Protein Fluorophores Index Melittin


The Fluorophore of Green Fluorescent Protein (GFP)

GFP is a fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequoria victoria, or from the sea pansy, Renilla reniformis. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. The gene for GFP has been isolated and has become a useful tool for making chimeric proteins of GFP linked to other proteins where it functions as a fluorescent protein tag. GFP tolerates N- and C-terminal fusion to a broad variety of proteins. It has been expressed in bacteria, yeast, slime mold, plants, drosophila, zebrafish, and in mammalian cells. As a noninvasive fluorescent marker in living cells, it allows for a wide range of applications where it may function as a cell lineage tracer, reporter of gene expression, or as a measure of protein-protein interactions.


Fluorescent Spectrum of GFP

Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively. Its emission peak is at 509 nm in the lower green portion of the visible spectrum. The GFP from the sea pansy exhibits a single major excitation peak at 498 nm. Crystals of GFP exhibits a nearly identical fluorescence spectrum and lifetime to that for GFP in aqueous solution.

For wild type GFP, exciting the protein at 395 nm leads to rapid quenching of the fluorescence with an increase in the 475 nm excitation band. This photoisomerization effect is prominent with irradiation of GFP by UV light. In a wide range of pH, increasing pH leads to a reduction in fluorescence by 395 nm excitation and an increased sensitivity to 475 nm excitation.

Some mutants of the GFP gene have been produced which have increased fluorescence. The major excitation peak has been red-shifted to 490 nm with the emission staying at 509 nm. This is better for use with standard optical filter sets, as the mutant GFPs have excitation range more compatible with the commonly available optical filters. This also is more useful for confocal laser scanning applications. The corrected excitation and emission spectra of GFP and some of its variants (termed as BlueFP, CyanFP, GreenFP and YellowFP variants) are shown here:

More details on GFP mutants later on this page.


The Fluorophore Center of GFP

In GFP, the fluorophore originates from an internal Ser-Tyr-Gly sequence which is post-translationally modified to a 4-(p-hydroxybenzylidene)- imidazolidin-5-one structure:

The fluorophore itself is a p-hydroxybenzylidene-imidazolidone. It consists of residues Ser65- dehydroTyr66 - Gly67 of the protein. The cyclized backbone of these residues forms the imidazolidone ring. The fluorescence is not an intrinsic property of the Ser-Tyr-Gly tripeptide. The amino acid sequence Ser-Tyr-Gly can be found in a number of other proteins as well. This peptide is neither cyclized in any of these, nor is the tyrosine oxidized. None of these proteins has the fluorescence of GFP.

Two resonant forms of the fluorophore (p-hydroxybenzylidene-imidazolidone) can be assumed:

In the 3-dimensional tructure of GFP, some basic residues appear to form hydrogen bonds with each of these oxygen atoms:

These basic residues act to stabilize and further delocalize the charge on the fluorophore.

The fluorophore is generated by a sequential mechanism in an auto-catalytic process. No co-factors or enzymatic components are required. The reaction is initiated by a rapid cyclization between Ser65 and Gly67 to form an imidazolin-5-one intermediate which is followed by a much slower rate-limiting oxygenation of the Tyr66 side chain by O2 on a timescale of hours. Gly67 is required for formation of the fluorophore, no other amino acid can replace Gly in this role. The reaction is thermosensitive. The yield of formation of the fluorophore decreases at temperatures greater than 30 oC. Once produced, GFP is quite thermostable, though.

Molecular oxygen is needed for formation of the double bond between two carbons on the tyrosine to form an extended aromatic system. In contradiction with this requirement, however, oxygen must be excluded from interactions with the fluorophore itself, or else collisional quenching of the fluorescence or damaging photochemistry will occur. The observed low bimolecular quenching rate (0.004 M-1s-1) suggests that GFP gives up efficient fluorophore formation for stability and higher quantum yields once fully formed.


The 3D-Structure of GFP

GFP is comprised of 238 amino acids. The crystal structure of recombinant wild-type GFP, 1gfl has been solved by multiwavelength anomalous dispersion phasing methods using seleniomethionyl-substituted protein. The table below contains links to entries in the PDBsum database for 1gfl and and other GFP variants, the structrure of which have also been subsequently determined:

PDBsum Link GFP Variants
1bfp blue variant of green fluorescent protein
1c4f green fluorescent protein s65t at ph 4.6
1ema green fluorescent protein from aequorea victoria
1emb gfp from aequorea victoria, gln 80 replaced with arg
1emc green fluorescent protein from aequorea victoria, mutant
1eme green fluorescent protein from aequorea victoria, mutant
1emf green fluorescent protein from aequorea victoria, mutant
1emg gfp (65-67 replaced by cro, s65t substitution, q80r)
1emk green fluorescent protein from aequorea victoria, mutant
1eml green fluorescent protein from aequorea victoria, mutan
1emm green fluorescent protein from aequorea victoria, mutant
1gfl structure of green fluorescent protein
1yfp structure of yellow-emission variant of gfp
2emd green fluorescent protein from aequorea victoria, mutant
2emn green fluorescent protein from aequorea victoria, mutant
2emo green fluorescent protein from aequorea victoria, mutant
2yfp structure of yellow-emission variant of gfp

GFP has a cylindrical fold. Two protomers associate together to form a dimer in the crystal and also in solution at low ionic strengths, <100 mM. Dimerization has an effect on the excitation spectra and energy transfer of GFP. The dimer is involved in physiological interactions with aequorin for efficient energy transfer. The structure is comprised of two regular beta-barrels as shown in the figure:

Eleven strands on the outside of cylinders form the walls of the structure. The cylinders have a diameter of 30 and a length of 40 . Small sections of alpha-helix form caps on the ends of the cylinders and an irregular alpha-helical segment also provide a scaffold for the fluorophore which is located in the geometric center of the cylinder. The strands of beta-sheet are tightly fitted to each other like staves in a barrel. The structure forms a single compact domain. It does not have clefts for easy access of diffusable ligands to the fluorophore. This folding motif, with beta-sheet outside and helix inside, represents a new protein class which has been named the beta-can.

This structure is very resistant to denaturation by heat and denaturants, and provides overall stability. Denaturation of GFP requires treatment with 6 M guanidine hydrochloride at 90 oC, or pH outside the range 4-12. Renaturation occurs within minutes following reversal of denaturing conditions by dialysis or neutralization.


Here is a coordinate file for 1gfp from the PDBsum database. Follow the link to PDB and download the file for use in Rasmol.

To inspect the fluorophore in the geometric center of the cylinder, type the following commands at the Rasmol command prompt:

select ser65
color yellow
spacefill
select tyr66
color red
spacefill
select gly67
color blue
spacefill

Observe that the fluorophore is highly protected. The environment around the fluorophore includes both apolar and polar residues, and a number of charged residues. Phe46 and Phe64 are near the fluorophore and separate the single tryptophan, Trp63 from close contact with the fluorophore. You might want to examine these residues in Rasmol as well.


The Excited State Dynamics of GFP

The excited state dynamics of GFP have been studied using steady state and time-resolved fluorescence spectroscopies.. The results suggest that proton transfer is involved in interconversions within two ground and two excited states. The extended set of polar interactions around the flourophore could accomodate proton rearrangements. The most likely direct effects is associated with:

Mutations at Ser65 and Glu222 results in loss in the 400 nm absorption bands. Thus, the 400 nm band may arise from the abstraction of the Ser65 hydroxyl proton by Glu222.

The tightly wound cylindrical fold and the central location of the fluorophore can explain the observed protection of the fluorophore from collisional quenching by oxygen and hence reduction of the quantum yield. Photochemical damage by the formation of singlet oxygen through intersystem crossing is reduced by the structure.


Fluorescence of GFP Mutants

The environment in the vicinity of the fluorophore can explain the fluorescence and behavior of existing mutants of GFP. Most of the polar residues in the pocket form a hydrogen-bonding network on the side of Tyr66. Atoms in the side chains of Thr203, Glu222, and Ile167 are in van der Waals contact with Tyr66. The mutation of these residues has direct steric effects on the fluorophore. Mutation also changes the electrostatic environment if the charge is changed. Furthermore, mutated residues near the fluorophore have direct effects on the absorption and emission spectra. Some mutations have significant wavelength shifts and most suffer a loss of fluorescence intensity.

Random and directed point mutations have resulted in various changes in fluorescent behavior. Here are some observations:

Removal of more than 7 amino acids from the C-terminus or more than the N-terminal methionine leads to total loss of fluorescence. All non-fluorescent mutants fail to exhibit absorption spectra characteristic of the intact fluorophore. The last seven residues are disordered and they loop back outside the cylinder. These residues do not form a stave of the barrel. Their presence is not essential and addition of further residues is easily tolerated. As for the role of N-terminal, the first strand in the barrel begins at residue 10. Thus barrel formation does not require the N-terminal region. The N-terminal segment is, however, an integral part of the cap on one end of the protein, and is essential in folding events and in protecting the fluorophore. Extensions at the N-terminus does not disrupt the structure of the protein.


Protein Fluorophores Index Melittin

gmocz@hawaii.edu