Formation of the HcRed Fluorophore

The far-red fluorescent protein, HcRed, was discovered through site-directed and random mutagenesis efforts on a non-fluorescent chromoprotein (hcCP) isolated from the Indo-Pacific Anthozoa species, Heteractis crispa, during a search in reef corals for naturally occurring GFP analogues emitting fluorescence in longer wavelength regions. Although HcRed shares only approximately 21 percent amino acid 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. The fluorescence emission spectrum of HcRed is shifted to longer wavelengths by almost 140 nanometers (645-nanometer peak; emitting in the far red) 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 HcRed fluorescent protein fluorophore, which features a similar imidazoline ring system to EGFP, but substitutes glutamic acid for serine as the first amino acid residue in the tripeptide sequence.

The tutorial initializes with an image of the pre-maturation HcRed fluorophore tripeptide amino acid sequence (Glu64-Tyr65-Gly66) stretched into a linear configuration so that the glutamic acid 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. 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 Glu64 in close proximity to the amino nitrogen of Gly66. 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. 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, unlike several related red fluorescent proteins, such as DsRed and eqFP611, HcRed does not appear to transition through a green fluorescent intermediate state that often occurs at this point in the maturation sequence. The red fluorophore is formed (indicated by a red glow surrounding the affected structural elements) after a second oxidation step, involving the alpha-carbon and amide nitrogen of Glu64, further increases the extended pi-bonding electron resonance system to include the carboxyl group of Cys63. Adding this acylimine moiety to the fluorophore results in a greater degree of electron delocalization during excitation, which contributes to the dramatic red shift of emission wavelengths observed in HcRed fluorescent protein.

A notable feature of the HcRed fluorophore is the mixed cis coplanar and trans non-coplanar ensemble of molecular conformations that are assumed by the Tyr65 phenoxy substituent due to increased mobility (compared to GFP, DsRed, and eqFP611) of the fluorophore within the cavity of the beta-can structure. This surprising degree of flexibility can be attributed to a lack of steric hindrance preventing rotational motion, as well as favorable molecular interactions with surrounding amino acids and water molecules for each isomer. The cis coplanar structure is most likely responsible for the red fluorescent properties of HcRed, while the alternative trans non-coplanar conformation results in reduced pi-bonding and more closely resembles the non-fluorescent structure of the parent chromoprotein, hcCP. In the tutorial, after the Fluorophore Maturation State slider has been translated to the far-right (mature) position, use the HcRed and hcCP radio buttons to toggle between the conformations of the Tyr65 phenoxy substituent.

As discussed above, HcRed was genetically engineered through site-directed and random mutagenesis of the parent chromoprotein originally isolated from the Heteractis crispa reef coral. A total of 6 amino acid substitutions were necessary to create a red fluorescent species that matured rapidly and efficiently at 37 degrees Celsius. However, similar to other reef coral proteins, the resulting red fluorescent HcRed displayed a tendency to form obligate tetramers when expressed in bacteria. Additional mutagenesis efforts resulted in a brighter dimeric variant, which has been denoted HcRed-2 and is commercially available (under the tradename HcRed1), but a monomeric version of the protein has not yet been discovered. In order to generate a species of the protein that is useful in creating fusion products for localization studies, a tandem dimer expression vector of HcRed (two head-to-tail identical copies of the protein) has been constructed. When fused to a gene product that itself forms biopolymers (such as actin or tubulin), the HcRed tandem dimer forms an intramolecular dimer complex that apparently does not interfere with the biological activity of the resulting chimera. In the future, continued mutagenesis investigations and unique strategies, such as the tandem dimer concept, should collectively yield a useful cadre of fluorescent proteins that enable imaging in the far-red and near-infrared portions of the visible light spectrum.