Real-time monitoring of the in vivo redox state transition using the ratiometric redox state sensor protein FROG/B.

The intracellular redox state is one of the key factors regulating various physiological phenomena in the cell. Monitoring this state is therefore important for understanding physiological homeostasis in cells. Various fluorescent sensor proteins have already been developed to monitor intracellular redox state. We also developed fluorescent redox sensor proteins named Oba-Q and Re-Q, the emissions of which are quenched under oxidized and reduced conditions, respectively. Although these sensors were useful to visualize the redox changes in the cell over time, they have the weakness that their emission signals are directly influenced by their in situ expression levels. To overcome this problem, we developed a redox sensor protein with a single excitation peak and dual variable emission peaks. This sensor protein shows green emission under oxidized conditions and blue emission under reduced conditions. We therefore named this sensor FROG/B, fluorescent protein with redox-dependent change in green/blue. By using this sensor, we successfully measured the changes in intracellular redox potentials in cyanobacterial cells quantitatively caused by light/dark transition just by calculating the ratio of emission between green and blue signals.