Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Fluorescent Protein Technology

Kindling Fluorescent Protein (KFP1) Chromophore Formation

A useful optical highlighter, the Kindling fluorescent protein (KFP1), has been developed from a non-fluorescent chromoprotein isolated in Anemonia sulcata, and is now commercially available. Kindling fluorescent protein does not exhibit emission until illuminated with green or yellow light in the region between 525 and 580 nanometers. Low-intensity light results in transient red fluorescence (kindling) with excitation and emission maxima at 580 and 600 nanometers, respectively, which slowly decays upon cessation of illumination as the protein relaxes back to its initial non-fluorescent state (exhibiting a half-life of approximately 50 to 60 seconds). Irradiation with intense blue light quenches the kindled fluorescence immediately and completely, allowing tight control over fluorescent labeling. This interactive tutorial explores the molecular rearrangement that occurs during the maturation of the KFP1 chromophore, as well as the mechanism of photoswitching through conformational changes to the substituted imidazolinone ring system.

Investigations into the mechanism of fluorescent protein photoswitching suggest that a cis-trans isomerization of the hydroxybenzilidine chromophore moiety is a key event in the switching process. The fluorescent state represents the cis conformation, whereas the trans isomer is adopted by the chromophore in the non-fluorescent state. In addition, these conformational changes are apparently accompanied by varied protonation states of the chromophore that further determine the fluorescent properties. Furthermore, the light-induced photoswitching is probably a manifestation of chromophore planarity and structural rearrangements of internal amino acid residue side chains within the chromophore cavity. These collective features may constitute a fundamental mechanism that is common to all photoactivatable and reversibly photoswitchable fluorescent derivatives.

Contributing Authors

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