PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Reflections on the GFP Nobel Prize by William W. Ward, Ph.D. On October 8, 2008, the Nobel Prize for Chemistry was awarded to Green-Fluorescent Protein (GFP) researchers Osamu Shimomura, Marty Chalfie, and Roger Tsien. This is an account of the decades of research that preceded this award. Fluorescence in the outer rim of the bioluminescent jellyfish, Aequorea Victoria, had been noted as far back as 1848, but GFP, the protein, was first studied in the very early 1970's by Harvard University scientists, Jim Morin and J.W. Hastings, and by John Wanpler and Milton Cormier of the University of Georgia. The Harvard group concentrated on the bioluminescent hydroid, Obelia, while the Georgia group studied the bioluminescent sea pansy, Renilla reniformis. Both groups recognized, as early as 1971, that the green fluorescent glow in these coelenterates came from a remarkable protein--one named "green-fluorescent protein" by Jim Morin. Very soon thereafter, in 1973, I joined the Cormier group, spending my next four years of postdoctoral research studying the physical properties of Renilla GFP. At about that same time, Frank Prendergast began working on structural properties of Aequorea GFP in the Mayo Clinic laboratory of John Blinks. Soon thereafter, Tony Campbell began his studies of photoproteins and GFP molecules at the Cardiff Medical School in Wales. Dr. Blinks spent a number of years in the 1960's, as did Princeton's Osamu Shimomura and Frank Johnson, isolating, from the jellyfish Aequorea, the calcium triggered, blue light-emitting, photoprotein, aequorin (a protein discovered and named by Shimomura in 1962). In those years, Blinks concentrated on applications of aequorin as an intracellular calcium ion indicator. Shimomura focused his attention on the organic chemistry of aequorin and its tightly bound chromophore (a small molecule he later named coelenterazine). Prendergast worked on the biochemistry of Aequorea GFP, becoming the first to purify and correctly characterize Aequorea GFP in 1978. Meanwhile, in Georgia, I was the first to purify and characterize Renilla GFP in 1979 (1). I showed Renilla GFP, unlike Aequorea GFP, is an obligate dimer of identical 27 Kdal subunits. Most importantly, I demonstrated for the first time, in 1978, with a dual phototube luminometer, that Renilla luciferase forms an electrostatically stabilized complex in dilute solution with Renilla GFP (2). This complex produces green light (not blue light) by radiation-less energy transfer from luciferase-bound luciferin. The GFP-luciferase complex is completely disrupted by the addition of sodium chloride to the reaction buffer, leading to blue light production. Furthermore, pure GFP molecules I painstakingly prepared from distantly related coelenterates, but ones having identical spectral characteristics, failed to generate green light under the same experimental conditions. This was the first unequivocal demonstration of radiation-less energy transfer in the field of bioluminescence. Kazuo Hori, in the Cormier lab, was first to synthesize an active luciferin (coelenterazine) molecule. Later, Russ Hart, another synthetic organic chemist who joined the Cormier lab in 1975, would show that dark variants of luciferin could transfer excitation energy quite efficiently to Renilla GFP--the final proof of radiation-less energy transfer. As a controversy had arisen about the nature of the bound chromophore of photoproteins, I performed crucial experiments to show that the here-to-fore unidentified choromophores in the coelenterate photoproteins, aequorin, mnemiopsin and berovin, are all identical to the Renilla luciferin Dr. Hori had characterized by chemical synthesis. In 1977, I joined the Biochemistry and Microbiology Department at Rutgers University, Cook College, as an Assistant Professor, where I began an intensive research program on Aequorea GFP. This research, like that of Dr. Shimomura, required mounting a major jellyfish collecting program nearly 3000 miles from New Jersey, at the University of Washington's marine lab, Friday Harbor Laboratories (FHL) in the San Juan Archipelago. Along with as many as 8 colleagues and students, I made this trip for 17 summers, hand-collecting and hand-dissecting as many as 100,000 jellyfish in 3 weeks time each summer. Every summer, Dr. Dennis Willows, FHL director for most of that time, graciously accommodated our conspicuous, sloppy crew of jellyfish collectors. He must have realized more value in what we were doing than we ourselves recognized. After reducing the collected material to a manageable volume at FHL and transporting the precious material on dry ice back to Rutgers, we then spent 6-person months each year purifying the GFP to homogeneity. We accomplished much in the years that followed. Our group was the first to demonstrate (3) the spectroscopic identity of Renilla and Aequorea GFP in the denatured state, providing a convenient means to calculate molar extinction coefficients for all future GFPs based on the very careful work I had done with Renilla GFP in the Cormier lab. Those calculations remain, to this day, the absolute standard for determining molar extinction coefficients and fluorescent quantum yields for all genetic variants of Aequorea GFP. Breaking with the Nobel Prize winning experiments of Christian Anfinsen, we showed that Aequorea GFP can be reversibly denatured, but only if removed from the denaturing agent very rapidly (Anfinsen had stressed slow removal on thermodynamic and kinetic grounds). We needed to remove the denaturant quickly, as two otherwise fully buried cysteine residues in the protein become slowly oxidized when the protein is denatured, thus preventing proper refolding. While working on this problem, we discovered that the GFP chromophore, previously thought to be "rock stable," is quite easily (and reversibly) perturbed upon dimer formation and upon exposure to organic solvents and extremes of pH. Our discovery that Aequorea GFP forms reversible dimers at high protein concentration provided a new, and highly revealing "tail is wagging the dog," (or, as I like to put it, "your Camelions are mating") interpretation for some of the FRET-based (fluorescence resonanance energy transfer) assays already being marketed by Aurora Biosciences, notably the calcium-sensing Camelion. If blue-emitting and yellow-emitting forms of GFP (on opposite ends of a FRET construct) are able to self-associate (head to tail with another FRET construct) in the absence of calcium, as we clearly demonstrated they would (4), the calcium-free background of the Camelion assay would rise, making data interpretations much more difficult. This problem was later corrected by mutagenizing the contact interface of the dimeric pair, but only because we raised the issue in public. We were first to obtain Aequorea GFP crystals that diffract X-rays, and along with Doug Prasher, V.K. Eckenrode, Prendergast and Cormier were first to complete the primary sequence of Aequorea GFP in 1992 (5). One year later, we became the first to identify, correctly, the chromogenic hexapeptide of GFP (6). Our lab was the first to show an oxygen requirement for the "greening" of recombinant GFP and, in collaboration with others, we were the first to synthesize GFP in a cell-free system. In another collaborative effort, we were first to demonstrate the safety of GFP in rat-feeding experiments (7). Our lab collaborated in the 1994 blockbuster cover story in Science that showed the first successful cloning of GFP; a paper, with 3400 citations at last count, and one considered to be among the 10 most cited papers in all of biotechnology (8). As I had purified Aequorea GFP to homogeneity and as I possessed the most convincing spectral data from native Aequorea GFP, I was in the best position to validate, for the Science paper, the identity of the native and recombinant proteins. In addition to those I have already acknowledged as pioneers in GFP research, others throughout the decades of the 1980's and 1990's, including Cormier, Shimomura, Prendergast, Campbell, K. Ward, G. Phillips, F. Tsuji, Hart, and Prasher all made significant contributions to our understanding of the structure and function of GFP. But by 1993, Doug Prasher, with assistance from V.K. Eckenrode, W.W. Ward, F.G. Prendergast, M.J. Cormier, C.W. Cody, and W.M. Westler made two of the most crucial advances with GFP. Together we deduced the entire primary structure of Aequorea GFP (5) and the correct structure of the chromogenic hexapeptide (6). In particular, Doug Prasher cloned the gene. Prasher's cloned gene failed to produce a fluorescent product as it contained, at one end, twenty additional amino acids that interfered with the proper folding of the protein. Having lost his funding and, therefore, unable to fix the small error in this gene, Doug gave the gene to Marty Chalfie and Roger Tsien who capitalized brilliantly on the applications that followed the repair of Prasher's gene. The "magic" of GFP is that the cyclic tripeptide, or the fluorescent chromophore of GFP, is part of the primary sequence of the protein. Isolated from the protein, as we had shown in the 1970's, this chromophore is non-fluorescent. But sequestered within the can-shaped barrel of the protein (a structure determined by George Phillips), that same chromophore becomes brilliantly fluorescent. So, unlike other chromoproteins (like hemoglobin) that need multiple genes (one or more genes to make the protein and many more to make the chromophore), GFP needs only one gene. Remarkably, that gene, transfected into and expressed in other cells, tissues, organs and organisms, almost invariably expresses its brilliant fluorescence. GFP is now widely used as a marker for gene induction and countless other cellular and physiological processes. At present, nearly 20,000 research papers have been written about GFP, virtually all about how GFP is utilized to solve basic cellular, biochemical, and biomedical questions (9). An additional 20,000 research papers cite one or more of these original works. There is an even bigger take home message that should be communicated through the GFP story. That message is to use the GFP story to uncover, for every reader of this article and every decision maker in our society, what really drives scientific inquiry. I would argue that the driving force for most scientific inquiry is the aesthetic appreciation science brings to the investigator. In particular, research on bioluminescence epitomizes this scientific thirst for the art and the aesthetics of science. More than 5 decades ago, as a high school freshman, I took the Kuder Preference Test, a binary choice test that evaluates the sorts of activities a young student appreciates and might gravitate toward in a career choice. My top two categories came out "scientific" and "artistic." I did not doubt the scientific, but I wondered, then, about the artistic. Now having lived most of my life as a scientist studying bioluminescence, I know that Kuder was telling me the truth. I am strongly motivated by the beauty and artistry of bioluminescence. I would argue that the love of art and aesthetics drives good science and that good science gives rise to good practical applications. It is not important what the scientific project is, so long as it is approached with passion accompanied by good observational skills. Virtually every major scientific discovery comes about purely by accident within basic research projects not foreseen to have practical applications. Such is the case with bioluminescence and GFP--such is the case with most other scientific discoveries. Literature Cited
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