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Transcript
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.