Mechanisms and Applications of Redox-Sensitive Green Fluorescent Protein-Based Hydrogen Peroxide Probes

Genetically encoded hydrogen peroxide (H₂O₂) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available "toolbox" for monitoring cellular H₂O₂ changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔC_R offer considerably improved H₂O₂ sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H₂O₂ levels.

The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain.

The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H₂O₂ levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H₂O₂ probes, roGFP2-based H₂O₂ sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.