Ramachandran Plot

History:

Ramachandran was born into a Tamil family in Ernakulam. In 1939, he completed his BSc Physics and then
went on to work in the Electrical Engineering Department at the IISc Bangalore in. Electrical engineering,
however did not interest him as much as physics so he switched to the Physics department and went on to do his
Masters and PhD under Sir CV Raman. Ramachandran returned to the IISc Bangalore as an assistant professor
of Physics in 1949. In 1952, he became the HOD of physics department at Madras University. While he was
there, he shifted his focus from crystal physics to the composition of biological macromolecules. Ramachandran
and Gopinath Kartha hypothesized the triple helical structure of collagen using X-ray diffraction, published in
Nature Magazine in 1954, gaining the recognition of "Madras group". i

Ramachandran's interest in examining various polypeptide conformations led him to develop a useful tool, the
Ramachandran plot. He aimed to address issues at a more fundamental level and establish a standard for
assessing protein structure.

The Ramachandran plot was developed in the early 1960s by Gopalasamudram Narayana Ramachandran, an
Indian physicist and biophysicist, and his student, Venkatachalam S. Ramakrishnan. Ramachandran had become
interested in the problem of protein folding, specifically the angles of the peptide bond in proteins, and began
exploring ways to visualize the possible conformations of these bonds.

They were interested in understanding the conformational preferences of amino acid residues in proteins. They
calculated the energetically favourable angles for the peptide bond and plotted these values on a graph. They
also used model peptides and calculated the allowed conformations of the peptide backbone using bond angle
constraints and steric considerations. They found that the allowed conformations clustered around certain
regions of the phi-psi space. This led to the invention of the Ramachandran Plot.
 A new 3D Model Of Ramachandran Plotii

The Ramachandran plot is a two-dimensional plot that shows the distribution of phi and psi angles for each
amino acid residue in a protein. The plot is divided into different regions, each representing a different type of
conformational preference. The most common regions are the alpha-helix, beta-sheet, and random coil regions.
The calculations made by them between 1962 and 1963 resulted in the Ramachandran plot, which first appeared
in a paper titled "Stereochemistry of Polypeptide Chain Configurations" published in the Journal of Molecular
Biology.

Features Of Ramachandran Plot:

The four quadrants that make up the Ramachandran plot represent the various possible phi () and psi () angle
pairings for each amino acid residue. Each amino acid residue is shown as a dot in the graph.

How psi and phi angles are measured

The Ramachandran plot's four quadrants are as follows:

• The 1st quadrant of the plot is known as the left-handed alpha-helical region. It contains combinations
of phi and psi angles consistent with left-handed helical secondary structures, which are uncommon but
have been seen in a few proteins.
• The 2nd quadrant of the plot, known as the "beta-sheet region," comprises combinations of phi and psi
angles that represent the beta-sheet secondary structure. Quadrant II also shows the biggest region in
the graph.
• The 3rd quadrant of the figure, known as the alpha-helical area, comprises combinations of phi and psi
angles that represent the right handed alpha-helical secondary structure.
• The lower-left quadrant of the figure is known as the polyproline II (PPII) area. There are essentially
no delineated regions in Quadrant IV. Due to steric conflict, this conformation (between -180 and 0
degrees, between 0-180 degrees) is not preferred.
 Ramachandran Plot With Validation Contours

Moreover, the Ramachandran plot includes contour lines that show how frequently various phi and psi angle
combinations appear in high-quality protein structures. These contour lines indicate the permitted regions of
conformation for each amino acid residue. Outlier zones, which are found outside of the contour lines and are
typically connected to structural mistakes or faults in protein models, are places where the combinations of phi
and psi angles are uncommon or prohibited in high-quality protein structures.

Emerging Applications:

Protein structure prediction:

One can utilise the Ramachandran plot to evaluate the accuracy of a protein structure prediction. By analysing
the torsion angles of amino acid residues, it is possible to predict the possible conformations of a protein
structure. This information can be used to generate structural models. The structural stereochemical property is
predicted by the Ramachandran plot, which is produced by a variety of programmes and servers.
To assess the stereochemical quality of a predicted model, PROCHECK (a software) looks at the geometry of
the entire model and the geometry of each individual residue. Model validation, which can be done using
Ramachandran plot analysis, comes after protein structure prediction, refinement, and model quality
assessment. Protein structures must be stored in a specific file format, and servers use quantum mechanics to
determine the percentage of preferred, permitted, and forbidden regions. The computation considers the
backbone's phi and psi angles, hydrogen bonds' energies, the planarity of peptide bonds, and steric conflicts. iii

PROCHECK Working

Understanding protein dynamics:

The dynamics of protein structures can also be understood using the Ramachandran plot. One can spot
conformational changes in a protein structure and how these changes impact protein function by examining the
distribution of phi and psi angles in the Ramachandran plot over time. A method called RP-MDS, which
combines the Ramachandran plot (RP) with molecular dynamics simulation (MDS), was developed by
researchers Benjamin Tam, Siddharth Sinha, and San Ming Wang to evaluate the structural change for large-
sized proteins (MDS). While MDS offers a numerical estimate for the variant-caused primary structural change,
the Ramachandran plot captures the variant-caused secondary structure change.iv

Drug Discovery:

To create small molecules that can bind to particular areas of a protein structure, the Ramachandran plot is used
in drug discovery. It is feasible to determine which parts of a protein are reachable by small molecules and
create substances that can bind to these parts by evaluating the Ramachandran plot.

Enzyme catalysis:

To comprehend the mechanism of enzyme catalysis, one can use the Ramachandran plot. It is possible to
pinpoint the essential amino acid residues involved in catalysis and how they contribute to the reaction process
by measuring the torsion angles of amino acid residues in the active site of an enzyme.
Protein sequence analysis:

The Ramachandran plot is used to analyse protein sequences. By comparing constraints and plots of different
protein sequences, it is possible to identify conserved or variable regions of the protein structure, infer
functional constraints, and predict the effect of mutations.

Protein engineering:

Protein engineering is the process by which a researcher alters a protein sequence by adding, deleting, or
swapping nucleotides in the gene that codes for the protein to produce a modified protein better suited than the
original protein for a given use or application.

Protein engineering can make the use of the Ramachandran plot to enhance the stability, activity, and specificity
of proteins. It is possible to find mutations that can enhance the stability and functionality of a protein by
looking at the torsion angles of amino acid residues.

Conclusion:

The Ramachandran plot is useful for comprehending the conformational space accessible to amino
acid residues in protein structures. We looked at the various uses and emerging applications of the
plot. Even though, it has been 60 years since the Ramachandran Plot came out, it is continued to be in
use today and is used as a first step in elimination of proteins/amino acid that are not sterically viable.

i
https://en.wikipedia.org/wiki/G._N._Ramachandran
ii
https://europepmc.org/article/pmc/3061398
iii
https://unacademy.com/content/csir-ugc/study-material/life-sciences/ramachandran-plot-in-biology/
iv
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744649/