GFP Structure
GFP- beta barrel with chromophore shown
in green

The crystal structure of GFP was solved in 1996. It has a unique soda can shape. Eleven beta-strands make up the beta-barrel and an alpha-helix runs through the center. The chromophore is located in the middle of the beta-barrel, it is occasionally referred to as the “light in the can.”

Another representation of the GFP beta-barrel. Beta sheets are green, helices red and connecting loops black.

another view

In June 2003, GFP was the protein databank's (pdb) molecule of the month

 

The chromophore of GFP is responsible for its fluorescence. It has the following structure where the R groups are the first 64 and last 170 residues of GFP. GFP catalyzes the formation of its own chromophore. It is proposed that Arg96 plays a crucial role in this catalysis. Two different mechanisms have been proposed for its action, see below.

 

 

The biggest difference between green fluorescent protein and its red analog, DsRed, is that the chromophore of DsRed has an extra double bond (drawn in yellow) which extends the chromophores conjugation and causes the red-shift.

 




Table 1. List of amino acid side chains with close contacts (less than 5 Å) to the fluorophore. The fluorophore is defined as the 7 atoms of the phenol of Tyr66, the 6 atoms of the imidazolidone, and the bridging methylene between the rings. The following amino acid side chains would be expected to have the most direct effects on fluorescence and perhaps fluorophore formation. The atom names are taken from the Brookhaven Protein Data Bank nomenclature.



Protein Fluorophore Distance



residue atom residue atom (Å)




Arg 96 NH2 Tyr 66 O 2.7
Gln 94 NE2 Tyr 66 O 3.0
His 148 ND1 Tyr 66 OH 3.3
Gln 69 CD Tyr 66 O 3.4
Glu 222 OE2 Tyr 66 CE2 3.5
Val 150 CG2 Tyr 66 CE1 3.6
Phe 165 CE1 Tyr 66 CD1 3.6
Thr 203 CG2 Tyr 66 CE2 3.6
Ile 167 CD1 Tyr 66 OH 3.7
Thr 62 CG2 Tyr 66 CG 3.7
Tyr 145 CE2 Tyr 66 OH 3.7
Ser 205 OG Tyr 66 CE2 4.0
Val 61 CG1 Tyr 66 CE2 4.4
Gln 183 NE2 Tyr 66 O 4.8
Val 68 CG2 Ser 65 C 4.9



 

Figure 2. Stereo view of a monomer, with colors that vary slowly as a function of the distance along the polypeptide chain. The termini and C atoms of every 20th amino acid are marked just to the upper right of each atom. Figure produced by RasMol.


 
Figure 1. The overall shape of the protein and its association into dimers. Eleven strands of -sheet (green) form the walls of a cylinder. Short segments of -helices (blue) cap the top and bottom of the 'b-can' and also provide a scaffold for the fluorophore which is near geometric center of the can. This folding motif, with -sheet outside and helix inside, represents a new class of proteins. Two monomers are associated into a dimer in the crystal and in solution at low ionic strengths. This view is directly down the two-fold axis of the non-crystallographic symmetry. Figures 1 and 6 were produced with Ribbons.






Figure 4. Model of the fluorophore and its environment superposed on the MAD-phased electron density map at 2.2 Å resolution. The clear definition throughout the map allowed the chain to be traced and side chains to be well placed. The density for Ser65, Tyr66 and Gly67 is quite consistent with the dehydrotyrosine - imidazolidone structure proposed for the fluorophore. Many of the side chains adjacent to the fluorophore are labeled. Figures 4 and 5 were produced with O43.






Figure 5. Stereo view of the fluorophore and its environment. His148, Gln94 and Arg96can be seen on opposite ends of the fluorophore and probably stabilize resonant forms of the fluorophore. Charged, polar, and non-polar side chains all contact the fluorophore in some way.





 
Figure 6. The dimer contact region. The two polypeptide chains associate over a broad area, with a small hydrophobic patch (in the yellow box) and numerous hydrophilic contacts. The two-fold symmetry axis is in the plane of the page, and is marked by the red arrow. The polar residues are marked with red atoms for oxygen and blue for nitrogen.



 

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