The Prize
The background story of almost every Nobel Prize awarded includes the biographies of one or more people who did lots of research but didn't get the prize. The New York Times published an article today about Douglas Prasher, who first cloned and sequenced Green Fluorescent Protein (GFP).
Prasher didn't share the Nobel Prize awarded to Osamu Shimomura, Martin Chalfie, Roger Y. Tsien, who worked to make GFP into the versatile tag that scientists use to visualize the inner workings of live cells and organisms. The GFP Chemistry Nobel Prize committee could only award three of the hundreds of people who developed GFP to its current state of usefulness. Prasher didn't pursue the scientific process of making GFP useful, and acknowledges that the winners deserved the prize over him, telling the Times reporter: "They worked their butts off over their entire lives for science, and I haven't." But his part is an interesting and keen choice for the New York Times human interest story, because science stories focus most often on the "winners", and in fact every major advance in science involves whole teams of research techs, students, contributors, most of whom don't share credit or fame, in fact some don't even eek a living out of it. The Times report explores the funding Prasher didn't get, as well as his difficulties finding employment and depression.
The paper doesn't go into the broader picture of the GFP protein discovery, which is interesting in itself and also helps one understand the process of scientific discovery.
The Long History Behind the Making of Useful GFP
The history of GFP research in the past 50 years traces the history of biology itself over the last half a century. In 1961, Osamu Shimomura discovered the protein while purifying and characterizing aequorin. Shimomura came to the US from Japan, when as a teenager, he was only 12 kilometers from the Nagasaki bomb explosion. After piecing together his education and life, he worked in a lab in Japan isolating and characterizing another protein, thereby earning his Ph.D. Shimomura was recruited to Princeton by Frank Johnson, co-author of the 1962 paper that first mentioned GFP. 1 Their paper gave a nod to the history of bioluminescence research to that point:
"In experiments that have become classic in bioluminescence, Dubois (1885, 1887), first prepared from a luminous elaterid, Pyrophorus, and a luminous clam, Pholas, respectively, crude extracts containing a substrate, luciferin, and an enzyme, luciferase, which luminesced on mixing in aqueous solution containing dissolved oxygen."
The 1962 paper gives the reader a view into some of the techniques used by cell biologists and biochemists at that time. It also give insight into the sort of fortitude of successful researchers. It's perspective that's useful to understanding how science research works.
Thousands and Thousands of Jellyfish
Shimomura collected tens or hundreds of thousands (the accounts vary) of the Aequoria jellyfish to learn about GFP and aequorin. Non-scientists probably can't imagine the tedium of collecting tens of thousands of jellyfish. And that's only the start. How would you figure out how to extract of luminescent parts of the jellyfish "squeezate" without destroying it? How would you determine a method for purifying a protein via repeated chromatography? How many trial and errors would finally lead to the chemical conditions in which a protein glows and at what wavelength? Scientists had to work through many questions before making GFP useful.
In 1962, the GFP protein find was incidental to his aequorin study. The authors only mentioned it in footnote number three:
"A protein giving solutions that look slightly greenish in sunlight though only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of Mineralite has also been isolated from squeezates. No indications of a luminescent reaction of this substance could be detected."
In later work Shimomura et al went on to determine the emission spectrum of GFP and learned that the protein absorbs light in the blue spectrum emitted in Aequorea victoria by the calcium activated aequorin, that is, it exists because of aequorin. Then they found that it emits its own fluorescent green light. Over the next few decades, many other scientists advanced the work, as Roger Tsien wrote in review paper of the GFP in 1999:2
"Morin & Hastings found the same color shift in the related coelenterates...and were the first to suggest radiationless energy transfer as the mechanism for exciting coelenterate GFPs in vivo. Morise et al purified and crystallized GFP, measured its absorbance spectrum and fluorescence quantum yield, and showed that aequorin could efficiently transfer its luminescence energy to GFP when the two were coadsorbed onto a cationic support. Prendergast & Mann obtained the first clear estimate for the monomer molecular weight. Shimomura proteolyzed denatured GFP, analyzed the peptide that retained visible absorbance, and correctly proposed that the chromophore is a 4-(p-hydroxybenzylidene)imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring...The crucial breakthroughs came with the cloning of the gene by Prasher et al and the demonstrations by Chalfie et al and Inouye & Tsuji that expression of the gene in other organisms creates fluorescence."
Traditions of giving credit in papers varies widely across labs, but in general the more generous inclination to list all contributing authors on a paper runs counter to that you might see at the Academy Awards, where winners give thanks a whole list of people including their mothers, wives and extended families. Behind each of the papers noted by Tsien was a who team of scientists, advisors, and I'm sure familial support that went unmentioned.
Rainbows of Fluorescent Proteins
Prasher's cloning and sequencing contribution defined GFP research. Sequencing in the late 1980's was laborious, much more so than it is today, where the most tedious parts of it were automated. Prasher spent years accomplishing his research, but then didn't get the funding to take the work forward from there. As Tsien and Chalkie acknowledged, their work depended on his, and Prasher passed his results on to Chalfie and Tsien and moved to another lab. In the light of the Nobel prize, Prasher's current job as a driver seems like a dreary "human interest" tale typical of the New York Times or NPR.
But this is actually typical for both "successful" and "unsuccessful" scientists. Progress moves in fits and starts -- fits and starts of research progress, of funding, and of luck, layered with varying dispositions of the people who read the grants, support the researchers, and whose labs the funding ends up in. Science might have more than it's share of unrewarded contributors.
Of the three prize winners, Chalfie lab constructed GFP to be used as a reporter protein in C. elegans, a transparent roundworm used as a model organism for research. C. elegans were put to use as a model organism in 1974, long after the discovery of GFP. Because the worms are transparent, Chalfie saw the potential to use GFP in genetic experiments, and to use it in place of other reporters like beta-lactamase which was used extensively at the time. Chalfie first noted his positive result in the October 1993 edition of the humble Worm Breeder's Gazette and went on to publish the research in Science.
In his 1998 review of GFP protein Tsien wrote:
"Unfortunately, Aequorea GFP genes are the only GFP genes that have been cloned... Painstaking research like that undertaken by the pioneers of Aequorea and Renilla GFP would be needed before cloning efforts could begin. It is unclear whether any investigators or granting agencies are still patient enough to undertake and fund such long-term groundwork."
(A little understandable lobbying for continued funding there.)
Rewarding, But Only One Award
While many scientists worked on GFP, many of the same scientist would have imagined its current utility. GFP was became important as technology changed the nature of science research, as the questions that scientists asked changed over time, and as successive bench developments proved the protein's potential.
Tsien's lab wrote another review of the protein in 2002, and by that time at least 30 other fluorescent proteins had been cloned and sequenced, thanks to continued funding and increased evidence of their utility. High throughput methods of sequencing and cloning accelerated work and allowed faster identification than in Prasher's Wood's Hole days. In 2005 Tsien wrote another review advising researchers how to choose the most appropriate fluorescent proteins among all that were available. Today, Martin Chalfie has said, uses of fluorescing proteins are only limited to scientists imaginations of what they want to do.
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1Osamu Shimomura, Frank Johnson, and Yo Saiga."Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea'", Journal of Cellular and Comparative Physiology October, 1962
2 Tsien, R. "Green Fluorescent Protein" Annual Review of Biochemistry Vol. 67: 509-544 July 1998.
3 Tsien, R. "A guide to choosing fluorescent proteins Nature Methods" 906 Vol. 2 No. 12, 905 - 909 (2005)
