Friday, January 11, 2013

Remembering GNR

This week has been filled with chatter - scientific, nostalgic and gossipy - it has been a week commemorating 50 years since the Ramachandran map was published.  Most modern biologists are familiar with this plot, which defines the allowed torsion angles in a polypeptide chain.  This probably sounds incomprehensible to many readers of this blog, but the work (deserving of a Nobel prize and now a standard plot in many biology textbooks) laid the foundation for determining the spatial arrangement of amino acid chains that eventually form proteins.

Proteins are the objects that scurry around (or insert themselves in specific locations) in a cell and perform most of the important functions in a cell.  The specific task of a protein depends on its structure, hence it is very important to understand what these proteins look like.  The sequence of amino acids in a protein is defined by the genetic sequence (which can be easily determined nowadays).  But the way in which this linear chain of amino acids will fold into a three dimensional, active and stable protein is hard to deduce.

GN Ramachandran (generally called GNR by his colleagues) and his students approached this problem from a theoretical viewpoint and their calculations (plotted on a map called the Ramachandran map) described the kinds of angles that the links in the main chain (formed by the amino acids) could adopt and the angles that were not allowed.  This map, a completely new concept at the time (in the 1960s) continues to be relevant and useful, not just to theoreticians but to everyone who experimentally tries to solve a protein structure.  The manner in which the plot was worked out, in the University of Madras, with a remarkable idea, a lot of initiative and hard work and nothing other than a simple calculator and log tables, is an inspiring story.  Professor C. Ramakrishnan (CR), who actually carried out the calculations was present at the meeting and shared old memories with the audience, in what can only be termed the "CR style".  Armed with his thesis, original models and quirky wit, he spoke to a fascinated audience about the "birth" of this map.  Time flew by; I hoped and wished he had been able to speak for longer.

The meeting, which was large and diverse, allowed people from many different areas of structural biology to come together, mingle for a few days, exchange notes and thoughts.  Perhaps not very different at the outset from many meetings (though gigantic in scale with over 700 participants), it grew warmer and more animated with time as science melded with bittersweet memories, nostalgia and humour.  There was a place for everyone and everything.  For potentially unending conversations with fluent and famous scientists, for broken but interesting conversations with scientists who spoke English haltingly, for wacky snippets exchanged over beer, for strains of Russian folk songs spontaneously and wonderfully sung by one of the speakers, for new and jazzy T-shirt designs, for the chink of crockery at a candle-lit poolside dinner on a warm Bangalore night.

The meeting draws to a close today and people will go their own ways.  For me, it was nice to be able to observe a part of the events, to hear a few people I wanted to, to meet old friends and to see the Ramachandran map through CR's eyes once more.  Several years ago I had interviewed CR about his involvement with the Ramachandran map (part of which was published in Resonance, ).  As this article is not easily accessible to readers outside India, I give below an excerpt from the original interview.

CR- My experience was that many people knew of a scientist called Ramachandran.  They knew only one thing about him, namely that he was almost close to getting a Nobel Prize.  But what more did they know about him?  Everybody said “Oh, he has worked with collagen.”
But what is GNR’s contribution to proteins? 

Proteins are complicated molecules, but they need not be studied in a complicated way.  There is a simple way to understand the various arrangements that proteins can take.

Everybody had the impression that the Ramachandran Map is the one which can be used for the prediction of protein structures.  I want to dispel that idea from their minds.  Ramachandran Map is a tool which can be used for testing a structure- this is now being done extensively.  It is not one which can be used for predicting whether it is an α helix or β sheet.

SV- How did you decide to enter research?

CR- Going back to my college days, I did what we called B.Sc. Honours, in Physics.  I did not have any background in Biology except in the school days, which was more descriptive in nature, and which did not go into my head very much.  I did not have much exposure to Chemistry because that was also over by the Intermediate stage.

In Madras University Physics department they had a course called ‘one year M.Sc.’, which was a specialization in Crystallography and Biophysics.  There were just 5 or 6 seats available for that course.  That was the department of which GNR was the head.

When I wanted to join the M.Sc. course, the only condition GNR put for me was that I continue research.  So, he was clear that this course is not for anybody except people who want to do research.

Though honestly, sincerely, I was not having any idea of research either in University or laboratories, nor had I been exposed to research in my earlier days, I said ‘Yes”, with the idea of joining the course, because if I said “No”, he may not give me the seat.

In that one year, when I was in the department, I was exposed to a research atmosphere.  After that year, he said “Now that it’s over, are you ready to join research?  I’ll give you a scholarship.”  The scholarship was Rs. 200 per month, which was a large amount.  Being familiar with the research atmosphere in the department, I thought “I can also do research.”

After joining came the question “Which branch do you want to do research in?”  I found that there were many people working in Crystallography and not many working in Biophysics.  So I said I would like to work in Biophysics and definitely not in Crystallography.  Any person who has asked this question would be very curious to know why I said I didn’t want to work in Crystallography.  But GNR didn’t ask anything.  He said “I am glad.  Now you can begin.”  That is how my whole future was set.

My fascination has always been with Mathematics.  So when I joined the department the first thing which attracted my attention was an electrical desk calculator, which was called a Marchette calculator.  It could do wonderful things like addition, subtraction, multiplication and division.

SV- When you began work, with collagen, what was the approach?

When I joined the department, in June 1960, it was buzzing with activity on collagen because there was going to be a symposium at the Central Leather Research Institute (CLRI) in December. Many students were working on the structural aspects. The amino acid composition of collagen was known but not the sequence.  Crick had proposed a Gly-Pro-HyPro arrangement, with one hydrogen bond for three residues.  GNR had proposed Gly-X-HyPro (where X is any other amino acid), with two H-bonds for three residues (wherever Pro occurs, the H bond will break).  The X-ray diffraction patterns agreed with both structures though there were some small things that the two-bonded structure showed better.

My job was to do a Fourier transform of both structures and to find out what should be the intensities of a collagen X-ray pattern for each.  I had to find out where the X-ray intensities will be for the entire space, and how much.  ‘Where’ was easy, ‘How much’ was difficult.

But collagen gave birth to the Ramachandran Map.  Crick’s objection to GNR’s structure was that when you have close packing, some of the Van der Waal’s distances will be violated.  This question was not approached from the collagen point of view by GNR, but from a generalized point of view.  He said “This is not just an isolated problem with collagen, this is a problem which is going to be involved in any molecule which has peptides and amino acids.”

At that time we didn’t have a computer.  Everything was developed ab initio.  Prof. Sasisekharan (a student with GNR at that time) was doing a literature search of peptide structures to develop the criteria (for bond lengths).

Regarding my work, some easy geometrical methods existed which were used to calculate the helical parameters of a polypeptide chain.  Peptides being planar was known from Chen and Pauling’s work from crystal structures.  Pauling had also given all the peptide dimensions.  Pauling focussed more on the α helix.  But GNR focussed on one level below that.  The initial idea was, if you take a pair of peptide units, how do you mathematically develop a helix out of it?  Then came the question of defining the orientation.  It can be angles between the planes, it can be angles between the two bonds, it can be anything.  In this case you need the angles of rotation.  The alphabet for the conformational study was a simple system of 4 atoms linked to each other, where you can have a rotation about the middle bond.

When the angles had to be defined there was no starting point.  GNR said “We will take the fully extended chain as the starting point.”

Torsion angles were known in Chemistry but they were used to describe preferred arrangements; to go from one conformation to the other was also known.  But to take two planar units and go from one conformation to another was not known..

It was a rigid body rotation.  Each peptide is a rigid body but the totality of the picture is not a rigid body.  So actually it was very difficult at that time to imagine an angle of rotation without having a defined initial position.  What was needed was to apply the principles (of physics and mathematics) to the actual case of a peptide, and to transform it in a way that you can work with.

SV- How long did it take you?

CR- About one and a half years.  From January 1961, it took about 3-4 months to get the ideas crystallized.  Journals were not easily obtained.  There was no computer.  The Ramachandran map formulation was based on the formulation of the matrix.  There was no book which gave the matrix explicitly.  For about two months I had to search for books.  Finally someone told me “You refer to Classical Mechanics by so- and- so, for the matrix.  I rushed but that book was not available in the library.  Then I had to find out who has taken the book, find out where he is and then, finally get the book.  There the matrix was available.  It took time because the library was in another campus and buses were not easily available. After getting the matrix I knew I was at home because matrix is something connected with Mathematics.

We had only two calculators in the department, one was mostly occupied by the crystallographers.  There was only one person who could service the calculators, and if something went wrong, somehow we had to find him.  There were no phones in those days so we had to go to his house.  Once it so happened that we could not get him.  Fortunately there was one more calculator in CLRI, so I used to sit there for some time.

SV- The amount of information that has been represented in a simple, two-dimensional way is impressive.

CR- Any mathematics or physics person always thinks of plotting.  When you have two parameters variable, it becomes an X-Y plot.  It is easy to perceive and to communicate, but if you are going to work with it you must remember that the two ends are the same; the top and the bottom are the same.  It is almost like a latitude and longitude: Φ and ψ are the latitude and longitude, which go from 0º to 360º each way.  In essence, it is something like a globe.

SV- Were you under any pressure while doing fundamental research?

CR- From the pressure point of view, both GNR and myself were in resonance that we must do the work for work’s sake.  It was not a question of application, it was a question of doing the work.  I didn’t have that pressure but I had the pressure of time, because GNR will say “Go and do it” and I have to do it because I really don’t know, from the next day onwards, when he is going to call me and ask for the results.

SV- What was GNR like, as a Professor and as a person?

CR- I can say only a few things- his sincerity to the work in the department was extraordinary. Whatever he wanted to do he would put his whole body and soul into it.  But the negative point was that he was very impatient.  He would think that everything should be done very fast and correctly, in the right way.  Though he appeared very strict, he had a very good heart.  He would give credit to whatever one did at every place.

He had a knack of seeing things correctly, and was very imaginative.  Though there may be people like him, I have not come across any..

SV- When did you realize the impact of the Ramachandran Map?

CR- For me, it was a continuous process because right from the beginning I have been using it.  I could see that whenever I had a problem about structure or conformation I could immediately use it.  It was my day to day tool.  At the same time one cannot exactly say how much it can be used.  It all depends upon the person (using it).

Now we have come far away from the Ramachandran Map.  As a tool it is has served its purpose and to that extent it was an amazing learning experience.  The main lesson was “Don’t think whether it will be finally useful or not, whether it will be really earthshaking or not, whether you can publish it in this journal or that, but start work, plan it carefully, execute it correctly and present it such a way that other people can understand”.

1 comment:

prof prem raj pushpakaran said...

Ramachandran's contribution in nurturing the indian science and the biophysics field in general is praiseworthy. I feel IISc did a wonderful work in organizing the event to commemorate 50 years of Ramachandran's Map.

Prof Prem Raj Pushpakaran

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