Download GREEN FLUORESCENT PROTEIN (GFP) GFP is produced by the

GREEN FLUORESCENT PROTEIN (GFP)
GFP is produced by the jellyfish Aequoria victoria. It is
responsible for shifting the blue light emission of the
bioluminescence system to green light. That is, the
bioluminescence of the jellyfish appears green (508
nm), even though the light emitting molecule of the
bioluminescence system (Aequorin) emits blue light
(466 nm).
GFP is an 11-stranded β-barrel protein threaded by an
α-helix running up the axis of the internal cylinder.
The fluorophore responsible for light absorbtion and
emission (i.e. fluorescence) is attached to the α-helix
and is buried in the center of the β-barrel cylinder.
The fluorophore [4-(p-hydroxybenzylidene) imidazolidin-5-quinolone] is covalently
attached to the peptide backbone and is also conformationally stabilized by hydrogen
bonds with multiple R-groups and to bound water molecules (W).
The fluorophore formes spontaneously from residues Ser65-Tyr66-Gly67 in the native protein.
Thus, the GFP gene contains all the information necessary for the posttranslational
synthesis of functional GFP, no additional enzymes or prosthetic groups are involved. A
hypothetical scheme for fluorophore formation is shown below. Note the requirement for
molecular oxygen in the final step.
GFP has a major excitation peak at
395 nm that is about three times higher
than a minor peak at 475 nm. The 475nm peak arises from GFP molecules
containing deprotonated or anionic
chromophores, whereas the 395-nm
peak represents GFPs containing
protonated or neutral chromophores).
The latter would be expected to
deprotonate in the excited state,
because phenols almost always
become much more acidic in their
excited states. During most light
absorption/emission cycles, the proton
transfer reverses. However,
occasionally the proton does not
return to the chromophore, so the
neutral chromophore is
photoisomerized to the anionic form.
Wild-type GFP folds fairly efficiently when expressed at or below room temperature, but
its folding efficiency declines steeply at higher temperatures. Presumably this natural
temperature sensitivity is of no consequence to the jellyfish, which would never encounter
warm water in the Pacific Northwest. Temperature sensitivity is restricted to the folding process.
GFP that has matured properly at low temperature is stable and fluorescent at temperatures
up to at least 65ºC.
REFERENCES
Protein Data Bank accession numbers: 1EMA, 1GFL.
Green Fluorescent Protein : Properties, Applications, and Protocols
by Martin Chalfie (Editor), Steven Kain (Editor)
The Green Fluorescent Protein
Roger Y. Tsien (1998)
Annual Review of Biochemistry 67:509–44.