If you think you know it, then you do not know it, and if you know that you cannot know it, then you know it.

In one of his Mathematical Philosophy (MATPHIL) reports, G. N. Ramachandran elaborated on this interesting paradox from Kena Upanishad, describing the Divine force of the Universe, which conveys perpetual doubt and indefiniteness.

Prof. Gopalasamudram Narayana Ramachandran, affectionately known as GNR, was the finest molecular biologist whose seminal work in structural biology had put India on the world map of modern biology. The structural Biology era started with Linus Pauling and his colleague’s discovery of the  -helical structures of polypeptides, which set the stage for our present-day understanding of protein function. The term molecular biology was coined in 1938 by Warren Weaver, a mathematician, and Director of the Natural Sciences Division at the Rockefeller Foundation, defining an amalgam of  Physics, Chemistry, and Biology. Still, the molecular biology revolution started with the discovery of the double-helical structure of DNA by Rosalind Franklin, Francis Crick, James Watson, and Maurice Wilkins in the early 1950s. During that period, most of the work in molecular biology was done in UK and USA. Following this, a new structural fold comprising a coiled-coil triple helix was described by GNR in a series of papers published between 1954 and 1956 in Nature with his student Gopinath Kartha. Later in the 1950s and 1960s, he applied stereochemistry principles to generate a two-dimensional plot that accurately described allowed conformations of proteins. The map, now famously known as the Ramachandran plot, has become an inalienable part of molecular biology and biochemistry textbooks.

Passionate X-ray crystallographer

After graduating from St. Joseph’s College, Trichy, in 1939, Ramachandran joined the Electrical Engineering department at the Indian Institute of Science (IISc), Bangalore, and later moved to the Physics department. GNR studied crystallography all by himself while working for D.Sc. degree under the supervision of Sir. C. V. Raman. While transferring GNR to the Physics department from the Electrical Engineering department at the IISc, Raman, who was the Director of IISc at that time, said, “I am admitting Ramachandran into my department as he is a bit too bright to be in yours…”.  After completing his doctorate, GNR moved to Cavendish Laboratory, Cambridge, where he worked on mathematical theory to determine elastic constants of cubic crystals from diffuse X-ray diffraction and obtained his second doctorate. After returning from Cambridge in 1949, he joined as an assistant professor at IISc, where he nurtured the X-ray diffraction laboratory. At the initiative of Dr. A. Lakshmanaswamy Mudaliar, the  Vice Chancellor of Madras University at that time, GNR joined Madras University in 1952 as a founding member of the Physics department. Ramachandran started cutting-edge research in crystallography that included phase determination when anomalous dispersion is present, the probability distribution of X-ray intensities, crystallographic statistics, and so forth. He derived the correct formula to calculate the X-ray phase angles using Bijvoet differences, which occur during anomalous X-ray scattering. In 1971 he returned to IISc and established the Molecular Biophysics Unit (MBU), now a leading center of research in structural biology.

The Triple helix

It has been known for a long time that form and shape of all mammals are dependent on collagen protein which makes up a third of all proteins in their bodies. Collagen is vital for structural integrity; imperfection or instability in its structure causes many disease conditions. Ramachandran and Kartha used X-ray diffraction data on the collagen derived from the Kangaroo tail tendon to delineate its structure. The structural model had three parallel left-handed helical polypeptide chains, side-by-side coiled coils packed together in a hexagonal array. The triple helical structure of collagen has been described as “braided in the manner of the pigtail of a long-haired maiden from Madras.” The collagen structure was alluding to the structural biologist of that time, and the work of GNR put India on the world map of structural biology. Ramachandran mentioned that he got the idea of a triple-helix coiled coil model from the rotation and revolution motion of the moon. 

Despite his ground-breaking research, recognition did not come easily to GNR. Other leading structural biologists of that time, like Alexander Rich and Francis Crick, who were also working on collagen structure, did not accept the model citing steric issues and discrepancies in number of inter-chain hydrogen bonds. As collagen contains hydroxyproline residues GNR in his model had more than one hydrogen bond, which were mediated by water molecule. Now the controversy is well settled in favor of the ‘Madras model’ of Ramachandran after the high-resolution crystal structure of collagen (PDB id 4OY5 [0.89 Ã…]) showed that there are, on average, 1.5 hydrogen bonds in the structure. Credit for collagen structure eluded him during his lifetime, but now the record has been set straight in favor of GNR. The basic three-dimensional structure of collagen unraveled by GNR helped us understand the molecular basis of its function and diseases caused by changes in its native structure.  

The controversy on the collagen structure based on steric issues made GNR more determined, and he started working on formulating a general stereochemistry rule for protein structure that resulted in Ramachandran Plot, which has immensely benefitted protein science and drug discovery research.

Ramachandran plot

Proteins are made of twenty naturally occurring amino acids joined together like pearls in a necklace. It is called its primary structure, where amino acid residues are joined by rigid planer peptide bonds. This linear protein chain further folds into secondary (a-helix and b-strand), tertiary and quaternary structures acquiring a unique three-dimensional shape that dictates its function. Understanding a protein’s function or any dysfunction leading to disease conditions necessarily requires knowledge of its three-dimensional structure.

Ramachandran working with Ramakrishnan and Sasisekharan analyzed all the crystal structures of amino acids and observed that nonbonded atoms were closer to each other than the sum of their respective van der Waals radii. Using this and Pauling’s a-helix information, GNR used mathematical calculation (without computers…!!!) to arrive at allowed rotations of rigid planer peptide planes that will lead to sterically acceptable contacts between them. The 2D plot of these pair of allowed angles, Phi-Psi (f –y), gave birth to the Ramachandran plot. The Ramachandran plot was proved correct later when the first crystal structure of a protein was determined. The plot remains valid today when around two lakh protein structures have been experimentally determined and their coordinates deposited in the Protein Data Bank (PDB). For accuracy and acceptability, all experimentally determined structures of proteins as well as predicted structures, must confer to this map. Like the Raman effect, Bose-Einstein statistics, and Chandrasekhar limit, the Ramachandran plot has immortalized the scientist behind it. 

Describing Ramachandran plot Prof. Dame Janet Maureen Thornton, a senior scientist and director emeritus at the European Bioinformatics Institute(EBI), EMBL, had written, “It never fails to excite me, when I see the Ramachandran plot and realize how much of the beauty and order of protein structures is encapsulated by this plot. I also think that this major discovery highlights the importance of clear thought and vision that do not always need expensive equipment and huge teams of people”.

Medical Imaging

G N Ramachandran (GNR) also made seminal contributions that made a deep impact to the field of three-dimensional medical imaging. In 1971 he published two exciting research papers on Projection Reconstruction; one titled ‘Reconstruction of substance from shadow: 1. Mathematical theory with application to three-dimensional radiography and electron micrography’. In another publication co-authored with A. V. Lakshminarayanan published in the same year in Proceedings of National Academy of Sciences, USA, he suggested using convolutions in the spatial dimension. Both opened the window for creating three-dimensional (3D) images of human anatomy using two-dimensional (2D) images, which could provide depth information. This led to the development of the Computed Tomography scan (CT-scan), where several 2D X-ray images obtained at different angles and depths, called projections, are used to construct 3D images on a computer.

Magnetic Resonance Imaging (MRI) is another imaging technique that uses a projection reconstruction algorithm developed by Ramachandran. In the first publication describing MRI in Nature in 1973, Paul Lauterbur cited Ramachandran’s algorithm as a method for projection reconstruction. Lauterbur, who received Nobel Prize in Physiology in 2003, had initially called the new imaging technique zeugmatography derived from the Greek word zeugma meaning “that which is used for joining.” MRI uses linear gradient in a homogeneous magnetic field of a Nuclear Magnetic Resonance (NMR) spectrometer so that different points in the object experience a slightly different magnetic field. This leads to the NMR spectrum as a projection of the 2D object into a 1D spectrum. In MRI, several projections are acquired, and images are reconstructed using the projection reconstruction algorithm of Ramachandran.

Both CT-scan and MRI have benefited enormously from G N Ramachandran’s 1971 papers, and he is rightly credited for having a deep impact on the ‘Medical Imaging’ field. In a nutshell, Ramachandran’s contribution has revolutionized human radiology helping with a high-throughput, efficient, and accurate diagnosis, which has become a boon for the modern medical sciences.  

Personal life and Vedanta

G N Ramachandran was born on October 8,1922 in Ernakulam in Kerala. He was the eldest son of G.R. Narayana lyer and Lakshmi Ammal. His father was a Professor of mathematics who taught him mathematics.  After retiring from MBU, he continued as a Professor of Mathematical Philosophy at IISc until 1989. In 1999 GNR was awarded the 5th Ewald Prize by the International Union of Crystallography for his outstanding contributions in crystallography. He spent his final years in Chennai, where he passed away on April 7, 2001.

Syadvada and Saptabhangi of Jain philosophy had a profound impact on Ramachandran. In one of his papers published in the journal Current Science in 1982, he described their usage in Boolean Algebra. In addition to applying mathematics to biological problems, Ramachandran used mathematics to look into divinity.  In ‘Vedanta and Epistemology’ he wrote, “There is a close analogy of these ideas of modern logical analysis with the concepts in Indian philosophy of the ‘Infinite’ (Ananta), which is one of the attributes of Brahman (Absolute Reality). The Upanishads contain many statements to the effect that this Reality has contradictory properties … The logical similarity to the example of infinity in mathematics is very close. In the case of mathematical infinity, we have to say that although it is a number, it does not belong to the class of finite numbers, and therefore it can have contradictory properties such as being both greater than and smaller than itself.”

Though his contributions like the Collagen triple-helix structure; Ramachandran plot; and Tomography should have got not one or two but three Nobel prizes, he remains a shining jewel missing in the Nobel crown.