Fantastic Fluorescence:Brainbow and The Nobel Prize 2008

In my childhood, I used to be fascinated by the mysterious glow of fireflies. Later I learned that it was due to a reaction between a substance called Luciferin and an enzyme, luciferase, a phenomenon called bioluminescence. This kind of glow is not limited to land creatures. Creatures living at the bottom of oceans too emit light.

Osamu Shimomura of Japan was given the task of isolating the substance which let the marine mollusk Cipridina glow when it was crushed and mixed with water. He succeeded, and on the wings of his publication, was recruited by the Princeton University, in the United States. There he began studying Aequorea Victoria, a marine jellyfish which glowed green when agitated. The jellyfish had an umbrella like shape and its outer rim glowed green. He chopped off the outer edges, crushed it, and filtered it to obtain a ‘squeezate’. He noticed one day that the ‘squeezate’ glowed blue when he poured some into the sink. He understood that it was the calcium ions (Ca++) in the seawater present in the sink that made it glow blue. It was christened aequorin.

During the extraction process they also chanced upon another protein called GFP, for green fluorescent protein. It glowed green when excited with ultraviolet light or light in the blue spectrum. The structure of GFP is barrel shaped (also likened to beer can shape), and consists of 238 amino acids. The chromophore or light emitting part is in the interior of the ‘beer can shaped’ molecule, in a region called the alpha helix region (of the molecule); while the exterior of the molecule was comprised of beta pleated sheets of the GFP molecule. Shimomura and colleagues showed that the blue color emitted by aequorin donor excited the GFP acceptor in an energy transfer process. The photons in the blue wavelength were absorbed by the GFP chromophore and photons of green wavelength were emitted - a phenomena called (bio)fluorescence. Fluorescence differs from luminescence from the fact that in fluorescence light of another wavelength is emitted than the one absorbed and luminescence means emission of light.

The whole story struck a chord in Martin Chalfie’s ears. He thought what if he could harness the gene that codes for GFP and bind it to the segment that coded for a protein of interest? He worked with a roundworm, Caenorhabditis elegans, a simple organism with only 959 cells and yet a complete organism for it could procreate, had a brain and even one third of its genes were related to humans. It was translucent and hence studying its interior was easier. He contacted Douglas Prasher who was also hot in the trail for the GFP genes. Douglas Prasher did as he promised. He sent the GFP gene to Chalfie once he got hold of the gene. Chalfie introduced it behind the promoter of the gene that coded for proteins in C elegans’ touch receptor neurons. The neurons were cleanly delineated, that too in a live worm and ‘real-time’! GFP, being a natural gene product, is non toxic.

Roger Tsien wanted more. He knew from earlier studies that the ‘chromophore’ had 3 key amino acids: serine, tyrosine and glycine in position 65, 66 and 67 respectively in the 238 amino acid long GFP molecule which formed the chromophore. He used DNA technology to alter the amino acid sequence so as to obtain GFP variants that would absorb and emit light in different part of the electromagnetic spectrum. This way he obtained cyan, yellow and blue. He obtained DsRED, a red GFP-like protein extracted from coral, from two Russian researchers and modified it so that it was stable and of desired molecular weight.

It was all set by now. Researchers now modified mice genetically and introduced the gene for red, cyan and yellow GFP. They expressed the corresponding proteins in their brain and what we got is a riot of colors, the ‘brainbow’, short for brain and rainbow. Like the elementary colors, these colors when combined in different proportions, produce many colors, just as a color printer does using them (cyan, yellow and red). One could now visualize the neural circuitry in much the same way as seeing electronic circuits. Disease detection and progression in Alzheimer’s disease, cancer and Parkinson’s disease are some potential clinical applications. Watching biogenesis of HIV1 (the virus that causes AIDS) in live cells in real time is now an easy meat. GFP can also be engineered to recognize heavy metals like cadmium (a cancer causing chemical), explosives like TNT and Arsenic (a water pollutant causing Arsenicosis).

Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry, 2008. Sadly, Doug Prasher was left out, despite his outstanding contribution in this field. He is now driving a van at $10 an hour to meet his living expenses. He is not alone. This year’s Nobel in Physiology or Medicine too left out Robert Gallo, an HIV pioneer. So, not totally a happy ending.

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References:

The Nobel Prize in Chemistry 2008
The Man Who Missed the Nobel Prize
Stuart Cantrill (2008). Nobel Prize 2008: Green fluorescent protein Nature Chemistry DOI: 10.1038/nchem.75