The GFP chromophore is encoded by the primary amino acid sequence, and forms spontaneously without the requirement for cofactors or external enzyme components (other than molecular oxygen), through a self-catalyzed protein folding mechanism and intramolecular rearrangement. Thus, genetically-encoded GFP provided for the first time the ability to label specific proteins inside the living cell without the need for exogenous synthetic or antibody-labeled fluorescent tags. When coupled to the astonishing advances in live-cell imaging instrument technologies that have occurred over the past decade, the fluorescent proteins have truly ushered in a new era in studies of cell biology and physiology. This interactive tutorial explores the molecular rearrangement that occurs during the formation of the enhanced green fluorescent protein (EGFP) chromophore, which substitutes threonine for serine at position 65 in the amino acid sequence of the wild-type protein.
The tutorial initializes with an image of the pre-maturation EGFP chromophore tripeptide amino acid sequence (Thr65-Tyr66-Gly67) stretched into a linear configuration so that the threonine residue is positioned in the upper left-hand side 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 self-catalyzed intramolecular rearrangement of the tripeptide sequence that occurs during chromophore maturation.
The first step in EGFP chromophore maturation is a series of torsional adjustments in the polypeptide backbone that relocate the carboxyl carbon of the Thr65 residue 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 (termed a para-hydroxybenzilidine moiety). The result is a highly conjugated π-electron resonance system that largely accounts for the spectroscopic properties of the protein.
Early studies of the structure-function relationships in the GFP chromophore region by Roger Tsien's laboratory showed that mutations altering the first amino acid in the chromophore, Ser65, to cysteine, leucine, alanine, or threonine simplified the excitation spectrum to a single peak ranging from 471 to 489 nanometers. For example, changing the Ser65 to threonine (the S65T mutation) stabilized the hydrogen-bonding network in the chromophore, resulting in a permanently ionized form of the chromophore absorbing at 489 nanometers. The resulting GFP-S65T mutant was a distinct improvement over wild-type GFP (wtGFP) for applications as a fluorescent marker in living cells because it had a well-defined absorption profile with a single peak at 489 nanometers. In addition, the GFP-S65T derivative is about five-fold brighter than wtGFP, and it matures more rapidly, allowing fluorescence to be detected at earlier time points after cell transfection. The GFP-S65T variant was further modified by replacing phenylalanine for leucine at position 64 (F64L), which improved the efficiency of protein maturation at 37° C, yielding EGFP (enhanced GFP). This enhanced variant features an excitation spectral profile that overlays nicely with the 488-nanometer argon-ion laser line and is similar in profile to fluorescein and related synthetic fluorophores that are readily imaged using commonly available filter sets designed for fluorescein (FITC). Furthermore, EGFP is among the brightest and most photostable of the Aequorea-based fluorescent proteins. The only drawbacks to the use of EGFP as a fusion tag are a slight sensitivity to pH and a weak tendency to dimerize.
Continued engineering of EGFP has yielded several additional green variants with improved characteristics. Among the best of these is the derivative called Emerald, which has improved photostability and brightness. Until recently, there was no commercial source for plasmids encoding Emerald (it is now available from Invitrogen), so there has been limited use of this high-performance green variant. Emerald fluorescent protein contains the S65T, and F64L mutations featured in EGFP, but also has four additional point mutations that further improve the efficiency of maturation and folding at 37° C, and increase the intrinsic brightness. Although Emerald is more efficient than EGFP in folding and developing fluorescence in mammalian cells, it has a fast photobleaching component that might affect quantitative imaging in some environments. Addition of the monomerizing A206K mutation, which is applicable to virtually all Aequorea GFP derivatives, may reduce or eliminate the fast photobleaching component in Emerald.
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.