In the cell, several biological processes involve changes of the reduction-oxidation (redox) state, a misbalance of which can result in the production of free radicals and/or other Reactive Oxygen Species (ROS). Although, cells physiologically produce a certain amount of ROS (e.g.: respiratory chain in mitochondria), excessive production has the potential to significantly damage cellular homeostasis, mainly through oxidative modifications of DNA/RNA and proteins. In several publications, this has been linked to diseases and aging.
In order to protect themselves from non-physiological ROS concentrations, cells employ antioxidant molecules, mainly utilizing thiol-groups to catalytically detoxify themselves from free radicals. Generally, glutathione is the most abundant antioxidant molecule employed by the cell. It reduces ROS through the glutathione-peroxidase enzyme.
A modified GFP protein as redox reporter
The redox-sensitive GFP (roGFP) protein is a GFP variant that can be used as a biosensor for redox changes. In roGFP, two surface-exposed cysteines were introduced into the β-barrel (β-sheet reach region) of standard GFP at positions 147 and 204.
Importantly, roGFP has two different and redox-dependent excitation peaks at 405 nm and 488 nm, and one single emission peak at 510 nm. While cysteine oxidation results in an increase of the 405 nm excitation peak, cysteine reduction increases excitation at 488 nm.
Consequently, the ratio between emission at 510 nm and excitation either at 488 nm or 405 nm can be used as an indicator of the relative amount of oxidized/reduced roGFP. This ratio reflects the redox state of the cell or cellular compartment in which roGFP is present, with the ratios for the two excitation maxima shifting in opposing directions depending on reduction or oxidation.
The benefits of roGFP
Based on published data, roGFP has quite a few advantages:
- It is genetically encoded and hence can stably be transfected in cells
- It can be used in living cells (yeast and mammalian) with no evident negative effect
- In living cells, it can monitor redox changes in real time
- The ratiometric detection is not affected by protein concentration or sample excitation
- It can be targeted to intracellular compartments (e.g.: mitochondria, ER, endosomes, etc)
- It can monitor redox changes also in physiological conditions
- Generally, it is pH independent
- It is less invasive than other “static” methods
There are several variants of roGFP, though two versions are widely used: roGFP1 and roGFP2. Although both contain the same cysteine additions, roGFP1 is based on wild-type GFP whereas roGFP2 on eGFP. The main differences are that roGFP2 has a more efficient excitation at 488 nm than at 405 nm and offers a higher signal yield. On the other hand, roGFP1´s dynamic range extends more into the reduced range and is less pH-sensitive than roGFP2.
How roGFP can be employed
roGFP is usually expressed as fusion protein. Through this fusion, roGFP can be targeted to specific intracellular compartments and report for specific ROS in real-time. For instance, roGFP2-Orp1, the fusion of roGFP2 to the thiol peroxidase enzyme Orp1 is usually employed as a H2O2-sensitive probe and can be used to determine how chemicals induce or drugs decrease production of ROS in cells (Application Note 252).
In yeast, a specific roGFP biosensor targeting mitochondria can be used to compare ROS production in normoxic and hypoxic conditions (Application Note 310).
Additionally, the expression of the glutathione-roGFP fusion complex (Grx1-roGFP) makes it possible to detect the glutathione-specific redox potential. For instance, the lab of Bruce Morgan, Junior Professor for Cellular Biochemistry at the University of Kaiserslautern, Germany, uses this approach to understand the role glutathione plays in cellular redox homeostasis and to better understand whether specific intracellular organelles preferentially store its oxidized or reduced form. Read here Prof. Morgan´s full interview.
The best platform for ratiometric roGFP detection
BMG LABTECH´s microplate readers represent the ideal platform for the detection of roGFP-based biosensors in living cells. roGFP detection benefits considerably from sensitivity of detection in fluorescence intensity, measurements from the bottom of the plate, and from automated compound injection. “The CLARIOstar is sensitive enough that even without chemical perturbation, we are able to detect baseline sensor signals. For the redox community this is very, very important”, said Prince Saforo Amponsah about the role the CLARIOstar plays in his research. Watch below Prince´s full video interview.
In fact, the CLARIOstar and PHERAstar FSX gained a reputation as go-to plate readers in the roGFP community. The following features make them the ideal detection systems for roGFP-based assays:
- Highest sensitivity on the market in fluorescence intensity in their respective class
- Open air optic system for bottom reading
- Automatic focus adjustment (resolution: 100 μm)
- Built-in, programmable reagent injectors with extremely low filling and dead volume
- Temperature control up to 45 °C
- Excitation and emission spectral scans (only CLARIOstar)
- Atmospheric Control Unit (ACU) for active and independent control of O2 and CO2 (only CLARIOstar)