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Why A Little Green Signaling Protein Prompted This Year's Nobel Prize in Chemistry — The Nobelists Share Their Story

ARTICLE IN BRIEF

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This year's Nobel laureates in chemistry discuss their journey toward discovering green fluorescent protein.

Martin Chalfie, PhD, chair of Columbia University's department of biological sciences, knows several Nobel laureates. In fact, when his childhood friend H. Robert Horvitz, PhD, a scientist at MIT, won the coveted prize in 2002, Dr. Chalfie took part in the weeklong festivities in Sweden.

Six years later, Dr. Chalfie will be returning to Stockholm — this time to collect his own prize. He is one of three scientists to win the 2008 Nobel Prize in Chemistry for their work in developing green fluorescent protein (GFP) to track changes in cells or organisms. Roger Tsien, PhD, professor in the departments of pharmacology and chemistry and biochemistry at the University of California-San Diego, and Osamu Shimomura, PhD an emeritus professor at the Marine Biological Laboratory in Woods Hole, MA, shared the prize.

In 1962, Dr. Shimomura identified GFP and figured out which part of the protein is responsible for its fluorescence. He found that calcium ions activate a jellyfish protein called aequorin. Once activated, aequorin produces blue light, but if the green fluorescent protein is nearby, the two proteins together yield a bright green signal. The same green light occurs if GFP alone is illuminated with ultraviolet or blue light. Thirty years later, this light-converting molecule was cloned by Douglas C. Prasher, PhD, a scientist at Woods Hole.

CLONING GENES IN THE ROUNDWORM

Dr. Chalfie's research model was the microscopic roundworm C. elegans that proved elegant in its simplicity and transparency. It has only 1,000 cells. When Dr. Chalfie heard that a scientist at Woods Hole was working on cloning GFP, he realized the possibilities immediately. What if a bioluminescent protein could cast its light in an organism that was already transparent? By 1988, Dr. Chalfie was interested in cloning genes and he wanted to know where genes were being expressed in the C. elegans. Dr. Chalfie was particularly interested in a handful of nerve cells that controlled the sensation of touch. He spent days knocking out genes or damaging them and testing the animal to see if its reaction to touch was disrupted.

When he heard about GFP at a lecture, the Columbia University scientist called Dr. Prasher at Woods Hole. He described what he was doing and said he was eager to get his hands on the gene, when it was cloned. Dr. Prasher said he would be happy to collaborate. But a few years later when he cloned GFP, and kept to his collaborative word, Dr. Chalfie was nowhere to be found. He had followed his new bride, also a scientist, to Utah and spent the year there on sabbatical. Dr. Prasher said he thought he had just dropped out of science.

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DR. MARTIN CHALFIE

The Woods Hole scientist published the details of his cloned gene in 1992 in the journal Gene. Around that time, a chemical engineering student had arrived at Columbia interested in fluorescence and Dr. Chalfie's idea of using it as a marker. He searched an online database and was shocked to find a new paper in the journal Gene on the cloning of the protein — by Dr. Prasher. They ran to the university's library to read it and Dr. Chalfie phoned him right away.

Dr. Chalfie called and cleared up any miscommunication. And Dr. Prasher, true to his word, sent him the DNA for the gene. His new student, Ghia Euskirchen, had her project. When the DNA arrived, they designed a way to get it into bacteria. A month later, they had green bacteria. And the rest is, well, a colorful history.

‘TRAINING MOLECULAR SPIES’

Dr. Tsien was one of the next callers to inquire about the clone. He saw it this way: “We were building and training molecular spies.” The GFP enters a cell and reports back in real time. And in Dr. Tsien's hands, that would come to mean many different colors. He could harness the power of these light-producing molecules to create a range of beautiful colors, allowing scientists to tag different cells and watch how they interact in living color. “We can teach cells how to make their own dyes for us,” he said recently. He is now trying to use these luminescent proteins to tag cells in humans and begin to think about ways to design cancer treatments using these proteins as an escort for targeted medicines.

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DR. ROGER Y. TSIEN

Since the GFP molecule came on the scene, its acclaim at helping shape scientific discoveries has paralleled the microscope. Only GFP allows scientists to study living cells and organisms.

As for Dr. Chalfie, he has used GFP to help unravel touch cells in roundworms, which is part of a larger story about the biology of the senses in general. In fact, it helped Dr. Chalfie and his colleagues identify a protein that is a sensor for touch. Dr. Prasher's collaboration with Dr. Chalfie resulted in a 1994 paper in Science. When Dr. Prasher's grant money ran out, he went to work for NASA in Alabama, moving far afield of GFP. When NASA cancelled his research project, he found himself unemployed. A year later, he took a job driving a shuttle van for a local car dealership in Huntsville, AL.

Meanwhile, Dr. Tsien was busy building GFPs that were brighter and could give off a strong signal. The colors were extraordinary. He then designed molecules that bind calcium and the colors changed as intracellular calcium levels increased. He also used it to detect activity of key enzymes.

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DR. OSAMU SHIMOMURA

For years, Dr. Chalfie's friends would say his GFP work was worthy of a Nobel Prize. But this year, as in past years, he would wonder who among his circle would win. On the Wednesday morning in early October when the announcements went live, he remembers waking at about six in the morning and hearing the faint sound of a phone. He lives in an apartment building in Manhattan and wondered why his neighbors were not answering their phone. He knew from friends and colleagues that the call comes in before 6 AM, shortly before they announce it to the world. At 6:10, he got out of bed wondering who won this year. He opened his laptop and typed in the Nobel's Web site. He saw his name. “I think it actually happened,” he yelled to his wife. His 16-year-old daughter awoke and they joyfully danced on air for a few minutes before the phone started ringing off the hook.

The first call didn't come from the Nobel committee. It came from a reporter from a radio station in Guatemala. A few minutes later, his answering machine basked at the news: “This is Stockholm calling,” the voice said. “If we don't get you, check your e-mail.”

Meanwhile, Douglas Prasher was already in his car, listening to National Public Radio on the way to the dealership. He heard the words “green fluorescent protein” and “Martin Chalfie” and “Nobel.” He knew instantly what was going on. “I was so thrilled,” he said during a telephone interview. “It was wonderful news.” The news got even better later that week when Dr. Chalfie and Dr. Tsien invited Dr. Prasher to Stockholm to share in the celebration that he had sparked about 15 years earlier.

Dr. Chalfie said that he wishes that there was not a three-person limit on the number of scientists who could win the Nobel Prize in each of the categories. “Cloning GFP was essential to this entire project,” said Dr. Chalfie. “Without it, neither my work nor Roger's work would have been possible.”

But Dr. Prasher has no ill feelings at all. He loves that GFP has gotten its credit and that he's played an important role in the story. And he has no apologies for the turn his life has taken outside of science. “I enjoy what I do. I didn't know that I enjoy talking to people.”

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REFERENCES

• Shimomura O, Johnson FH, Y Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 1962;59:223–229.
    • Prasher DC, McCann RO, Cormier NJ. Cloning and expression of the Cdna coding for aequorin, a bioluminescent calcium-binding protein. Biochem Biophys Res Comm 1985; 126:1259–1268.
      • Prasher DC, Eckenrode VK, Cormier MJ, et al. Primary structure of the Aequorea victorea green fluorescent protein. Gene 1992;111:229–233.
        • Chalfie M, Y Tu, Prasher DC, et al. Green fluorescent protein as a marker for gene expression. Science 1994;263:802–805.
          • Heim R, Prasher DC, Tsien RY, et al Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 1994;91:12501–12504.
            • Heim R, Cubitt A, Tsien RY. Improved green fluorescene. Nature 1995;37:663–664.