The highly unique photophysical properties of wild-type green fluorescent protein (wild-type GFP) were thoroughly investigated during the mid-1990s, and served as a foundation for the creation of the first useful optical highlighter designed specifically for photoactivation studies. Termed PA-GFP (for Photo Activatable Green Fluorescent Protein), this optical highlighter was developed by improving the photoconversion efficiency of the native chromophore from a predominately neutral form to a species that is anionic in character. By replacing the threonine at position 203 with a histidine residue (T203H) in wild-type GFP, researchers produced a variant having negligible absorbance in the region between 450 and 550 nanometers, thus dramatically enhancing contrast between the non-activated and activated species.
The tutorial initializes with the GFP chromophore depicted in the neutral phenol state surrounded by histidine 203 and glutamic acid 222 within the beta-barrel structure, thus representing the native dark state of PA-GFP. In order to operate the tutorial, click on the Photoactivation Control (the 405 nm Laser button) to irradiate the chromophore and expel carbon dioxide from the glutamic acid residue. Loss of carbon dioxide disrupts the excited state proton transfer (ESPT) chain and leads to a dramatic change in the chromophore protonation state (to the anionic phenolate form). The result is a shift of the absorption spectrum that yields green fluorescence upon excitation with 488-nanometer light.
PA-GFP is optimally excited at 400 nanometers, but has negligible absorbance in the region between 450 and 550 nanometers. However, after photoactivation with violet light, the absorption maximum of PA-GFP is shifted to 504 nanometers, increasing green fluorescence when excited at 488 nanometers by approximately 100-fold and providing very high contrast differences between the converted and unconverted pools. When PA-GFP-labeled proteins are photoactivated inside the living cell, the diffusion of the newly fluorescent proteins provides a direct measure of the mobility of the labeled proteins. The major drawback in the use of PA-GFP is that the non-activated form is not readily distinguishable before photoactivation, making it difficult to identify the regions that are expressing the fluorescent protein.
Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.