RoGFP

The reduction-oxidation sensitive green fluorescent protein (roGFP) is a redox sensitive biosensor. Two cysteines were introduced into the beta barrel structure of the GFP. The oxidation state (reduced dithiol or the oxidized disulfide) of the engineered thiols determines the fluorescence properties of the sensor. Originally, different roGFP versions were presented to allow the "in vivo" imaging of reducing compartments such as the cytosol (roGFP2). The cysteines introduced at the amino acid positions 147 and 204 produced the most robust results. roGFP2 preferentially interacts with glutaredoxins and therefore reports the cellular glutathione redox potential. The specificity of roGFP2 for glutathione is further increased by linking it to the human glutaredoxin 1 (Grx1).
By expressing the Grx1-roGFP fusion sensors in the organism of interest and/or targeting the protein to a cellular compartment, it is possible to measure the glutathione redox potential in a specific cellular compartment in real-time and therefore provides major advantages compared to other invasive static methods e.g. HPLC.
In addition, roGFPs are used to investigate the topology of ER proteins, or to analyze the ROS production capacity of chemicals.