UC San Diego

Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 and roGFP2 with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H2O2-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid. Placing positive charges near cysteine residues increases sensitivity to oxidation by H2O2. We designed a series of roGFP variants with outward-facing lysine residues placed in close proximity to the key cysteine residues. The double lysine mutant RoGFP2-F223KA206K (roGFP2-FA) was used in three distinct applications for roGFPs; The protein was expressed in primary neurons; the redox indicator was evaluated for sensitivity to environmental toxicants; and finally the potential for clamping roGFP2-FA in a particular redox state following equilibration using formaldehyde was investigated. Sensitivity of selenocysteine to oxidation was harnessed to further optimize roGFPs. By employing the 3'UTR of gluthathione peroxidase and an opal codon at each of the cysteine residues of roGFP we were able to create seleno- GFPs, which have a greater sensitivity to oxidation. A rational approach was taken for the discovery of the green fluorescent protein based singlet oxygen sensors (SOS- GFP). There was a strong response to singlet oxygen in particularly for mutations expressing histidines at positions 147 and 204. These double histidine mutants were evaluated for specificity and sensitivity to singlet oxygen. The most reactive/sensitive mutations were then used in biological settings to sense singlet oxygen generated from a nearby source