Credit: © iStockphoto.com / dra_schwartz

The very aptly named green fluorescent protein — or GFP as it is almost universally known — is a barrel-shaped protein made up of 238 amino acids. Threaded through the long axis of the β-sheet barrel is an α-helix that contains a chromophore that is responsible for the emission of green light when GFP is exposed to either blue or ultaviolet light. This particular property, coupled with the fact that GFP is well tolerated by many different organisms, has led to its use as a fluorescent tag for monitoring biological processes at the cellular level.

The story of GFP begins in the oceans with the jellyfish Aequorea victoria, which has the unusual property that its outer edges glow green when it is agitated. In the early 1960s, Osamu Shimomura collected raw material from thousands of these jellyfish and extracted a small amount of a blue luminescent protein, which was subsequently named aequorin. During this process he also found another substance that glowed green when exposed to ultraviolet light — this was the protein that later became known as GFP. Shimomura and colleagues went on to show that the green glow produced by the jellyfish arises from an energy-transfer process in which the aequorin donor excites the GFP acceptor, which then emits green light.

In the early 1990s, Martin Chalfie and co-workers demonstrated that GFP could be expressed in organisms other than Aequorea victoria — such as Escherichia coli and Caenorhabditis elegans — and this was the breakthrough that paved the way for the practical implementation of GFP as a fluorescent tag for studying biological processes. It was generally thought that a number of steps requiring other proteins would be needed to produce the chromophore in GFP, but these experiments proved this to be wrong. Significantly, this result meant that GFP could be used as a universal tag, because no other auxiliary agents were needed to induce fluorescence. By engineering the genetic machinery of C. elegans so that it would produce GFP when a protein with a specific activity was expressed inside a cell, Chalfie was able to see cellular processes in a whole new light — albeit a green one!

The further development of GFP was based on a greater understanding of the molecular structure of the protein and specifically the chromophore responsible for its colourful name. Roger Tsien and co-workers explained how three amino acids in the peptide backbone of GFP — namely serine, tyrosine and glycine in positions 65, 66 and 67, respectively — react in the presence of oxygen to form the fluorescent chromophore p-hydroxybenzylideneimidazolinone. With this more detailed description of GFP, Tsien went on to develop other GFP derivatives with different spectral characteristics and increased stability. Not only could the brightness of the fluorescence be enhanced, but also the colour of emission could be tuned. Today all the colours of the rainbow can be found in a range of GFP and GFP-like proteins.

Apart from the obvious biomedical implications, GFP sensors have also been developed that can detect chemical species such as metals ions and small molecules. So not only has GFP enabled scientists to see biological processes in a whole new light, but many other chemical opportunities await. The future for GFP is a bright one.

 FURTHER READING

The Nobel Prize in Chemistry 2008