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.
The tutorial initializes with an image of the pre-maturation ZsYellow chromophore tripeptide amino acid sequence (Lys66-Tyr67-Gly68) stretched into a linear configuration so that the lysine residue is positioned at the extreme left end of the window. Oxygen atoms are colored red, nitrogen atoms blue, carbon atoms white, and the black dashes at the peptide termini indicate continuation of the backbone beyond the portion illustrated. In order to operate the tutorial, use the Chromophore Maturation Control slider to transition through the intramolecular rearrangement of the tripeptide sequence that occurs during chromophore maturation. The first step is a series of torsional adjustments that relocate the carboxyl carbon of Lys66 in close proximity to the amino nitrogen of Gly68. Nucleophilic attack on this carbon atom by the amide nitrogen of glycine, followed by dehydration, results in formation of an imidazolin-5-one heterocyclic ring system, similar to the mechanism in GFP chromophore formation. Green fluorescence emission may transiently occur in this protein when oxidation of the tyrosine alpha-beta carbon bond by molecular oxygen extends conjugation of the imidazoline ring system to include the tyrosine phenyl ring and its para-oxygen substituent. However, spectroscopic investigations have yielded evidence that the green species is formed by a dead-end route to a incomplete chromophore product, rather than a true intermediate state. Proceeding with the maturation sequence to form the yellow chromophore (indicated by a yellow glow surrounding the affected structural elements), a short-lived acylimine moiety is attacked by the terminal amino group of Lys66 to form a novel partially unsaturated six-membered piperidine ring, simultaneously cleaving the polypeptide backbone between the 65 and 66 positions.
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.