Formation of the eqFP611 Fluorophore

The far-red fluorescent protein, eqFP611, was isolated from the sea anemone Entacmaea quadricolor and displays one of the largest Stoke's shifts and red-shifted emission wavelength profiles of any naturally occurring Anthozoan fluorescent protein. Although eqFP611 shares only approximately 23 percent sequence homology with the Aequorea victoria green fluorescent protein, enough critical amino acid motifs are conserved to form a very stable three-dimensional beta-can barrel structure, a consistent motif in fluorescent proteins. The fluorescence emission spectrum of eqFP611 is shifted to longer wavelengths by almost 105 nanometers (displaying a maximum at 611-nanometers) when compared to the enhanced green fluorescent protein (EGFP). This interactive tutorial explores the series of molecular re-arrangements that occur during the formation of the eqFP611 fluorescent protein fluorophore, which features a similar imidazoline ring system to EGFP, but substitutes methionine for serine as the first amino acid residue in the tripeptide sequence.

The tutorial initializes with an image of the pre-maturation eqFP611 fluorophore tripeptide amino acid sequence (Met63-Tyr64-Gly65) stretched into a linear configuration so that the methionine residue is positioned at the extreme left end of the window. Oxygen atoms are colored red, nitrogen atoms blue, carbon atoms white, the sulfur atom yellow, and the black dashes at the peptide termini indicate continuation of the backbone beyond the portion illustrated. Note that the maturation sequence occurs within the specialized environment provided by the central interior of the unusually stable beta-can barrel structure created by the folded protein. Perhaps the most important feature of all fluorescent proteins is that the fluorophore is fully encoded in the amino acid sequence, and is autocatalytically formed during maturation through a cyclization reaction between residues buried deep within the shielded environment of the barrel. During and after fluorophore maturation, the final structure and its intermediate states are stabilized by multiple interactions, including van der Waals forces and hydrogen bonds, with neighboring amino acid residues and water molecules that are not illustrated in the tutorial.

In order to operate the tutorial, use the Fluorophore Maturation State slider to transition through the intramolecular re-arrangement of the tripeptide sequence that occurs during fluorophore maturation. The first step is a series of torsional adjustments that relocate the carboxyl carbon of Met63 in close proximity to the amino nitrogen of Gly65. 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. Green fluorescence emission from the immature fluorophore (indicated by a green glow surrounding the affected structural elements) occurs 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. Proceeding with the maturation sequence to form the red fluorophore (indicated by a red glow surrounding the affected structural elements), a second oxidation step involving the alpha-carbon and amide nitrogen of Met63 further increases the extended pi-bonding electron resonance system to include the carboxyl group of Phe62. Adding this acylimine moiety to the fluorophore results in a greater degree of electron delocalization during excitation, which partially accounts for the dramatic red shift of emission wavelengths observed in eqFP611 as well as DsRed and related fluorescent proteins.

Crystallographic studies indicate that eqFP611 forms a tetramer exhibiting 222 symmetry, which is similar to that observed for the closely related DsRed fluorescent protein and also the non-fluorescent chromoprotein Rtms5. However, within the beta-can fold, the eqFP611 fluorophore adopts a unique coplanar configuration in which the Try64 phenoxy moiety is positioned trans rather than cis (as in DsRed) to the imidazoline ring. In addition, the bi-cyclic ring system and its substituents, which probably have a only limited mobility inside the protein, participate in numerous hydrogen bonds (at least nine) and a variety of non-polar van der Waals interactions with closely neighboring water molecules and amino acid residues. The extended hydrophobic methionine side chain fills a deep pocket that is also present in DsRed and Rtms5. Ultimately, the combined interactions between the fluorophore and neighboring aliphatic and aromatic amino acid side chains (and water molecules) may be helpful in elucidating the mechanism behind the unusual fluorescent properties of the eqFP611 protein.

As mentioned above, the Anthozoan fluorescent protein eqFP611 exhibits bright far-red fluorescence with an excitation peak at 559 nanometers and a maximum emission wavelength of 611 nanometers, to produce one of the largest recorded Stoke's shifts (approximately 52 nanometers) of any fluorescent protein. The quantum yield of fluorescence in eqFP611 is approximately 0.45, while the absorption spectrum has an extinction coefficient of 78,000 reciprocal moles per centimeter at 559 nanometers, values that combine to render the protein approximately as bright as EGFP. During the in vivo fluorophore maturation process, which occurs in approximately 12 hours, the protein passes through a green intermediate state as described in the tutorial. After maturation, however, only a small fraction of this green species (less and 1 percent) can be detected. In contrast to other Anthozoan fluorescent proteins, eqFP611 has a reduced tendency to oligomerize at lower concentrations as evidenced through electrophoresis and single-molecule experiments, although at high concentrations, the protein does form tetramers. Site-directed mutagenesis efforts have yielded functional dimeric variants of eqFP611, and continued efforts may finally lead to a monomeric far red fluorescent protein from this species.