I. Introduction

Green Fluorescent Protein (GFP) is naturally fluorescent protein in which the chromophore (fluorophore) is derived from posttranslational cyclization of a serine-tyrosine-glycine tripeptide of GFP, followed by dehydrogenation of the tyrosine. Thus, GFP requires no exogenous moiety for fluorescence, making it a tremendously useful marker in in vivo studies. Since its original discovery in the jellyfish Aequorea victoria, it has proven valuable in a plethora of biochemical, cellular, and developmental investigations.

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II. GFP Structure

Wild type GFP contains 238 amino acids, folded into a series of 6 alpha helices and 11 beta strands, connected by loops. The strands form a classical beta barrel, a cylindrical beta sheet with anti-parallel strands.

The fluorescent moiety of GFP protein is the ser-tyr-gly derived chromophore. This is buried deep within the the beta barrel, interrupting the helix that runs through the center of the barrel. It is thus protected from interactions with solvent by the beta strands. This likely accounts for the notable stability of GFP fluorescence.

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III. The GFP Chromopore

Wild type GFP exhibits two absorption maxima (395nm and 475nm). The major absorption peak at 395nm represents the non-ionized form of the chromophore, whereas the chromophore absorbs maximally at 475nm in its ionized form. This ionization is induced by UV light, and a return to the neutral state of the chromophore occurs over time. The transitions between neutral and ionized states of the chromophore are induced by interactions with GFP residues.

Two key residues that interact with the non-ionized chromophore are glu²²² and ser²⁰⁵. glu²²² forms a hydrogen bond with the serine-derived portion of the chromophore, and ser²⁰⁵ hydrogen bonds with the tyrosine-derived portion of the chromophore indirectly through a water molecule.

The main chain carbonyl oxygen of thr²⁰³ also interacts with the chromophore through the same water. glu²²² and ser²⁰⁵ are linked by an additional hydrogen bond. Two additional residues (his¹⁴⁸ and asn¹⁴⁶) are found in the vicinity of the chromophore, and share a hydrogen bond.

There thus exists a complex hydrogen bonding network in the neighborhood of the chromophore. This network permits the transfer of protons between the chromophore and residue sidechains, the direction of which determines the ionization state of the chromophore. The model for this process is as follows. In the non-ionized isomer that absorbs maximally at 395nm, glu²²² and ser²⁰⁵ help to buffer and neutralize the chromophore. However, when exposed to UV light, glu²²² donates a proton to the chromophore through the H-bond network involving ser²⁰⁵ and water, thereby generating the ionized isomer that absorbs maximally at 475nm.

The hydrogen bond pattern in the chromophore vicinity is thought to change, stabilizing the chromophore in its ionized state. his¹⁴⁸ now stabilizes the tyrosine -derived portion of the chromophore by hydrogen bonding directly to the phenolic oxygen. The side chain of thr²⁰³ now hydrogen bonds directly to this oxygen as well. Although the main chain carboxyl oxygen of thr²⁰³ no longer bonds to the water, the carbonyl of asn¹⁴⁶ now does, as it loses its H-bond to his¹⁴⁸. The H-bond between glu²²² and ser²⁰⁵ is lost in the ionized isomer.

The following two buttons allow the visualization of the hydrogen bonding networks in the neutral and ionized isomers.

neutral state

ionized state

The reestablishment of the original hydrogen bond network allows the chromophore to return the proton back to glu²²² in time, regenerating the neutral isomer.

Support for this model comes from the structure of a GFP mutation (ser65-->thr65). This mutation produces an altered conformation of residues near the GFP chromophore site. The hydrogen bond between glu²²² and ser²⁰⁵ does not form, thus preventing the proton transfer back to glu²²² through the hydrogen bond network. The thr65 mutant thus maintains the chromophore in an ionized state, and GFP-thr65 absorbs maximally at ~475nm.

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IV. References

Brejc, K., Sixma, T. K., Kitts, P. A., Kain, S. R., Tsien, R. Y., Ormo, M., Remington, S. J.: Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci USA 94: 2306-2311 (1997).

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