Nobel prize A glowing green Nobel The molecule that revolutionised and illuminated cell biology started with a . Lewis Brindley tells the story of this year’s Nobel prize for chemistry PHANTATOMIX / SCIENCE PHOTO LIBRARY PHOTO SCIENCE / PHANTATOMIX

38 | Chemistry World | November 2008 www.chemistryworld.org As collected his – a protein that produces Hole Oceanographic Institution in one thousandth jellyfish of the day, blue light in the presence of calcium In short Massachusetts. on a hot summer afternoon in 1961, he ions.  The 2008 Nobel Prasher was the first to envisage had no idea that the painstaking work Shimomura realised that something prize for chemistry how GFP might function as a would eventually net him a Nobel else was converting this blue light into was awarded to Osamu fluorescent tag. Being small enough to prize. But Shimomura’s discovery the green glow. Eventually he isolated Shimomura, Martin not hinder the action of other proteins of green fluorescent protein (GFP) a second protein, which was able to Chalfie and Roger and responsive to ultraviolet light, later that year set in motion a series of absorb both blue light and ultraviolet Tsien, for discovering Prasher reasoned that GFP would be developments that revolutionised the light and then fluoresce green. This and developing green perfect for tracking cancer cells. By way we look inside cells. protein was referred to as green fluorescent protein 1992, he had isolated the GFP gene The two men who share the Nobel fluorescent protein (GFP) – it was a (GFP) and published a paper detailing the prize in chemistry with him this simple name that stuck.  GFP was first sequence of the 238 amino acids that year are and Roger For the next 17 years Shimomura discovered in jellyfish make up the protein. Tsien. Each played a critical role in continued to harvest jellyfish to and can now be found in On hearing of Prasher’s results, turning an unusual jellyfish protein identify the chemical makeup of the cell biology labs across Chalfie decided to instruct his into compounds that are routinely glowing proteins. ‘We needed a lot of the world graduate student, Ghia Euskirchen, found in cell biology labs across the material, so our schedule required us  This glowing protein to begin work putting the GFP gene world and continue to provide to catch around 50 000 has given us a whole into the genetic code of E. coli bacteria. new insights into diseases jellyfish every summer,’ new way to examine the Only one month later Euskirchen such as cancer, HIV and says Shimomura. ‘So inner workings of cells, succeeded – producing E. coli cells Alzheimer’s disease. in total we probably providing insights into a that glowed brightly despite having collected over range of diseases none of the other jellyfish enzymes The great jellyfish hunt 850 000.’ that were thought to be needed. Shimomura was From this great Many organisms fluoresce born in Japan in 1928, collection, Shimomura was finally naturally, such as fireflies or tropical and his early life was able to characterise the chemical fish, but the majority of these blighted by war. He was only 12 structure of the GFP chromophore processes involve other components kilometres from the atomic bomb that in 1979 and lay the groundwork for that are used up during the struck Nagasaki – close enough to be future discoveries. luminescence. Chalfie found that GFP temporarily blinded by the flash. was unique – the protein itself was all His determined pursuit of a Seeing through it that was needed to convert ultraviolet scientific career was eventually In 1988, Martin Chalfie, a biological light into a bright shade of green. rewarded with a PhD from Nagoya scientist from Columbia University in University for discovering the protein New York, attended a seminar during In the genes responsible for the luminescence of a which GFP was briefly mentioned. Chalfie says that luck played a role sea-dwelling firefly. This work caught Chalfie remembers being so excited by in winning the race to express the eye of Frank Johnson at Princeton the idea of a fluorescent protein that the gene. ‘At least three University in the US, who recruited he didn’t listen to any other speakers other teams had tried to him to study the green-glowing that day. express GFP before us, but jellyfish Aequorea victoria in a project ‘The reason I was so excited their attempts all failed. that would last more than 20 years. was because I was working on a We later found out that Every summer, Shimomura would millimetre-long roundworm called this was because they had sail out into the bay of Friday Harbor, C. elegans – which is transparent,’ used a slightly different San Juan Island, Washington, when says Chalfie. ‘I realised that if I could process.’ the sea became thick with jellyfish. express the fluorescent protein in the Whereas Chalfie’s team During 1961, he extracted the faintly roundworm, I could watch cellular amplified only the precise glowing rings from around 10 000 processes as they happened under the sequence, others cut appropriate of the mouse-sized jellyfish and microscope.’ sized fragments from Prasher’s clone. squashed them through a filter. After Chalfie needed to find the gene However, this left small sequence isolating a protein from the glowing responsible for making GFP, and fragments attached to either end of ‘squeezate’, Shimomura was surprised it didn’t take long to find someone the strand that interfered with the to find that it emitted blue light, who was working on it – a biologist correct formation of the protein. not green. This, it turned out, was named Douglas Prasher at Woods Despite the success he achieved, AP PHOTOS AP

From left: Roger Tsien, Osamu Shimomura and

AP PHOTOS AP Martin Chalfie PHOTOS AP www.chemistryworld.org Chemistry World | November 2008 | 39 Nobel prize

in 2007, dyed neurons in mice in a kaleidoscope of 90 different colours – giving them the chance to look for unique patterns. AFP/GETTY IMAGES IMAGES AFP/GETTY Bittersweet prize The rules of the Nobel prize limit it to a maximum of three people. And, like many other important discoveries, more scientists contributed to the development of GFP. This year the man who cloned and sequenced the GFP gene, and who first imagined its potential, Douglas Prasher, missed out on this most prestigious of accolades. What’s much worse is that Prasher’s career was cut short. He failed to find funding for his research and dropped out of science entirely. He is now working as a bus driver. ‘That Doug could not be recognised certainly lends a bittersweet element to the prize,’ Chalfie says. ‘I think it Chalfie was still surprised by the time and green was not always ideal The brainbow was a must have been an agonising decision advances that GFP led to. ‘I certainly for practical studies. unique, colour-coded for the Nobel committee, and they realised that attaching fluorescent ‘We found that a specific amino acid map of the brain could easily have given the prize to tags to a subset of cells could be could change the primary colour of Doug instead of me.’ important. But what I never could emission, but other amino acids also Tsien also acknowledges Prasher’s have guessed was all the wonderful played smaller roles,’ he explains. By contribution saying: ‘It’s a shame adaptations and modifications that gradually making mutations in the that Doug has not been recently in a others have come up with, which gene to carefully change the amino position to do science. I hope all this really made GFP into a useful and acids, Tsien was able to tweak GFP to publicity will result in Doug once effective tool.’ create a broad palette of colours and again being able to do something for It wasn’t until Roger Tsien became improve overall brightness. which he is really talented.’ involved in 1994 that explanations for The new colours were an important Whether biology features too GFP’s behaviour were revealed. Tsien step forward, as they allowed prominently in the Nobel prize for was already well-known in the field scientists to image multiple different chemistry is a question raised almost for giving unprecedented views inside cells simultaneously and study how every year. ‘Our work certainly cells, for example, developing dyes different systems interact in real time. falls on the boundary line between that change colour in contact with chemistry and biology, but we calcium ions. Over the brainbow didn’t do this work for the purpose Jeremy Sanders, who supervised One thing Tsien had not yet managed of winning a Nobel prize – we Tsien during his PhD at the University to do was produce light towards the did it because it was scientifically of Cambridge, UK, says the work red end of the spectrum – this is very important and interesting at the time,’ Tsien did before his research on GFP useful as it can penetrate biological says Tsien. ‘opened up a whole new world for tissue more easily. The turning point Chalfie agrees and places his work looking at what was happening inside came when Sergey Lukyanov at the firmly on the border between the living cells’. ‘At some point during Shemyakin Institute of Bioorganic sciences. ‘I think that the barriers the 1980s, nearly a third of all papers Chemistry in Moscow, Russia, between the different science published in the field of physiology discovered a GFP-like protein in departments are becoming more cited Tsien’s work.’ coral, which was termed DsRED. and more porous. And I think the To begin with, Tsien charted how ‘The red proteins from coral were Swedish Academy have certainly the chromophore of GFP was formed larger and had a tendency to form acknowledged that here.’ Without in the 238 amino acid chain. The tetramers, so they needed the chemistry of GFP, the vast range secret lies in the way the protein folds extensive modifications of important biological applications itself – wrapping up into a barrel-like to make them user- would have been impossible. shape, with three key amino acids (a friendly,’ Tsien serine-tyrosine-glycine sequence) says. This resulted Lewis Brindley is a freelance folded tightly together in the centre. in a new series of science writer based in Tsien showed how these three delicious sounding Oxford, UK amino acids could react together proteins with with ambient oxygen to create the names like mPlum Further reading fluorescent chromophore. and mStrawberry –  O Shimomura et al, J. Cell. Comp. Next, he set to work making indicating the different Physiol., 1962, 59, 223 improvements. ‘The wild type of shades.  D C Prasher et al, Gene, 1992, 111, 229 GFP was made for the jellyfish’s own Perhaps the most vivid  M Chalfie,et al, Science, 1994, purposes, so it obviously wasn’t ideal demonstration of the 263, 802 for ours,’ says Tsien, noting that GFP new family came when a Harvard  R Heim et al, Proc. Natl. Acad. Sci. tended to lose its ability to glow over University team, led by Jeff Lichtman USA, 1994, 91, 12501 40 | Chemistry World | November 2008 www.chemistryworld.org