Among the most unique features of wild-type green fluorescent protein (wt-GFP) is the fact that illumination with either ultraviolet or blue-cyan light gives rise to green fluorescence having a maximum wavelength at approximately 507 nanometers. The bimodal absorption spectrum of wt-GFP features a large peak at 395 nanometers (the A band) and a much smaller peak at 475 nanometers (the B band). The lesser B band corresponds to the anionic chromophore, which demonstrates normal photophysics, whereas the predominant A band corresponds to the neutral chromophore that would normally be expected to emit blue light (peaking at approximately 450 nanometers) upon excitation in the ultraviolet. However, when excited with light ranging from 370 to 400 nanometers, the tyrosine residue in the neutral chromophore of wt-GFP becomes a strong acid and transfers a proton through a novel hydrogen bond network to generate an excited state anion (a process known as excited-state proton transfer; ESPT). It is the anionic form of the chromophore that emits green light.
The tutorial initializes with a drawing of the pertinent residues involved in wt-GFP ESPT surrounding the tyrosine 66 residue of the chromophore. The residues comprising this proton shuttling network include His148, Asn146, Thr203, Ser205, Glu222, and Ser65 (the latter is part of the chromophore). The dark state hydrogen bonding scheme is illustrated by default. In order to operate the tutorial, click on the 390nm Laser button to initate illumination of the chromophore with ultraviolet light and automatically cycle through the excited-state proton transfer network. Alternatively, use the Photocycle slider to examine each step individually. The Reset button re-initializes the tutorial.
In wt-GFP, the transfer of a proton from the excited state phenolate in the chromophore to glutamic acid 222 in the polypeptide backbone occurs through an organized hydrogen bond network that is capable of rapidly generating the anionic species to produce green emission. Details of this proton transfer reaction have been investigated using a variety of techniques, which have verified that the protonation state of Glu222 is altered as depicted in the tutorial. The ESPT pathway is fully reversible and constitutes a miniature photocycle. As a result, GFP is unique in that it is the only biological system for which ESPT has been demonstrated and affords the opportunity to study the fundamental physics of the process in a precisely defined experimental system.
Adam Rainey and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.