Fig. 1.

The H₂O₂ sensing mechanism of HyPer (A) and roGFP2-Orp1 (B). The sensing moieties of both probes (OxyR and Orp1 respectively) are colored in red and the fluorescent reporter moiety (i.e. cpYFP and roGFP2) in green or blue. (A) HyPer consists of a cpYFP fused to two halves of the OxyR regulatory domain. Upon the reaction of OxyR with H₂O₂, an intramolecular disulfide is formed which triggers a conformational change and thus modifies the fluorescence of cpYFP. (B) The roGFP2-Orp1 probe senses H₂O₂ via Orp1. An intramolecular disulfide is formed in Orp1 which is transferred to roGFP2 by thiol-disulfide exchange. The introduction of the disulfide into roGFP2 changes its fluorescence. Both probes are reversible in cellulo due to the reduction by glutathione/glutaredoxin(GSH/Grx) and/or thioredoxin(Trx) (dashed lines).