Nathan Silva and David Marcey
II. GFP Structure
The GFP Chromopore
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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.
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.
III. The GFP
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.
residues that interact with the non-ionized chromophore
glu222 forms a hydrogen bond
with the serine-derived portion of the chromophore,
hydrogen bonds with the tyrosine-derived portion
of the chromophore indirectly through
a water molecule.
The main chain carbonyl oxygen of
also interacts with the chromophore through the same water.
and ser205 are
linked by an additional hydrogen bond. Two
additional residues (his148
are found in the vicinity of the chromophore, and share a hydrogen
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
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.
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
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
is lost in the ionized isomer.
two buttons allow the visualization of the hydrogen bonding networks
in the neutral and ionized isomers.
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
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
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).