Formation of the GFP Fluorophore

Formation of the GFP Fluorophore - Java Tutorial

Among the most remarkable attributes of the original green fluorescent protein (GFP) derived from the Aequorea victoria jellyfish, as well as the more recently developed palette of color-shifted genetic variants, is that the entire 27 kiloDalton polypeptide structure is essential for the development and maintenance of fluorescence in this very remarkable family of proteins. The principle fluorophore (often termed a chromophore) is a tripeptide consisting of the residues serine, tyrosine, and glycine at positions 65-67 in the sequence. Although this simple amino acid motif is commonly found throughout nature, it does not generally result in fluorescence. This interactive tutorial explores the molecular re-arrangement that occurs during the formation of the enhanced green fluorescent protein (EGFP) fluorophore, which substitutes threonine for serine at position 65 in the amino acid sequence.

The tutorial initializes with an image of the pre-maturation EGFP fluorophore tripeptide amino acid sequence (Thr65-Tyr66-Gly67) stretched into a linear configuration so that the threonine 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 and electrostatic forces, as well as 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 self-catalyzed 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 Thr65 in close proximity to the amino nitrogen of Gly67. 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. Fluorescence (indicated by a green glow surrounding the affected structural elements) occurs when oxidation of the tyrosine alpha-beta carbon bond by molecular oxygen extends electron conjugation of the imidazoline ring system to include the tyrosine phenyl ring and its para-oxygen substituent. The result is a highly conjugated pi-electron resonance system that largely accounts for the spectroscopic properties of the protein.

The protective beta-can barrel provides a scaffold surrounding the fluorophore to maintain planarity and foster a wide range of potential interactions with trapped water molecules and amino acid side chains from the polypeptide backbone. Combined with the short portions of alpha-helix and loops at the ends of the barrel, the entire structure serves as a shield against environmental damage to the fluorophore. In this regard, the requirement for molecular oxygen as a fluorophore activation catalyst to form the extended aromatic system in fluorescent proteins is remarkable considering that oxygen must ultimately be excluded from regular interactions with the fluorophore to avoid collisional quenching of fluorescence. The generally low photobleaching rate of fluorescent proteins suggests that the design has evolved with a sacrifice of efficient fluorophore formation as a compromise for long-term stability and higher quantum yields.

Two fundamental aspects of the GFP fluorophore have important implications for the use of this molecule as an intracellular probe. First, the photophysics of green fluorescent protein as a fluorophore are quite complex and thus, the molecular structure can accommodate quite a bit of modification. A wide variety of amino acid substitutions have been successful in fine-tuning the fluorescence of native GFP to provide a broad range of fluorophores that emit colors ranging from the blue to the yellow regions of the visible spectrum. Second, it is important to note that GFP fluorescence is very dependent on the structure surrounding the fluorophore. Denaturation of the protein destroys fluorescence, and mutations in residues immediately adjacent to the fluorophore can significantly alter the fluorescent properties.

As mentioned above, the packing of amino acid residues inside the protein beta-barrel is very stable, which often results in relatively high fluorescence quantum yields for GFP and its derivatives (up to 80 percent). This consolidated protein structure also enhances resistance to changes in pH, temperature, and common denaturants, such as urea. Mutations in GFP that affect the fluorescence profile generally produce negative effects on this stability, often resulting in a reduction of quantum yield and enhanced environmental sensitivity. Although several of these defects can be overcome by additional mutations, fluorescent protein derivatives are usually more sensitive to the environment than native GFP. These limitations should be considered when designing experiments.