direction.
β-pleated sheets

β-pleated sheets

(a) The hairpin connection between antiparallel strands is topologically in the plane of the
sheet.
(b) A right-handed crossover connection between successive strands of a parallel sheet.
Nearly all such crossover connections in proteins have this chirality.
(c) A left-handed crossover connection between parallel sheet strands. Connections with this
chirality are rare.
Connections between adjacent polypeptide strands in β pleated sheets

In the early 1960’s, G. N. Ramachandran (University of Madras, India) and co-workers
computationally determined the phi and psi angles that avoid steric collisions, initially
treating the atoms simply as rigid spheres. They showed that the physically allowed angle
combinations (that avoid clashes) correspond largely to the secondary structures observed
in proteins: alpha helices, beta sheets, and turns.
Ramachandran and team also showed that the major effect of sidechains on the allowed phi
and psi angles is due to Cβ. Sidechains larger than that of alanine affect the allowed angles
by only a few percent.
The Ramachandran Plot shows the phi and psi angles actually observed in proteins.
Ramachandran Plot

Clashes: where non-bonded
atoms are overlapping, and
thus in physically impossible
positions. (This model
simulation allows two atoms
to overlap, unlike real atoms.)
Peptide Bond (Pink) Plane (Yellow) of a peptide
bond.

The Ramachandran Principle says that alpha
helices, beta strands, and turns are the most likely
conformations for a polypeptide chain to adopt,
because most other conformations are impossible
due to steric collisions between atoms.
Ramachandran Principle

The conformation angles (Φ and Ψ) of several secondary structures
NB: Gly, the only residue without a Cβ atom, is much less
sterically hindered than the other amino acid residues.

How many bonded atoms are required to constitute a dihedral (torsion) angle,
such as phi or psi?
a. None
b. 1
c. 2
d. 3
e. 4
f. 5
Answer: The phi dihedral (torsion) angle is defined by:
(1) the carboxy carbon from the previous amino acid;
(2) N in the amino acid containing the phi bond;
(3) Cα in the amino acid containing the phi bond; and
(4) the carboxy carbon of the amino acid containing the phi bond.

The number of phi and psi angles in an isolated amino acid (not in a polypeptide
chain) is:
a. None
b. 1
c. 2
d. 3
e. 4
f. 5
Answer: The phi angle involves the carboxy carbon of the previous amino acid in
a polypeptide chain. The psi angle involves the N of the subsequent amino acid.
Therefore an isolated single amino acid has neither phi nor psi angles

The number of atoms held into a geometric plane by a peptide bond is:
a. None
b. 1
c. 2
d. 3
e. 4
f. 5
g. 6

The peptide bond is unable to rotate because:
a. It is a covalent bond.
b. It is a non-covalent bond.
c. Rotation would cause clashes.
d. It is a partially double bond.
Phi and psi angles directly determine
a. Primary structure.
b. Secondary structure.
c. Tertiary structure.
d. Quaternary structure

Alpha helices are compatible with
a. All possible phi-psi angle combinations.
b. A limited range of phi-psi angle combinations.
c. A limited range of phi angles with all possible psi angles.
d. A limited range of psi angles with all possible phi angles.

Common secondary structures are energetically favoured because
a. They optimize main-chain hydrogen bonds.
b. They represent all possible conformations.
c. They maximize clashes between atoms.
d. They minimize clashes between atoms.
Overlap of van der Waals radii
a. Between two non-bonded atoms is called a clash.
b. Between two covalently bonded atoms is called a clash.
c. Occurring between two non-bonded atoms in a molecular model signifies an
energetically favourable interaction.
d. Is physically impossible between two non-bonded real atoms.

In a Ramachandran plot, the dots cluster
a. Where clashes occur.
b. Where clashes do not occur.
c. Where alpha helices occur.
d. Where beta strands occur.
e. Where alpha helices, beta strands, and turns do not occur.
f. Where the phi and psi angles are energetically favourable.

Molecular docking: Predict the preferred orientation of one molecule to a second, when bound to each other
to form a stable complex. Depending upon binding properties of ligand and target, it predicts the 3-D structure
of any complex.
Applications:
1. Drug design
2. Analysis of molecular interactions
3. Identification of role of phytochemicals in curing diseases
4. Aptamer selection
• Biosensor development
• Targeted drug delivery
Molecular Docking

Molecular Docking
Docking can be between:
• Protein - Ligand
• Protein – Protein
• Protein – Nucleotide
The ligand acts as signal
triggering molecule in case of
protein-ligand binding by
binding the specific site on the
protein where it is targeted.

Work Flow of Molecular Docking

Most relevant pharmacophore
features
Examples
hydrogen bond acceptors hydroxyl or carbonyl groups
hydrogen bond donors hydroxyl or amide groups
positively charged groups guanidines
negatively charged groups carboxylates
hydrophobic groups cyclohexyl or isopropyl groups
aromatic moieties phenyl ring or other aromatic ring
systems
Pharmacophore
A pharmacophore is best defined as the arrangement of these functional groups in an active compound that
are crucial for its activity. Pharmacophores can be derived from 2-D or 3-D molecular representations, but
3D pharmacophores are most popular (considering the fact that molecules are active in three dimensions).

Molecular Mechanics Quantum Mechanics
Structure-based Drug Design
or
Target-based Drug Design
(Docking Studies)
Ligand-based Drug Design
 Pharmacophore Mapping
Three-dimensional (3D) Structure of
Target Known (Receptor/enzyme)
Three-dimensional (3D) Structure of
Target NOT Known/available
Specify the binding site
surrounding the bound
co-crystal ligand
Homology Modeling of
Target protein / receptor
Search / Identify binding
site in Homology Model
Specify the binding-site
Docking studies
Pharmacophore Modelling

Structure Based Drug Design

This method will
 Begin with biologically active compounds.
 Describe what chemistry those compounds have
in common.
 Few new compounds that match this description.
 Compounds that match the description will also be active.
Initial two steps involving the process called ‘model building’ while the final two steps are
known as ‘database screening’.
Ligand Based Drug Design

70% hits was reduced
after primary assessment
99% hits was reduced
after molecular docking
Case Study
Stem and bark of Camptothecin acuminate
Camptothecin (CPT), targets DNA
Topoisomerase I
In order to predict structurally diverse novel
chemical entity as Top1 poison with better
efficacy, Ligand-based-pharmacophore
model was developed.
33% database was
reduced after screening

Heat Maps
 In heat maps the data is displayed in a grid where each row
represents a gene and each column represents a sample.
 The colour and intensity of the boxes is used to represent
changes (not absolute values) of gene expression.
Upregulated genes
Downregulated genes
Unchanged expression
Many of the methods for visualising and interpreting gene
expression data can be used for both microarray and
RNA-seq experiment. HM is one of them.

samples
proteins
Proteins are divided into two groups
(left dendrogram) roughly corresponding
to membrane-specific proteins and other
proteins.
Samples are divided into three groups
(upper dendrogram).
• The large cluster on the left
corresponds to samples quantified
with the URZB and URNB extraction
methods.
• The middle cluster includes samples
quantified with the TCA1 method
• The last one involves the TEAL
method
1847 proteins quantified on 19 samples extracted from one maize
leaf and according to 4 extraction methods based on label-free
shotgun proteomics

• Samples extracted using the URZB and
URNB methods have a relatively high
expression of the top cluster of proteins
compared to the lower cluster.
• On the other hand, the TEAL samples
(right part of the map) have a relatively
low for the top protein cluster and
relatively high for the bottom cluster.
• Finally, the TCA1 samples (middle cluster)
seem to exhibit intermediate quantities for
most proteins (nevertheless, proteins at the
bottom of the map are relatively more
abundant than proteins at the top).
Low values
High value
Intermediate values
Heat Map Analysis

Protein Secondary Structure, Ramachandran Plot, Molecular Docking, Pharmacophore, Heat Map

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