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Interactive Java Tutorials
Formation of the DsRed Fluorophore
The red fluorescent protein, DsRed, was discovered in the Anthozoan genus Discosoma during a search in reef corals for naturally occurring GFP analogues emitting fluorescence in longer wavelength regions. Although DsRed shares only approximately 26 percent sequence homology with the Aequorea victoria green fluorescent protein, enough critical amino acid motifs are conserved to form a similar very stable three-dimensional beta-can barrel structure. The fluorescence emission spectrum of DsRed is shifted to longer wavelengths by almost 75 nanometers (583-nanometer maximum) compared to the enhanced green fluorescent protein (EGFP). This interactive tutorial explores the molecular re-arrangement that occurs during the formation of the DsRed fluorescent protein fluorophore, which features a similar imidazoline ring system, but substitutes glutamine for serine as the first amino acid residue in the tripeptide sequence.
The tutorial initializes with an image of the pre-maturation DsRed fluorophore tripeptide amino acid sequence (Gln66-Tyr67-Gly68) stretched into a linear configuration so that the glutamine 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 Gln66 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. Green fluorescence emission (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 Gln66 further increases the extended pi-bonding electron resonance system to include the carboxyl group of Phe65. Adding this acylimine moiety to the fluorophore results in a greater degree of electron delocalization during excitation, which accounts for the dramatic red shift of emission wavelengths observed in DsRed fluorescent protein.
The DsRed fluorophore is similar in structure to that found in native green fluorescent protein, but is modified through the oxidation of a second backbone bond, as discussed above, to extend the conjugated pi-electron system to include the phenylalanine amino acid residue at position 65 (preceding the fluorophore) in the peptide chain. In addition, an unusual cis peptide bond configuration is adopted between Phe65 and Gln66, which positions the alpha-carbon atom of the glutamine residue in the same plane as the imidazolinone ring system, coinciding with the rest of the fluorophore (note that GFP features a normal trans configuration at this location). The cis orientation of the DsRed fluorophore, which has been elucidated through crystallographic diffraction analysis, is stabilized by reduced steric hindrance, enhanced electron delocalization, and increased hydrogen bonding with neighboring amino acid residues. The presence of such an unusual bonding configuration in DsRed (which is not present in GFP) suggests that isomerization around this bond may be a key step in fluorophore maturation.
All of the fluorescent proteins discovered to date display at least a limited degree of quaternary structure, exemplified by the weak tendency of native Aequorea victoria GFP to dimerize when immobilized at high concentrations, the obligate dimerization of fluorescent proteins isolated from Renilla, and the strict tetramerization motif of the orange and red fluorescent proteins isolated in reef corals and anemones. Coupled to the fact that DsRed undergoes a green state during the very slow (approximately two days) maturation period of the fluorophore, the protein has not found great utility as a genetically encoded fusion tag. However, the tetrameric DsRed protein features a very high extinction coefficient and quantum yield, as well as reduced photobleaching rates and greater resistance to changes in pH than Aequorea victoria fluorescent protein derivatives. These properties have led researchers to search for genetic modifications that alleviate the tendency of DsRed to form dimers and tetramers, an effort that should ultimately yield a high-performance monomeric red fluorescent protein.
Contributing Authors
David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, 37232.
Jennifer Lippincott-Schwartz and George H. Patterson - Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892.
Matthew J. Parry-Hill, Nathan S. Claxton, Scott G. Olenych, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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