Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process.
The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S(1) and S(0) energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.
Follenius-Wund
Anny
A
UMR CNRS 7034-Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, Université Louis Pasteur Strasbourg I, Faculté de Pharmacie, BP 24, 67401 Illkirch-Cedex, France.
Bourotte
Maryline
M
Schmitt
Martine
M
Iyice
Fatih
F
Lami
Hans
H
Bourguignon
Jean-Jacques
JJ
Haiech
Jacques
J
Pigault
Claire
C
D055592:Biophysical Phenomena; D001703:Biophysics; D004146:Dioxanes / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006860:Hydrogen Bonding; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D008956:Models, Chemical; D009154:Mutation; D013053:Spectrophotometry; D013696:Temperature
D004146:Dioxanes; D005456:Fluorescent Dyes; D007093:Imidazoles; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C025223:1,4-dioxane; D005990:Glycerol