A genetically encoded sensor for H2O2 with expanded dynamic range.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Hydrogen peroxide (H2O2) acts as a crucial second messenger in intracellular signaling.
• Redox-active thiolates in proteins are selectively oxidized by H2O2, modulating cellular pathways.
• Genetically encoded fluorescent probes like HyPer enable real-time monitoring of intracellular H2O2 levels.

Purpose of the Study:

• To develop an improved genetically encoded fluorescent probe with an enhanced dynamic range for detecting intracellular hydrogen peroxide.
• To overcome the limitations of existing probes, such as the original HyPer, in applications requiring a wider detection range.

Main Methods:

• Engineering of a novel ratiometric genetically encoded fluorescent probe, HyPer-2, based on the Escherichia coli OxyR protein.
• Introduction of a single point mutation (A406V in HyPer, equivalent to A233V in wtOxyR) to modify the probe's properties.
• Characterization of HyPer-2's dynamic range by measuring the ratio change (F500/F420) under saturating hydrogen peroxide conditions.

Main Results:

• HyPer-2 exhibits an up to sixfold increase in the F500/F420 ratio under saturating H2O2, compared to a threefold change for the original HyPer probe.
• The A233V mutation in the OxyR sensing domain, present in HyPer-2, stabilizes the dimer interface.
• This stabilization of the dimer by the mutation leads to a significant expansion of the probe's dynamic range.

Conclusions:

• HyPer-2 represents a significant advancement in genetically encoded probes for monitoring hydrogen peroxide.
• The enhanced dynamic range of HyPer-2 facilitates more precise real-time tracking of intracellular H2O2 signaling.
• This improved probe is expected to benefit a wide range of biological applications studying redox signaling.