Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Fluorescent Protein Technology

PA-GFP Chromophore Photoactivation

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.

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.

Contributing Authors

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