All the PAFPs mentioned above demonstrate irreversible photoconversion. In contrast, reversibly convertible PAFPs ensure repeated activation and quenching. For example, chromoprotein asFP595 (16) as well as its mutants called kindling fluorescent proteins (KFPs) (17,18) can be transformed from nonfluorescent to the red fluorescent state by irradiation with relatively intense green (or, for some variants, blue) light. After that, they spontaneously relax back to the dark state within tens of seconds or minutes. Alternatively, after photoactivation, most KFP variants can be instantly quenched by blue light. The proposed mechanism for these photoconversions is a light-induced trans-cis isomerization of the chromophore, which possesses higher fluorescence quantum yield in a cis conformation (18,19,20,21,22,23,24,25). The so-called Dronpa (26) represents another type of reversible PAFP. Initially, Dronpa fluoresces green upon blue light excitation. Upon intense irradiation by blue light, Dronpa is quenched to the nonfluorescent form. After that, Dronpa can be activated back to the green fluorescent state by a pulse of UV-violet light. Several similarities with KFPs’ photobehavior indicate that the mechanism for Dronpa photoactivation is likely cis-trans isomerization as well (3,27). Solutions of crystal structures (27,28) and biophysical studies (29,30) should help reveal the basis for this phenomenon.
As with other common fluorescent proteins, the oligomeric state of many PAFPs restricts the range of their applications. In most cases, attempts to use tetrameric GFP-like proteins to label cellular proteins result in disturbance of the studied protein's function, mislocalization, and aggregation effects. Only monomeric PAFPs: PA-GFP, PS-CFP, PS-CFP2, mEosFP, Dendra, Dendra2, and Dronpa are widely suitable for the labeling and tracking of the target proteins. Oligomeric PAFPs, such as Kaede, KikGR, EosFP, and KFP1 can be utilized mainly for photolabeling organelles and cells.Dendra2: a Monomeric Green-to-Red Photoconvertible Protein
In the present paper, we are focusing on the detailed description of Dendra2—an improved commercially available version of green-to-red convertible protein Dendra. Compared to Dendra, Dendra2 comprises a single A224V substitution, which leads to more complete maturation (chromophore formation) and a brighter fluorescence both before and after photoswitching. In contrast to all other monomeric PAFPs, which necessarily require UV-violet (e.g., 405 nm laser) light for activation, Dendra and Dendra2 permit the use of blue (e.g., 488 nm laser) activating light.
The key desirable properties of any fluorescent protein are: bright fluorescence, monomeric nature (making it suitable for fusion protein labeling), fast maturation (important for early signal detection and for overall brightness in a cell, which is dependent on the balance between protein maturation and turnover rate), high photostability (allowing prolonged and/or high intensity excitation for the visualization of minor amounts of the protein and for time series), and high pH stability (making the fluorescent signal independent of local pH changes and allowing targeting of the protein to acidic organelles without signal loss).
Among the photoactivatable proteins, Dendra2 provides a unique combination of advantageous properties: monomeric state suitable for protein labeling, fast and efficient maturation at 37°C in mammalian cells, high contrast photoconversion, bright fluorescence of both initial and photoactivated forms, high photostability of the activated red signal, and low phototoxic activation with the 488-nm laser commonly installed on all commercial confocal microscopes. The minor disadvantage that should be kept in mind is a relatively low pH stability of the activated red form (with a pKa, i.e., the pH at which fluorescence is 50% of maximal, being 6.9). Here we provide some practical recommendations concerning applications of Dendra2 for tracking proteins of interest.Primary Visualization of Dendra2
Nonactivated Dendra2 possesses excitation-emission maxima at 490 and 507 nm (Figure 1A). Therefore, the 488-nm laser line perfectly fits the excitation maximum ensuring easy detection of the green signal using confocal microscopes. Commonly used fluorescence filter sets for enhanced GFP (EGFP), fluorscein isocytothiocynate (FITC), and other green dyes are well suited to visualize Dendra2's green state. However, wide-field arc lamp excitation with a blue light can cause undesirable photoconversion of the whole visualized field and therefore should be very accurately applied for the preliminary visualization of Dendra2. Therefore, we recommend using strongly attenuated light from a mercury lamp and avoiding prolonged exposure of Dendra2-expressing cells.