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.
The tutorial initializes with an image of the pre-maturation kindling chromophore tripeptide amino acid sequence (Met63-Tyr64-Gly65) stretched into a linear configuration so that the methionine residue is positioned at the extreme left end of the window. Oxygen atoms are colored red, nitrogen atoms blue, sulfur atoms yellow, carbon atoms white, and the black dashes at the peptide termini indicate continuation of the backbone beyond the portion illustrated. In order to operate the tutorial, use the Chromophore Maturation Control slider to transition through the intramolecular rearrangement of the tripeptide sequence that occurs during chromophore maturation. The first step is a series of torsional adjustments that relocate the carboxyl carbon of Met63 in close proximity to the amino nitrogen of Gly65. Nucleophilic attack on this carbon atom by the amide nitrogen of glycine, followed by dehydration, results in formation of an imidazolin-5-one heterocyclic ring system similar to that observed in the native Aequorea victoria protein. Subsequently, oxidation of the tyrosine alpha-beta carbon bond by molecular oxygen extends conjugation of the imidazoline ring system to include the tyrosine phenyl ring and its para-oxygen substituent. The KFP1 chromophore is fully mature after a water molecule attacks the alpha-carbon of Met63, producing a chain break between Met63 and Cys62. Clicking on the Green He-Ne Laser button produces red fluorescence as the chromophore transitions from a trans to a cis conformation. Turning the laser off reverts the chromophore to a dark (trans) state.
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.