Real-time monitoring of peroxiredoxin oligomerization dynamics in living cells

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifica-tions, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Forster reso-nance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quater-nary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide-and thioredoxin-related quater-nary transitions to take place in cellulo and observe that the relation-ship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Further-more, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small mole-cules affecting the oligomeric state inside cells. Mutagenesis ex-periments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.