Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Fluorescent Protein Technology

zsYellow Fluorescent Protein Chromophore Formation

The yellow fluorescent protein, ZsYellow (originally referred to as zFP538), was discovered in the Anthozoan button polyp Zoanthus during a search in reef corals for naturally occurring GFP analogues emitting fluorescence in longer wavelength regions. Although ZsYellow shares only approximately 28 percent sequence homology with the original Aequorea victoria green fluorescent protein, enough critical amino acid motifs are conserved to form a similar very stable three-dimensional beta-barrel structure. One of the most unique features of the ZsYellow fluorescence emission spectrum is that the peak (538 nanometers) occurs almost midway between those of GFP (508 nanometers) and DsRed (583 nanometers), presenting an opportunity to investigate proteins emitting fluorescence in the yellow portion of the visible light spectrum. This interactive tutorial explores the molecular rearrangement that occurs during the formation of the ZsYellow fluorescent protein chromophore, which 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 sequence.

Formation of the additional double bond contained in the heterocyclic ring results in a greater degree of electron delocalization during excitation when compared to GFP, which accounts for the longer emission wavelengths observed in ZsYellow fluorescent protein. In fact, the degree of conjugation observed in ZsYellow is intermediate between that observed with GFP and DsRed (one double bond more than GFP, and one less than DsRed), which accounts for the positioning of emission wavelengths in the yellow region. Structural analysis by x-ray diffraction indicates that the novel heterocyclic ring system lies in a plane that is approximately parallel with the rest of the ZsYellow chromophore, a finding consistent with the extended conjugation mechanism of increasing emission wavelengths. In addition, cleavage of the peptide backbone between residues Phe65 and Lys66 results in the formation of a terminal carboxamide group at residue 65, which should be available for participation in hydrogen bonding to stabilize the chromophore.

Similar to all of the fluorescent proteins isolated from reef corals to date, ZsYellow exhibits a marked tendency to form tetramers when expressed in vivo, hampering the use of this protein as a fusion partner for localization investigations. Furthermore, the reduced brightness level of ZsYellow when compared to enhanced green fluorescent protein (25 percent of EGFP) also limits somewhat the utility of this reporter in fluorescence microscopy. The unique emission spectral profile of ZsYellow, however, should encourage the search for genetic modifications that alleviate the tendency to form tetramers while simultaneously increasing the quantum yield and extinction coefficient, an effort that could ultimately yield a high-performance monomeric yellow fluorescent protein.

Contributing Authors

Tony B. Gines, Kevin A. John, Tadja Dragoo and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.