The Chromophore of GFP

GFP is unique among fluorescent proteins in that its fluorophore is not a seperately synthesized prostethic group but composed of modified amino acid residues within the polypeptide chain.

Chemical Structure of the Chromophore

Upon limited papain digestion of GFP, a hexapeptide with the same spectral properties as denatured GFP is released. The amino acid sequences of the hexapeptides from Aequorea and Renilla are: [3]

Aequorea:	FSYGVQ
Renilla:	FSYGDR

Cody et al. [1] have determined the chemical structure of this papain derived chromophore peptide from Aequorea victoria GFP.

The chromophore itself is a p-hydroxybenzylidene-imidazolidone (green background). It consists of residues 65-67 (Ser - dehydroTyr - Gly) of the protein. The cyclized backbone of these residues forms the imidazolidone ring. The peptide backbone trace is shown in red.

Although the amino acid sequence SYG can be found in a number of other proteins as well, it is neither cyclized in any of these, nor is the Tyrosine oxidized, nor are these proteins fluorescent. This implies that the tendency to form such a chromophore is no intrinsic property of this tripeptide.

Biosynthesis of the Chromophore

Heim et al. [2] have proposed the following biosynthetic scheme for the chromophore. (R1 signifies residues 1-64, R2 residues 68-238.)

The first step is a nucleophilic attack of the amino group of Gly67 onto the carbonyl group of Ser65 (upper left). Subsequent elimination of water results in formation of the imidazolidinone ring (upper right). In a second step the Calpha-Cbeta bond of Tyr66 is oxidized to give a large delocalized pi-system (bottom). The proposed biosynthetic scheme is based upon the following observations:

Excitation and Fluorescence Emission Spectra of GFP

Denatured GFP is not fluorescent and its absorbtion spectrum is significantly different from native GFP [1], which implies that noncovalent interactions of the chromophore with its local environment have a great influence on the spectral characteristics and that fluorescence is mediated by amino acids close to the chromophore in the tertiary structure of GFP.

The excitation spectrum of native GFP from A. victoria (blue) has two excitation maxima at 395 nm and at 470 nm. The fluorescence emission spectrum (green) has a peak at 509 nm and a shoulder at 540 nm [3]. (Spectra after [4])

Recombinant A. victoria-GFP generated in E. coli has the same spectral properties [4], the excitation spectrum of R. reniformis-GFP is different, whereas its emission spectrum is the same [3]. The absorption spectrum of A. victoria-GFP, is not significantly affected by titration of external pH up to pH 10, which is just below the threshold of denaturation [2, 11].

Förster cycle

GFP is the first known example of a Förster cycle within the core of a protein [5,6,7]:

Depending on whether Tyr66 is in its hydroxyl form (upper left) or in its phenolate form (lower left), the fluorophore absorbs light at 395 nm or at 470 nm, respectively. As phenols are known to be more acidic in their excited state than in their ground state, it is postulated that the protonated excited form of the fluorophore (upper right) converts to the excited phenolate (lower right), which is the only fluorescent species and emits light at 509 nm. As a result, a cycle is formed, in which the flourophore absorbs a photon, then loses a proton, emits a photon and finally takes up a proton, returning to its original state.
Interestingly, excitation at 395 nm leads to a decrease over time of the 395 nm peak and a reciprocal increase of the 475 nm excitation band [6].

After the structures of wild type GFP [6] and of the S65T variant of GFP [7] have been solved by X-ray crystallography, some GFP variants, which have one excitation band of wild type GFP deleted and retain the other without a significant wavelength shift, can be understood in view of the 3-dimensional structure and the Förster cycle:
The fluorophore inside the protein is not solvent accessible. It interacts with the sidechains of many surrounding residues via hydrogen-bonds:

Click on the small picture for a larger illustration (8k) of some of the fluorophore's interactions with the surrounding sidechains. Hydrogen-bonds are shown in blue, except for the one presumably stabilizing the respective form of the fluorophore, which is shown in red.

Mutations affecting the pKa of Tyr66

Stabilization of the phenolate or hydroxyl form of Tyr66 by specific hydrogen-bonds can explain why disruption of these hydrogen-bonds leads to an altered absorption spectrum in GFP-variants:

Mutations of the Chromophore Forming Residues

There is a vast amount of different GFP variants described in the literature, many of which have quite interesting characteristics. But discussion of all of these is far beyond the scope of this project and besides, there is no rational explanation yet for quite a few of the observed features. So I will limit further discussion of GFP variants to those which bear a mutation in the fluorophore itself.

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Silke Jonda's PPS2 project
Structure and Function of GFP
updated 28.11.96