The phenomenon of photochromism (the ability to switch between fluorescent and dark states) has been observed in wild-type GFP and several yellow fluorescent protein derivatives at the single molecule level. However, none have demonstrated this phenomenon when measured in bulk. In single molecule studies, fluorescent proteins exhibited fluorescence for several seconds during illumination at 488 nanometers followed by an equally short interval without fluorescence, after which the fluorescence resumed. The on-and-off switching or blinking behavior was repeated many times before each fluorescent protein molecule ultimately photobleached. Unfortunately, photoswitching in most of the fluorescent proteins described above is stochastic, and cannot be used in quantitative experiments. However, some optical highlighters have been isolated (such as Dronpa) that can be reliably toggled on or off by illumination with different excitation wavelengths, and these are called photoswitchable fluorescent proteins.
The tutorial initializes with the chromophore of Dronpa fluorescent protein illustrated in the trans or dark state. Surrounding the chromophore are structural elements of the beta-barrel. In order to operate the tutorial, click on the left-hand side Photoswitch Control (the 405nm Laser button) to irradiate the chromophore with violet light, thus evoking a switch to the bright, fluorescent state (cis chromophore). Once photoswitched to the "on" state, Dronpa can be reverted to the dark (or "off") state by clicking on the 488nm Laser button in the Photoswitch Control panel.
The most prominent and well-studied photoswitchable fluorescent protein is named Dronpa (named after a fusion of the Ninja term for vanishing and photoactivation), which is a monomeric variant derived from a stony coral tetramer. Dronpa fluorescent protein exhibits an absorption maximum at 503 nanometers (arising from the anionic, deprotonated chromophore) with a minor peak at 390 nanometers (from the neutral, protonated chromophore). The anionic chromophore emits green fluorescence with a maximum at 518 nanometers and has a brightness level almost 2.5 times that of EGFP. Dronpa photoswitching occurs in part by interconversion between the deprotonated (on state; bright) and protonated (off state; dark) forms. Illumination at 488 nanometers drives Dronpa to the dark species after which the fluorescent protein can be subsequently switched back on by brief illumination at 405 nanometers. This cycle can be repeated several hundred times without significant photobleaching.
The primary mechanism of fluorescent protein photoswitching is thought to also arise from cis-trans isomerization of the hydroxybenzilidine (tyrosyl side chain) chromophore moiety that accompanies the changes in the protonation state. Similar to other highlighters, Dronpa is useful both for dynamics and superresolution studies. Variants of Dronpa with faster switching times, reversed photoswitching properties, and broader spectra have been reported, but application of these rather dim fluorescent proteins has so far been limited to evaluating their performance in superresolution imaging. Future versions will no doubt feature higher brightness and should prove useful in routine dynamics investigations.
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.