STRUCTURAL BASIS FOR DUAL EXCITATION AND PHOTOISOMERIZATION OF THE AEQUOREA VICTORIA GREEN FLUORESCENT PROTEIN

The 2.1-Angstrom resolution crystal structure of wild-type green fluor escent protein and comparison of it with the recently determined struc ture of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein, The two absorption maxima are caused by a change in the ionization st ate of the chromophore. The equilibrium between these states appears t o be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains, The predominant n eutral form of the fluorophore maximally absorbs at 395 nm, It is main tained by the carboxylate of Glu-222 through electrostatic repulsion a nd hydrogen bonding via a bound mater molecule and Ser-205. The ionize d form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein, Glu-222 donates its charge to the fluo rophore by proton abstraction through a hydrogen bond network, involvi ng Ser-205 and bound water, Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148, UV irradiation shifts the ratio of the two abs orption maxima by pumping a proton relay from the neutral chromophore' s excited stale to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the e quilibrium dark-adapted state of the chromophore, In the S65T structur e, steric hindrance by the extra methyl group stabilizes a hydrogen bo nding network, which prevents ionization of Glu222, Therefore the fluo rophore is permanently ionized, causing only a 489-nm excitation peak, This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluores cent indicators and UV-triggered fluorescent markers of protein diffus ion and trafficking in living cells.