Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Fluorescent Protein Technology

Excited-State Proton Transfer

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.

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.

Contributing Authors

Adam Rainey and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.