Shimomura muffles the light from the laboratory, takes a handful of a powder made up of dried bugs that he keeps in a jar marked with the Cypridina-1944 label, puts it in a mortar to which he adds water and begins to grind it. A soft blue luminescence soon bursts out of the bowl, intensifying when you apply more pressure. The radiance of this marine being –Cypridina luciferinis an ostracod, a class of microscopic crustacean – it lit the path of this 82-year-old researcher, from the dark days in postwar Japan to the 2008 Nobel Prize in Chemistry.Marine Biological Laboratory, and Massachusetts,Shimomura is the discoverer of the green fluorescent protein? GFP ?, “one of the most important tools of modern biology”, according to the Swedish Academy.
This protein is found inAequorea victoria, a bioluminescent jellyfish, that is, capable of generating its own light. Its discovery in 1961 revolutionized molecular biology, and now it is possible to manipulate this chemical lantern to illuminate the interior of the cell.
An accidental discovery
Thanks to Osamu Shimomura -and Martin Chalfie and Roger Y. Tsien, who shared the Nobel Prize with him-, GFP can be introduced into a living cell to observe its changes and thus understand, for example, the organization of neurons, propagation cancer or the interaction of proteins with each other. ? GFP was an accidental consequence of my work. My initial target was aequorin, the protein ofAequorea It produces blue light – it fluoresces in the blue part of the spectrum. MeI wanted to understand the chemical process of light emission by animals, key to science ?, says Shimomura.
Their adventure had started decades ago with theCypridina, very abundant in Japan. In the 1940s, Japanese soldiers used its glow to read maps at night on battlefields: all you had to do was add a little saliva to a handful of powder.Cypridinaground. Bioluminescence in this ostracode is produced by the oxidation of apigment called luciferinand the action of the catalytic enzyme luciferase. Determining the nature and functioning of both elements became the Holy Grail of biochemistry of the moment.
A bath against atomic radiation
Young Shimomura grew up in one of the most difficult periods in the history of his country. His father, an army colonel, moved the family away from Osaka during World War II, because he feared the city would be bombarded. He settled in a house … 10 kilometers from Nagasaki !: “During the first day of high school, we were told that there would be no classes because students would have to go to work in the war industry, so I ended up in a factory of planes on the outskirts of Nagasaki, “recalls Shimomura. And he adds: “The factory was attacked by US B-29 fighters with magnesium bombs and I saw many of my colleagues die. On August 9, 1945 the sirens sounded as usual.” From a hill he saw a single enemy plane dropping three small parachutes with elongated objects. “When I returned to work, an intense light flooded the interior of the building and blinded me temporarily n An Atomic Radiation Bath. Less than a minute later an explosion sounded and the shock wave gave me an earache. Then everything turned gray. During on the way home there was a black rain falling. As soon as I arrived, my grandmother took off my clothes and bathed me. Maybe that saved me from the radiation. “
During the postwar period there was no future for young people in Japan. Many professors had been killed in the bombings, so Shimomura was unable to graduate and, although he continued to study on his own, his attempts to enroll at the university were rejected. One day he traveled to Nagoya to ask a certain professor for a job, who happened to be traveling. Wandering disillusioned through the Faculty of Chemistry, he ran into Professor Yoshimasa Hirata, somewhat distracted and half deaf, who assumed that the young man wanted to work with him. “You can come to my lab to help me isolate and purify compounds,” he told the astonished Shimomura, who readily agreed.
On the first day, Hirata caught a handful ofCypridinadissected, he made it glow blue and said, “We don’t know anything about this. Start by isolating and studying the luciferin in this organism.” “I went to work with no help other than the little existing literature, almost all in English. I only knew that luciferin was the fuel that caused bioluminescence, but I did not know if it was a protein, a sugar, an amino acid or another type of unknown molecule.Of the tens of thousands of substances that make up theCypridina, I had to isolate a highly unstable one, which degrades rapidly when exposed to oxygen. “
Shimomura made his experiments in hydrogen chambers, a dangerous gas due to its explosive nature. Each attempt required a week of work, but although each sample was purer than the last, it failed to crystallize Luciferin. Until one afternoon, by chance, he left a small amount of the substance in a very acidic environment. The next day he was amazed that a layer of red crystals had formed in the solution. Eureka, she did it!
In the late 1950s, the Japanese scientist accepted a job offer from Princeton University and was soon fascinated by the luminescent flashes of theAequorea, very abundant on the North American Pacific coast. During a summer of work in a Vancouver laboratory, Shimomura and his wife Akemi – also an expert in marine biology – collected 9,000 jellyfish with nets to clean swimming pools, extracting the strips of bioluminescent organs with scissors, wrapping them in cotton handkerchiefs and They squeezed them to remove the luminous liquid, which could be on for several hours. But the couple stopped the reaction and separated the luciferin from the luciferase as soon as possible.
Finally, the Japanese biologist discovered that the key to bioluminescence of Aequorea was a photoprotein, which he named aequorin; and that, when activated with calcium, it emitted a blue light. “The jellyfish manages the concentration of this element in its cells to control light production,” explains Shimomura. “When they bother her,” she continues, “the calcium level rises and sets off the alarm, which looks like a flashing neon.” But one day in 1961, the scientist observed that, viewed under ultraviolet light, the light from the jellyfish took on a greenish hue due to the action of what he later called green fluorescent protein: GFP, which emits bioluminescence in the green zone. of the visible spectrum.
The one whoGFP hadrelationship with calcium levelIt is key, as experts know that the mobility of this element plays a crucial role in many biological processes that include muscle contraction, the transmission of nerve impulses, the release of neurotransmitters, cell division and insulin secretion. So the possibility of “applying fluorescence on a molecular scale to follow the calcium pathway allows us to improve our understanding of the mechanisms of numerous diseases.”
In the 1980s, Harvard neurobiologist Martin Chalfie wondered whether it would be possible to implant the GFP from the jellyfish into a kind of worm calledCaenorhabditis elegans-barely 1 mm long and widely used in biological research- to synthesize the protein and produce light. Thus, it could be observed in vivo which genes are involved in bioluminescence. Chalfie was right: lGFP could make creatures other than Aequorea glow. It was a perfect protein for the molecular biology revolution and was soon implanted in experiments with genes from various plants, frogs, fish, goats, mice, rabbits, monkeys, flies, and yeast.
The Chinese-American biochemist Roger Y. Tsien took things even further and set out to study the cell as if it were a city to spy on its inhabitants in their daily chores: it was about observing how protein molecules are born and how they modify, travel, cooperate, compete and even murder others. His research is like a cellular anthropology. Tsien wanted to invent visual techniques with fluorescent dyes that would allow neurophysiologists to see the brain without having to open the patients’ heads. “The dyes change their fluorescence intensity in the presence of free calcium ions inside the cell, just like the Aequorea jellyfish does to produce light. These calcium ions stick to proteins and make them act. This process can only be studied in living cells, “explains Gruber.
Science fiction creatures
Tsien described the structure of the GFP molecule, allowing him to combine the 238 amino acids of the protein and invent mutations. Thus he came up with the formula to create a super-bright synthetic protein, much more visible than the natural one, and dyes of all colors, so that, when proteins are studied, the interior of the cell looks like a painting of contemporary art.
Today, oncologists, immunologists, virologists, neurobiologists, cell biologists, and botanists use Tsien’s fluorescent proteins, which gleam happily inside all kinds of guinea pigs. Production has become massive in his company Aurora Biosciences, whose capital exceeds 1,500 million dollars. Some labs make science fiction creatures, like mice with green ears and tails, cats that radiate a soft iridescent blue, or pink rabbits. There are more than 24,000 published studies on GFP and its applications. Shimomura hears the figure, smiles and repeats the mantra with which he opened his classes: “Never give up. If you find an interesting topic, study it to the end. If you face difficulties, solve them. Do not be discouraged.”
Angela Posada-Swafford
