As visual creatures, humans believe what they see. We rely on our vision for macroscopic observations. Vast advances in microscopy now also enable visualization of cellular and sub-cellular structures. However, even the best microscopes cannot directly view molecular-level processes such as gene expression or protein interaction in vivo. In 1962, scientists found a solution in the most unlikely place – the green fluorescent protein (GFP) isolated from the bioluminescent jellyfish Aequorea victoria, native to the northern Pacific Ocean.

Ribbon diagram of green fluorescent protein, based on the determined X-ray structure.

Ribbon diagram of green fluorescent protein, based on the determined X-ray structure.

The  2008 Nobel Prize in Chemistry was awarded
in three equal parts for the discovery and development of GFP. Osamu Shimomura was recognized for discovering and characterizing GFP in 1962. Martin Chalfie first demonstrated that GFP could be expressed in other organisms without the aid of auxiliary proteins thirty years later.  Finally, Roger Tsien developed a diverse palette of GFP-related fluorescence proteins, with improved brightness, photo-stability, and other useful properties.

Discovery
Osamu Shimomura was initially interested in the protein aequorin, obtained from A. victoria, which emits blue light in response to calcium. However, the jellyfish appears bright green, not blue. The puzzle was solved in 1962 by Shimomura’s discovery of GFP, which has a peak excitation wavelength (460 nm) closely matching the peak emission wavelength of aequorin (470 nm).

Others concluded that GFP (the acceptor) absorbs the energy emitted by aequorin (the donor) in a process now known as Fluorescence Resonance Energy Transfer (FRET). Shimomura also identified the central chromophore, the specific chemical group responsible for GFP’s fluorescent properties (1).

At the time of Shimomura’s discovery, experts believed that auxiliary enzymes were required to form this central chromophore.  This would mean that if GFP were expressed in any organism other than A. victoria, it would likely be non-functional.

Martin Chalfie settled the issue by obtaining the gene for GFP from Douglas Prasher who had originally cloned it, and successfully expressed the flourescent protein in E. coli in 1992. He then expressed GFP in the nematode C. elegans, driven by a promotor for β-tubulin which is strongly expressed in six touch receptor neurons (1). This demonstrated the usefulness of GFP as a genetic marker, without the need for additional genetic manipulation. It was later expressed in yeast and, more importantly, mammals (1).

At this point, the mechanisms of GFP chromophore formation and fluorescence were still not understood, despite the obvious utility of the protein. Roger Tsien found that oxygen was all that was needed to activate the GFP, and since aerobic cells constitute the vast majority of biological research, the protein could be broadly expressed in these cells without additional enzymes. In addition, by inducing point mutations in the GFP gene, Tsien improved brightness and photo-stability, and created a colorful spectrum of variants which have proved invaluable in simultaneous labeling of multiple proteins. Tsien also engineered fluorescence proteins such as tdTomato and mCherry, which fluoresce in the orange-red part of the spectrum, based on the protein DsRed from the coral Discosoma. With the help of other collaborators, he also solved the crystal structure of GFP (1).

Properties
The original green florescent protein (GFP) found in Aequorea consists of 238 amino acids, of which residues 65-66-67 form a fluorescent chromophore in the presence of oxygen. The tertiary structure comprises a cylindrical eleven strand β-barrel threaded by an α-helix containing the fluorescent chromophore (1).

GFP is maximally excited by UV light at 400 nm, with another smaller peak at 470 nm (blue light). It emits photons with a sharp peak at 505 nm (green light) (1).  It is non-toxic when expressed at reasonable levels. Additionally, it can typically be fused to other proteins without changing either its fluorescence properties or the functional properties of the protein. These qualities make GFP extremely conducive to diverse biological applications.

Research Applications
Since its discovery, GFP has revolutionized biological research. Over 20,000 publications involving GFP have appeared since 1992, permeating essentially every area of biology (1).  Along with the discoveries being recognized this year with the Nobel Prize, concurrent advances in imaging techniques and data analysis have combined to maximize the utility of this versatile tool.

In the laboratory, scientists commonly fuse GFP to a protein of interest, in order to track the trafficking of the protein within a cell (1). For example, the oscillating movement of proteins during cell division in bacteria was observed by fusing MinC to GFP (5).

Expressing the GFP gene alone with a specific promoter is often used to label classes of cells in which that promoter is active. This is commonly used in neurobiology to label subsets of neurons, as first achieved by Chalfie in C. elegans. Currently, a modified form of GFP is being used to build a complete wiring diagram of the Drosophila brain (2). By concurrently expressing three or four fluorescent proteins, numerous individual cells can be viewed simultaneously, using a technique known as “Brainbow” (3). Another exciting application is specific expression of fluorescent proteins in tumor cells (4).

Fluorescence technology is also used to monitor the distance between two proteins that have been tagged using GFP-family proteins. In the technique known as FRET, the emission spectrum of one fluorophore (the donor) matches the excitation spectrum of the other (the acceptor). When the two tagged molecules are in close proximity (less than about ten nanometers) and the donor is excited by incident light at its excitation wavelength, the emitted energy is transferred to the acceptor and is then emitted at the acceptor’s characteristic emission wavelength. FRET can not only reveal if two molecules are close together and therefore interacting in some way, but can also provide an estimate of the exact distance between them, based on the decay rate of the signal (1).

Other techniques include monitoring the interaction of two proteins by tagging each protein with half of the GFP molecule, so that when they bind, GFP is completed and becomes functional. GFP has also been strategically fused with other molecules to construct pH or calcium sensors, enabling concentrations to be imaged at high spatial and temporal resolutions across populations of cells (1).

Conclusion

Shimomura’s initial discovery of GFP, followed by its expression in other organisms by Chalfie, and finally the development of a chromatically and functionally diverse spectrum of related florescent proteins by Tsien, have dramatically impacted biological research.  Aside from increasing the visual appeal of otherwise dull figures, GFP-family proteins have already shed light on numerous important findings.  The future looks even brighter.

References
1. The Royal Swedish Academy of Sciences, Scientific Background on the Nobel Prize in Chemistry (2008).
2. A. Chiang, “Building a wiring diagram of the Drosophila brain” (2008). Lecture delivered at the Marine Biological Laboratory, 30 June (2008).
3. J. Livet et al., Nature 450, 56 (2007).
4. M. Yang et al., Proc. Natl. Acad. Sci. U. S. A. 100, 14259 (2003).
5. D. M. Raskin, P. A. J. de Boer, Proc. Natl. Acad. Sci. U. S. A. 96, 4971 (1999).