Shining protein

Published : Nov 21, 2008 00:00 IST

One Japanese and two American scientists share the Chemistry Nobel for the discovery and development of green fluorescent protein.

THREE scientists, one Japanese and two American, share this years Nobel Prize in Chemistry for the discovery and development of the green fluorescent protein, GFP. They are Osamu Shimomura, 80, of the Marine Biological Laboratory, Woods Hole in Massachusetts, and Boston University Medical School; Martin Chalfie, 61, of Columbia University, New York; and Roger Y. Tsien, 56, of the University of California, San Diego.

Luminescence and fluorescence refer to light emission from cold bodies. The former results from chemical reactions or subatomic motions or stress on a crystal. In particular, it is referred to as bioluminescence when the luminescence occurs in living organisms, say a firefly, owing to chemical reactions within some part of the organism. Another bioluminescent organism is the jellyfish Aequorea victoria whose outer edge glows green when the jellyfish is agitated. Fluorescence is luminescence caused by external light stimuli as an optical phenomenon in which molecular absorption of a photon triggers the emission of another photon, usually of a longer wavelength.

In 1955, Osamu Shimomura, after having been devastated by the War and the atomic bombing of Nagasaki where he lived, was employed as an assistant by a professor at Nagoya University. He was given the task of finding out what made the remains of a crushed mollusc, Cypridina, glow when it was moistened with water. This was a problem considered to be difficult even for professional researchers and, apparently, the professor not wishing to spoil the career of a research student over such a problem, put Shimomura on this task.

However, in 1956, to the professors surprise, Shimomura isolated the glowing material. It was a protein that glowed 37,000 times more brightly than the crushed mollusc. This earned him a valuable gift of a Ph.D. from his professor even though he was not enrolled as a doctoral student. Armed with his results and the doctorate, he joined Princeton University under Frank Johnson, with whom he set about studying another bioluminescent organism, the jellyfish.

During the whole of the summer of 1961, Shimomura and Johnson gathered jellyfish at Friday Harbour, Washington. They chopped off the edges of the jellyfish and pressed them through a filter to get what they called a squeezate. Serendipitously, on one occasion, when Shimomura poured some of the squeezate into the sink, he saw a bright blue flash, not green as in the jellyfish. He reckoned that the sea water in the sink, which contained calcium ions, caused the squeezate to luminesce. Johnson and Shimomura collected squeezate from about 10,000 jellyfish and took it to Princeton. It took them a few months to purify just a few milligrams of the blue luminescent material from the liquid. They named this protein aequorin.

There were many difficulties and troubles. But somehow I found how to extract the protein, Shimomura says in the post-award interview with the Nobel Foundations website. And after finding that, what we needed to do to study that protein was to get large amounts of that protein. So, we collected huge numbers of jellyfish by going to Friday Harbour every summer, with a schedule of 50,000 per summer, in one or two months, he adds. They did this apparently for 19 summers and accumulated a total of 850,000 jellyfish.

In 1962, when Shimomura and Johnson published their isolation of aequorin, they mentioned that they had also isolated another protein, which was slightly greenish in sunlight, yellowish in the light from an incandescent bulb and green in ultraviolet light. This was a fluorescent protein (as against the luminescent aequorin), which they called the green protein, but later it came to be called green fluorescent protein (GFP).

In the 1970s, Shimomura started to investigate GFP more closely. Clearly, the 850,000 jellyfish must have come in very handy. He showed that GFP contained a chromophore, a special chemical group that absorbs and emits light, the basic mechanism of fluorescence mentioned earlier. Now why does the jellyfish emit only green light and not blue? The GFPs chromophore absorbs the blue light from aequorin and emits it in the longer-wavelength green light.

The important thing about a fluorescent protein like GFP is that it does not require a continuous supply of chemical energy through energy-rich molecules. One just has to illuminate GFP with blue or UV light. If one had to deal with luminescence, one would have had to inject a suitable chemical into the organism, a process that can disturb the cell and make it difficult to carry out microscopic studies, which now GFP enables. Indeed, GFP has become an extraordinary and widely used tagging tool in bioscience, using which scientists have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or the spreading of cancer cells.

An even more interesting use of GFP is that researchers can actually follow the processes inside individual cells, whose size is about 0.02 mm. The human body consists of billions of cells. The more one learns about a cell type (how it develops and functions), the greater the chance of developing effective cell function-specific drugs. This is beyond the function of an ordinary microscope, and it is even more difficult to follow the processes within in detail. Proteins regulate the chemical processes in cells. There are tens of thousands of different proteins, each with a different function. By identifying which protein is present in which cell, scientists can follow a given protein and watch its interactions with other proteins. That is, with GFP one can now track a single protein under a microscope.

Martin Chalfie, the second Nobel laureate in chemistry this year, actually heard about GFP only as late as 1988, at a seminar dealing with bioluminescent organisms at Columbia University. Chalfies bread-and-butter work was dealing with the millimetre-long roundworm called Caenorhabiditis elegans, one of the most studied organisms in the world. Although it has only 959 cells, it has a brain, it grows old and it mates. In addition, a third of C. elegans genes are related to human genes. But most importantly, from the perspective of GFPs potential use, it is transparent, which makes it easy to study the organism. So, during the seminar, Chalfie realised that GFP would be a fantastic tool for mapping C. elegans.

Although there are tens of thousands of proteins performing various functions in our bodies, they are all long-chained molecules made of 20 different amino acids that are linked together. It is the length of the chain, the sequence of the amino acids and the way the molecule folds up that distinguish one protein from the other. In general, each gene is associated with a protein. Whenever a protein is required by the cell, the gene is activated, which results in the production of the protein in the cell.

Chalfies idea basically was to connect the gene for GFP with various gene switches, or with genes from other proteins, so that he could study how gene switches activate and where the proteins are produced. The green light from GFP would act like a beacon shining on each step of the process. In order to be able to do this, Chalfie needed to know where the GFP gene resided in the genome of the jellyfish.

He found that Douglas Prasher at Woods Hole too had similar ideas and was going to isolate and clone the gene. The two decided to collaborate to see whether this would work in C. elegans. However, this collaboration did not actually come about immediately because Chalfie moved to some other university. In 1992, Chalfie revisited the problem with a graduate student and found by literature search that Prasher had already isolated and cloned the gene. Then they established contact and Chalfie got the gene from Prasher. His student, Ghia Euskirchen, then succeeded in producing GFP in Escherichia coli, the ordinary intestinal bacterium. When E. coli was radiated with UV light, they could see it glowing green under a microscope.

While many other researchers had similar ideas of using GFP and many had indeed tried with Prashers cloned gene before Chalfie, they did not succeed. The technique they used for amplifying the gene happened to be inappropriate. The construct they used for the amplification was that from Prashers clone they cut the appropriate DNA (deoxyribonucleic acid) fragment (that represented the peptide backbone of the protein causing the fluorescence).

The fragment had extra bits of sequences sticking either end. And, apparently, those sequences were poison, so it never got made. So extra DNA interfered with protein production. We did it in a different way. We simply amplified exactly the coding sequence from the clone, and didnt have any extra DNA. So when we put things in bacteria, it worked, explained Chalfie in his post-Prize interview. Thus, even though others had the chance to do it earlier, they failed because they just happened to follow a different procedure. It was just good fortune, remarks Chalfie.

Also, the fact that Chalfie worked with a transparent organism made significant difference in the use of GFP to study cell functions. The only reason that I worked on GFP is that, Chalfie remarked, for several years before I worked on GFP, I said that one of the great things about working with C. elegans was the fact that it was transparent, and so when I first heard that seminar describing GFP, and realised, I work on this transparent animal, this is going to be terrific! I will be able to see the cells within the living animal. That was really great. That really was the motivation.

In the early 1990s, scientists generally assumed that several steps were involved in the cells in the production of naturally fluorescing molecules and pigments that give flowers, fish and other organisms their colour and that each of these steps required a protein to control the chemical production. It was also generally believed that a few different proteins were needed to produce the chromophore in GFP, but Charlie and Euskirchens experiments showed that this premise was wrong. Nothing other than GFP was involved.

Roger Tsiens contribution to the GFP revolution was that he extended the colour palette with many new colours that glowed longer and brighter.

First, Tsien showed how the GFP chromophore was formed chemically. GFP actually consists of 238 amino acids linked in a long chain. The chain folds up into the shape of a beer can (see figure). It had been previously shown that three amino acids 65, 66 and 67 reacted chemically with each other to form the chromophore that absorbed UV and blue light to give out green light. Tsien showed this chemical reaction required only oxygen; he also showed how it could happen without the help of other proteins. And since molecular oxygen is ubiquitous in all aerobically living animal cells, he explained how GFP could easily fluoresce in every organism that it was expressed in and suggested that the mechanism was common to all aerobically living organisms.

Using DNA technology, Tsien exchanged different amino acids of GFP and created new variants of GFP. This resulted in the protein absorbing and emitting light in different parts of the spectrum. By experimenting with different amino acid sequences in GFP, Tsien could produce GFP mutants that shone more strongly and in quite different colours such as cyan, blue and yellow. This has enabled researchers today to mark different proteins with different colours and study how they interact.

One colour that Tsien could not produce was red. A red fluorescing molecule could be even more useful because red light penetrates tissues easily and would be especially useful to study cells and organs inside the body.

At the same time, two Russian researchers, Mikhail Matz and Sergei Lukyanov, were looking for GFP-like proteins in the most unlikely of places fluorescent corals and found six more proteins, one red, one blue and the rest green. The red protein, DsRED was, however, too large and heavy to become an easily usable tagging tool. It had four amino acid chains instead of one. Tsiens research group solved the problem by redesigning DsRED so that the protein was stable even as a one-chain molecule and also fluoresced, which could now be easily connected to other proteins. Starting from this smaller red protein, Tsiens group has developed many proteins that glow in frequencies in the neighbourhood of red. More workers have joined to enlarge this palette by adding new colours. After the nearly half a century since Shimomura discovered the first GFP, today there is a kaleidoscope of GFP-like proteins that shine in all colours of the rainbow.

There are many extraordinary things that have been done with these GFP and GFP-like proteins. For example, three of these proteins were used by researchers in a spectacular experiment. Mice were genetically modified to produce varying amounts of colours yellow, cyan and red within the nerve cells of the brain, like the colours used by a computer printer. The mouse brain ended up glowing in rainbow colours. The scientists could follow nerve fibres from individual cells in the dense network in the brain. The experiment was quite aptly named brainbow.

GFP can also be used for biotechnological applications, as has been done, for example, for the detection of arsenic in water wells. Researchers have genetically modified arsenic-resistant bacteria that will glow green in the presence of arsenic. There have been attempts to modify other organisms to fluoresce in green in the presence of the explosive trinitrotoluene (TNT), or heavy metals such as cadmium and zinc. These days, GFP has gone into glowing toys as well.

Shimomura started out with asking a basic question: what makes jellyfish shine? He was interested only in an answer to that.

I didnt know any use of the protein at that time, he says, until Chalfie discovered in 1994 that it can be expressed in living cells. So I had no idea of the applications of GFP for a long time. The point is that I dont do my research for application or any benefit. I just do my research to understand why jellyfish luminesce and why that protein fluoresces. There are many, many [undiscovered molecules in nature that emit light]. Interesting at least to me.

But that basic scientific curiosity of Shimomuras has led to results in an entirely different field in a completely unexpected way. And it seems to promise much more.

There are lots of cute things, says Tsien in an interview, that GFPs and RFPs have done. The one thing they have never done is to get us really long wavelengths, like excitation beyond 600 nanometres. That is truly red, deep red excitation and infrared emission. And that would be very important for going into whole animals that have a lot of blood in them, like you and me, and more particularly mice. Haemoglobin absorbs quite severely below 600 nm. So you want to be above 600 nm to avoid the blood absorbance as much as possible.

Besides, there is also the fundamental evolutionary question, slightly different from that of Shimomura, which remains unanswered. We now know what makes jellyfish shine. But why does it shine? Many organisms living in the sea use light from biofluorescent proteins to confuse their predators, to attract food, or to tempt a partner. But no one knows what has caused Aequorea victoria to evolve to produce aequorin and GFP.

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