Hundreds of Men
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 all 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 is like some but not all of the Nobel Prize awards, in that hundreds of people from many science disciplines developed GFP to its current state of usefulness. The committee could only choose three winners.
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 as well as insight into the fortitude of the researchers. It's perspective that's useful to understanding how science works, on many levels.
Thousands and Thousands of Jellyfish
People like to recount how Shimomura collected tens or hundreds of thousands (the accounts vary) of the Aequoria jellyfish used to conduct his GFP and aequorin experiments. Non-scientists might be able to imagine collecting tens of thousands of jellyfish. But that's only the start. Most people probably can't fathom what its like to try and figure out how to extract of luminescent parts of the jellyfish "squeezate" without destroying them, how to determine a method for purifying a protein via repeated chromatography, or how to deduce under what chemical conditions a protein glows and at what wavelength, information that makes a protein useful, etc.
In 1962, the GFP protein, not the focus of Shimomura's study, was a bit of a mystery. Scientists didn't know exactly how it accomplished its glowing or interacted with aequorin that was the focus of his study. Here it is mentioned in by the authors 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 a a 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 figured out that the protein absorbs light in the blue spectrum emitted in Aequorea victoria by the calcium activated aequorin, then emits its fluorescent green light. Over the next few decades others advanced the work as 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 credit-giving vary widely across labs, but in general the inclination to list co-authors runs the opposite of the instinct of the Academy Award winners to give thanks to their extended families. Behind each of these papers was a team of scientists, advisors, and support who went unmentioned.
Rainbows of Fluorescent Proteins
Prasher's contribution defined GFP research. Sequencing in the late 1980's was laborious, much more so than it is today. 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. Prasher passed his results on to Chalfie and Tsien and moved to another lab. In the light of the Nobel prize, Prasher's seems like a stark tale when written up by the New York Times or by relayed NPR. An incredible amount of time, years, decades in some cases, could be spent doing one thing, than poof, it doesn't work out or research moves on.
Like all progress, science 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. It has its share of unrewarded contributors. Prasher generously told the New York Times"They worked their butts off over their entire lives for science, and I haven't."
The 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, 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 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."
Rewarding, But Only One Award
Many of the early scientists who worked on GFP could have never foreseen 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. High throughput methods of sequencing and cloning accelerated work and allowed faster identification than in Prasher's day, or that anyone could have fathomed 40 years ago. Early researchers couldn't have imagined what an impact GFP would have on developmental and cellular biology. In 2005 Tsien wrote another review advising researchers how to choose the most appropriate fluorescent proteins among all that were available. Uses for the protein are now only limited, as Martin Chalfie put it, by scientists imaginations of what they want to do.
----------------------------------------------------
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)
Leave a comment