Photoswitchable cyan fluorescent protein for protein tracking

In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported(1-7), including photoactivatable PA-GFP(1), photoactivatable protein Kaede(2), the DsRed 'greening' technique(3) and kindling fluorescent proteins(6,7). So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.