Fluorescent Proteins - From the Beginnings to the Nobel Prize

The interest of man in fluorescent proteins can be traced back to the first century A.D. when the roman natural philosopher Pliny the Elder [1] (Gaius Plinius Secundus, 23 A.D. – 79 A.D.) described a glowing jellyfish (Pulmo marinus) in the Mediterranean Sea. In his eyes these animals emitted light of such intensity that it could almost be used as a torch (Bohn, 1855). Beside this jellyfish there is an abundance of other organisms attracting our attention because they glow in the dark. In fact they don’t do this to catch our admiration but, amongst other theories, to communicate with their conspecifics (fireflies exert a pull on their mates e.g.), attract prey (frogfish e.g.) or to frighten predators (bobtail squid e.g.). Some of these fascinating creatures are living in the world of darkness – such as the deep sea – where light is a raw property. Whereas only the upper 200 m of the oceans are penetrated by sunlight, underneath that border the only existing illumination is produced by organisms which are living there.

Using chemiluminescence and fluorescence to emit light, evolution developed one of the most impressive and powerful tools of recent biochemistry and cell biology which is based on the scientific curiosity of several persons. Their efforts and discoveries should be mentioned in the following.

With the upcoming interest in natural sciences after the Middle Ages, people like the Spanish physician and botanist Nicolás Monardes described a gleaming wood extract made of Lignum nephriticum in 1565. Later on this light could be ascribed to an oxidation product of a flavonoid which is found in this plant and showed that glowing was not only restricted to animals. More than three centuries later (1858) George Gabriel Stokes, an Irish mathematician and physicist, formed a name for that kind of natural occurring phenomena: Fluorescence. He thought up this term after the blue light emitting mineral fluorite and broadened the observation of radiating objects from animals over plants to stones.

The next path-breaking step in the triumphal procession of GFP had to wait for the invention of cloning techniques in the 1970s. It was the work of the American microbiologist Douglas Prasher who cloned the complete GFP gene for the first time in 1992 [3]. Before he prepared an A. victoria cDNA library together with his colleague Milton Cormier at the University of Georgia, knowing already the prospective of GFP. Unfortunately Prashers funding by the American Cancer Society ran out before he could start to express the recombinant GFP in bacteria.

Initial doubts that GFP would not work outside jellyfishes were erased by Martin Chalfie, an American biologist who could benefit from Prasher’s preliminary work. By introducing the GFP gene into E. coli and the nematode C. elegans he could show that there were no other jellyfish specific proteins or factors necessary to produce the green glow [4].

With that experience the door was opened for the green fluorescent protein to start its astonishing career in life sciences. GFP became the key to observe structures in living cells and organisms without the necessity of synthetic labels or fluorescent antibodies. In the case of immunofluorescence staining, the access of antibodies to accordant target structures inside the cell has to be guaranteed. This is achieved by permeabilizing the cells with detergents (e.g.), which inevitably induces cell death. Furthermore most antibodies have a preference for denatured antigens. Therefore immunofluorescence techniques use agents like para formaldehyde to denature cellular protein target structures. Altogether the use of GFP could overcome these non physiological conditions and paved the way for life cell imaging.

Another person who was realizing and developing the great potential of GFP was Roger Tsien. Coming from the Calcium regulation field, the American cell biologist was getting interested in tracking macromolecular interactions inside living cells. After studying and working in Harvard, Cambridge and Berkeley he finally settled down at the University of California, San Diego. As a Professor of Pharmacology, Chemistry and Biochemistry he improved the efficiency of GFP by taking advantage of a very common principle in evolution: Mutation. In 1994, he and his group established a single point mutant (S65T) of GFP with a much better intensity and photo stability than the wild type version. Besides a brighter glow the S65T mutant had another striking technical benefit. Whereas the wild type GFP had two excitation maxima at 395 nm and 475 nm, the mutated version had only one at 484 nm. By keeping its emission wavelength at 509 nm, the spectral characteristics of the "new" GFP fitted almost into classical FITC fluorescence properties (FITCex: 496 nm, FITCem: 520–530 nm). Because of its enhancements this GFP variant was called “enhanced” GFP or EGFP.

By doing structural research on GFP Tsien and coworkers developed further fluorescent derivates. With the GFP structure in their hands they established a variant (T203Y) which was shining in bright yellow and was therefore given the name "yellow fluorescent protein" or YFP. Cyan (CFP) and blue (BFP) forms followed [5].

The colour palette of fluorescent proteins was extended into the red range of the spectrum when the biochemist Sergey A. Lukyanov, who is a corresponding member of the Russian Academy of Sciences since 2003, discovered red fluorescing proteins in corals [7]. After buying some of these Cnidaria in a moscow pet shop Lukyanov studied the fluorescent behavior of these primitive animals destined for russian marine aquariums. Amongst others he identified a red fluorescing protein coming from Discosoma – a Corallimorpharia – which he called DsRed. Besides DsRed, Lukyanov and coworkers identified a whole palette of other glowing proteins in Anthozoa, which were optimized for biochemical use time after time.