Fluorescent Protein Technology

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.

A planar trans motif is found in the chromophore of the red fluorescent protein eqFP611, isolated from a sea anemone, and displays one of the largest Stokes shifts and red-shifted emission wavelength profiles of any naturally occurring fluorescent protein.

Although HcRed shares only approximately 21 percent amino acid sequence homology with GFP, enough critical amino acid motifs are conserved to form a very stable three-dimensional beta-barrel structure.

Upon illumination of the green species with ultraviolet light, the Kaede chromophore undergoes polypeptide chain cleavage between His62 and Phe61 to generate red fluorescence.

Investigations into the mechanism of kindling fluorescent protein photoswitching suggest that a cis-trans isomerization of the hydroxybenzilidine chromophore moiety is a key event in the switching process.

By replacing the threonine at position 203 with a histidine residue in wild-type GFP, researchers produced a variant having negligible absorbance in the region between 450 and 550 nanometers, thus dramatically enhancing contrast.

The most prominent and well-studied photoswitchable fluorescent protein is named Dronpa (named after a fusion of the Ninja term for vanishing and photoactivation), which is a monomeric variant derived from a stony coral tetramer.

Unlike photoactivatable fluorescent proteins, Kaede and Eos are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions for optical highlighting.

When excited with ultraviolet light, the tyrosine residue in the neutral chromophore of wild-type GFP becomes a strong acid and transfers a proton through a novel hydrogen bond network in a process known as excited-state proton transfer.