The Ramachandran Plot

GNR with wife Rajam, Dorothy Hodgkin

The double helix is, perhaps, one of the most iconic structures from the world of science.

In Chennai, not far from the banks of river Adyar, there’s an auditorium in the heart of a national research centre that appears wholly unremarkable, but for its name: Triple Helix.

The name of the auditorium, Triple Helix, holds the key to a David and Goliath story from the rarified realms of research. The plot, set in the early 1950s, involves a puzzle in structural biology. The cast spans continents.

A young scientist in Madras, now Chennai, is working on a problem that interests celebrated researchers from labs at the University of Cambridge, the King’s College in London, and Caltech. This elite group of Goliaths included the co-discoverer of the DNA double helix, and the proposer of the single helix structure of proteins.

The David in this story is Gopalasamudram Narayana Ramachandran, or simply, GNR.

Barely 30, GNR had just been made head of the lrtodirac2 (1)brand-new department of physics at the University of Madras. As a graduate student, he had worked in the field of optics under CV Raman at the Indian Institute of Science, Bangalore, and received the equivalent of a doctorate degree. Later, he received a PhD from Cambridge University in 1949, where he worked in the Cavendish Lab directed by Sir Lawrence Bragg, co-discoverer of X-Ray diffraction.

Diffraction was the technique being used to decipher the structures of biomolecules such as nucleic acids and proteins.

Though the structure of the nucleic acid, DNA, would be the first to be decoded, the structures of some proteins were puzzles. It was important to solve protein structures because the architecture of the biomolecules decides what they do in the body . Form in biomolecules is firmly wedded to function.

Protein-structure

Like a string of pearls, proteins are made of repeating units or monomers of amino acids (there are 20 essential amino acids in the human body). What links the monomer units is a peptide bond – formed by removing a water molecule from a pair of amino acids – so what you have is a polypeptide chain. These chains pleat or intertwine, to yield two dimensional structures – helices or sheets – which then fold into 3D shapes.

We know today, that incorrect folding of peptide chains causes proteins to function incorrectly, and folding errors manifest as diseases. ( Hyper-mobility syndrome, Brittle Bone Syndrome and other diseases result due to deviations from the prescribed structure of the collagen.) The foundation for this understanding was being laid in the 1950s when first-rate scientists worked to discover the architecture of biomolecules.

In 1952, GNR got a laboratory of his own. Studying biomolecules would be the lab’s theme, the crystal physicist had decided, but he didn’t know where to begin. He was fired up by the lectures of Linus Pauling in UK, the scientist who wrote the seminal book, The Nature of the Chemical Bond. (There are chemists who swear that this book reads like a thriller, besides being a masterpiece in chemistry.)  A former colleague from England, the renowned crystallographer JD Bernal, who was on a visit to Madras, told him that research groups were grappling with the structure of collagen – the most abundant protein in animals  present in their connective tissue  giving them strength and form – and no one had hit the mark yet, not even Pauling himself.

This, GNR decided, would be the problem he would work on. First, he needed to find a source of collagen. The shark fin collagen from the biochemistry department didn’t do it. Good quality pictures were essential to cracking the collagen puzzle. Leather, it occurred to GNR, was largely collagen.

Not far from the university campus was a new institute – the Central Leather Research Institute. GNR decided to pay his neighbors a visit.  Besides easy access to animal carcasses (and their body parts) the biochemists at this center had expertise to purify the material which the physicist wanted. The choices in GNR’s mind were: Kangaroo Tail Tendon or Beef Achilles Tendon. The deputy director of CLRI turned out to be a kindred soul, happy to help a fellow scientist. The beef sample was easy to obtain locally. But if kangaroo collagen was going to yield the best diffraction images – as the scientific literature said – the deputy director promised to get GNR samples from Australia.

Thus, GNR found himself some purified marsupial collagen to work with.

The only information available on the fibrous protein collagen was this: one-third of its total amino acid content was glycine. Using this fact, looking at the pictures he had taken, GNR made an intuitive leap. Every third monomer in the polypeptide chain is glycine, and so collagen must be a triple helix. He imagined the structure of collagen as three separate helical chains stacked in an array. GNR and his group published this prototype structure in the August 7, 1954 issue of the journal Nature.

Suddenly, top-notch molecular biologists in Cambridge, California, and elsewhere, began paying attention to the work of the “Madras group.” They began to refer to the model GNR proposed as the Madras helix.

GNR and his first post-doc continued to take XRay pictures from various angles to refine the prototype. With experimental data, they came up with the “coiled-coil” structure that could explain all available experimental data on collagen. In this new model, the three helical chains – originally thought of as separate – intertwined to form a second helix.  D Balasubramanian,The Hindu’s inimitable science columnist, describes the structure simply: the three helices were “braided in the manner of the pigtail of a long-haired maiden from Madras.”

This new model was inspired by astronomy, GNR has said in subsequent lectures. Think of the night sky: the rotating moon which revolves around the earth, always presents the same side to earth. In collagen, the smallest of amino acids, glycine, always faces the center of the triple helix. Maybe, GNR’s collagen model was subliminally inspired by his beloved wife Rajam’s everyday coiffure as well.

Whatever the source of inspiration — celestial or something closer home — the results appeared in the September 1955 issue of Nature.

The “coiled coil” model was a fundamental advance in understanding protein structure. Francis Crick (who cracked the structure of the DNA with James Watson) acknowledged this in the November 1955 issue of Nature: “Very recently Ramachandran and Kartha have made an important contribution by proposing a coiled-coil structure of collagen. We believe this idea to be basically correct but the actual structure suggested by them to be wrong.” Crick offered minor modifications. So did another group, based in King’s College, London.

In the end, the big names received the lion’s share of the credit for deciphering the structure of collagen. GNR seemed doomed to be a footnote in collagen literature. But he took the criticism aimed at the Madras triple helix “stereochemically unsatisfactory” and worked to find the underlying principles which determined what forms polypeptides chains could and could not take in a protein.

By doing this, in 1963, he came up with the very grammar of protein folding. To this day, researchers use the Ramachandran Plot (or map, diagram) to validate protein structures.

Like the Raman Effect, Heisenberg’s Uncertainty Principle, or Einstein’s Theory of Relativity, the Ramachandran Plot has rendered the scientist behind it immortal. You’ll find Ramachandran’s name in standard biochemistry text books, and in the latest papers on protein structure in prestigious journals.

GNR organized two international symposia in Madras, one in the winter of 1963 and another in the winter of 1967. Addressing the second symposium as the president, two-time Nobel winner Pauling, discoverer of the alpha helix structure of proteins, recalled how GNR’s team had pipped his group to the post in finding the collagen structure. (Gleefully, Jim Watson had written about  using Pauling’s principles of model-building to decode the structure of DNA before Pauling could. One cannot imagine GNR saying anything like this! )

GNR’s insight — essential requirement of glycine, and only glycine, at every third position in the sequence and its critical role in the structure of collagen has been proven unambiguously, writes GNR’s student, Manju Bansal, who is currently a professor at IISc. When a mutation alters this basic fact – that is if a glycine is replaced by non-glycine in the chain – it unravels the prescribed collagen structure and results in disease.

After close to two decades in Madras, in 1971, GNR returned to the Indian Institute of Science to set up the Molecular Biophysics Unit. An excellent teacher, he spawned a dynasty of scientists who address biological questions in structural terms.

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