The growing class of fluorescent proteins useful for detecting events in living cells and animals has almost single-handedly launched and fueled a new era in biology and medicine.
It took over thirty years, and the advent of recombinant DNA as well as vastly improved molecular biological approaches to see the pioneering work of Osamu Shimomura developed into a useful tool for live-cell imaging by Doug Prasher and Martin Chalfie. Just in the past decade, however, we have witnessed a truly remarkable expansion in the fluorescent protein palette, largely driven by the innovative studies from Roger Tsien's laboratory. Most of the fluorescent proteins that are commonly used today have been modified through mutagenesis to optimize their expression in biological systems. Continued efforts using directed evolution approaches will no doubt improve the spectral characteristics, photostability, maturation time, brightness, acid resistance, and utility of the fluorescent protein tags for cellular imaging.
The current thrust of fluorescent protein development strategies is centered on fine-tuning the current palette of blue to yellow variants from jellyfish, while simultaneously developing monomeric fluorescent proteins emitting in the orange to far-red regions of the visible light spectrum. We now have jellyfish proteins that span an 80-nanometer portion visible spectrum from deep blue to yellow-green, providing a wide choice of genetically encoded markers for studies in cell biology. Fluorescent proteins derived from Anthozoa species (corals and anemones), as well as other sources, span the entire visible spectrum and feature a wide range of useful properties. The unique optical highlighter properties of fluorescent proteins can allow the investigator to change the color or the emission state, providing unique opportunities to track the dynamic behavior of proteins in living cells and animals.
Still the "gold standard" in fluorescent protein technology, the enhanced version of GFP features a chromophore based on a para-hydroxybenzylidene substituted imidazolinone. The chromophore of the first reported red fluorescent protein extends conjugation into the polypeptide backbone to generate fluorescence in the longer wavelength regions. The ZsYellow fluorescent protein chromophore features a novel three-ring system and peptide backbone cleavage due to the substitution of lysine for serine as the first amino acid residue in the chromophore tripeptide. The final step in mKO chromophore maturations involves the formation of a novel five-member thiazole ring system when the Cys65 hydroxyl moiety attacks the carbonyl of Phe64 and cyclizes. In a manner similar to mKusabira Orange, mOrange chromophore maturation involves the formation of a novel five-member oxazole (rather than a thiazole) ring system.