Genetically-Encoded Redox Indicators

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Background

Principle of ratiometric redox-sensitive GFPs. The relative fluorescence intensity of the two excitation maxima of roGFPs shifts depending on the redox state: reduction causes a decrease in the excitation at 400 nm and an increase in the excitation at 480 nm (arrows). From [1].

Tracking reactive oxygen species (ROS) and in particular hydrogen peroxyde (H2O2) is particularly relevant for studying oxidative stress (involved in the development of a wide variety of pathologies, including malignant diseases, diabetes mellitus, atherosclerosis, chronic inflammatory processes, ischemia and reperfusion injury, and some neurodegenerative diseases). H2O2 has also a role of intracellular second messenger, controlling signaling cascades by selective oxidation of redox active thiolates in proteins.

The first genetically-encoded fluorescent indicators of redox changes were reduction-oxidation-sensitive GFPs (roGFPs). This family of indicators was created in 2004 by substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds [2, 3]. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. These probes are ratiometric because of the presence of these two excitation peaks: ratios of fluorescence from excitation at 400 and 490 nm indicate the extent of oxidation (and thus the redox potential) while canceling out the amount of indicator and the absolute optical sensitivity.

In 2006, a specific H2O2 indicator, called HyPer, was designed in the laboratory of Sergey A. Lukyanov [4]. HyPer consists of a circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H2O2-sensing protein, OxyR [5]. HyPer2, an improved version of the probe, was generated by a single point mutation A406V from HyPer corresponding to A233V in wild type OxyR [6].

HyPer-2 versus HyPer response on H2O2. (b) Typical profiles of the ratio F500/F420 change in individual cells expressing HyPer-2 (black line), HyPerN353S (blue line) or HyPer (red line). (c) Time of half-oxidation of HyPer and HyPer-2 in cells upon addition of H2O2. Error bars indicate SEM values. (d) Time of half-reduction of HyPer and HyPer-2 in cells upon addition of H2O2. Zero time corresponds to the peak of the ratio. Error bars indicate SEM values. Data on (c) and (d) are the results of five independent experiments for HyPer, 6 for HyPer-2, 6 for HyPer-NES and 4 for HyPer-2-NES. NES: nuclear export signal. From [6].

Summary of available sensors

Acronym Full Name Description Accession # Refs
Redox indicators
roGFP1 reduction-oxydation-sensitive GFP 1 [2, 3]
roGFP2 reduction-oxydation-sensitive GFP 2 [2, 3]
H2O2 sensors
HyPer [6]
HyPer-2 [6]

List of available constructs

Name Description Map Available from
Lab Company

References

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  1. Error fetching PMID 21251723: [Palmer2011]
  2. Error fetching PMID 14722062: [Hanson2004]
  3. Error fetching PMID 14985369: [Dooley2004]
  4. Error fetching PMID 16554833: [Belousov2006]
  5. Error fetching PMID 2256675: [Storz1990]
  6. Error fetching PMID 20692175: [Markvicheva2011]
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