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Of jellyfish and camgaroos

The discovery of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has revolutionized cell biological research. The ability of this protein to form a fluorescent chromophore in the absence of any additional factors has spurred wide applications of GFP as a genetically encoded marker and biosensor. GFP is used to follow cells or patterns of gene expression in living organisms or cells, and the fusion of GFP to other proteins allows researchers to monitor the whereabouts of its host proteins in virtually all subcellular compartments. Clever modifications of GFP and the use of fluorescence energy transfer (FRET) have allowed the development of blue, cyan and yellow derivatives, as well as probes that respond to changes in, for example, intracellular pH or calcium levels.

In the past, GFP has generally been fused in tandem to either end of its host proteins, and only in rare cases has it been inserted within its hosts. The seemingly monolithic structure of GFP, an 11-stranded β-barrel bearing the chromophore in its centre, indicated that gross alterations or insertions in GFP itself might disrupt its function. However, in a recent paper ( Proc. Natl Acad. Sci. USA 96, 11241–11246; 1999), Roger Tsien and colleagues showed that such structural changes are possible, and can actually be taken advantage of, opening up new avenues for the use of GFP as a research tool.

Figure provided by R. Tsien

The authors discovered that circular permutations in which the carboxy- and amino-terminal domains of GFP are swapped were tolerated, with interesting differences in their fluorescent properties. Interestingly, the authors were also able to insert an entire protein, the calcium-sensitive calmodulin (CaM), into GFP or YFP, the yellow cousin of GFP. The putative structure of a GFP–CaM hybrid is shown here. The fluorescence of this ‘camgaroo’ (with YFP/GFP carrying CaM in its ‘pouch’) increases more upon calcium binding than do any of the existing genetically encoded fluorescent probes, and is able to detect changes in calcium levels in living cells. One can now envisage inserting a wide range of sequences into GFP, such as specific cleavage or phosphorylation sites to monitor protease or kinase activities, or domains that bind ligands such as lipids, metal ions or amino acids. The future is bright, with even more exciting applications of GFP as a genetically encoded biosensor on the horizon.

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Marte, B. Of jellyfish and camgaroos. Nat Cell Biol 1, E174 (1999). https://doi.org/10.1038/15610

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