FROG/B reporter gives bright insights into redox biology

Fluorescence spectroscopy has contributed to advances in biochemistry that we now take for granted. Many biological and chemical processes of great importance in both nature and technology were uncovered using this versatile technique. The finding of the jellyfish Aequorea victoria green fluorescent protein (GFP) has revolutionized cell labeling and molecular tagging (1). In the few years since its discovery, GFP has become a reporter for gene expression, protein localization, and protein dynamics in living cells. Given that we have learned much about a plethora of biological events using this green glowing marker, the Nobel Prize in Chemistry given in 2008 to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien rewarded their seminal research. Further developments in molecular biology led to proteins that glow cyan, blue, and yellow. Remarkably, many events in living cells are followed in real time because the chemical environment modulates the fluorescence of genetically encoded GFPs. On this basis, Sugiura et al. (2) now estimate intracellular redox changes via a variant of GFP prepared by the rational design of the GFP mechanism, an important aspect of cell metabolism that pervades a wide range of biological events, such as photosynthesis in plants and cancer in humans. In the present study, Sugiura et al. (2) describe a GFP-based redox sensor, FROG/B, with an absorption peak around 400 nm, but more importantly, an emission spectrum sensitive to the redox state of the molecule. The relevant feature of FROG/B is that the maturation level of the chromophore does not alter the signal of the sensor because one chromophore perceives redox changes. Although currently many other genetically encoded GFP-based indicators report on different cellular events, even the redox status (e.g., rxYFP and roGFPs) (3), FROG/B can be used to monitor the redox status in cell populations containing only one type (e.g., HeLa cells) or many types (e.g., Anabaena vegetative and heterocyst cells).

Redox biology underwent great changes 2.3 billion years ago when living organisms had to adapt to a new life because oxygenic photosynthesis in cyanobacteria increased the level of oxygen in Earth’s atmosphere. The most significant innovation in the history of life promoted the arrival of aerobic respiration and the occurrence of complex multicellular life. However, the emergence of aerobic metabolic processes in the biosphere unavoidably led to the production of reactive oxygen species (ROS) as by-products (4). ROS bear a resemblance to Janus, the ancient Roman god that presided over war and peace. On the one hand, ROS cause oxidative damage to proteins, DNA, and lipids. Hence, many mechanisms combat increased levels of ROS during abiotic stress conditions. On the other hand, cells purposefully generate ROS as signaling molecules to control many processes, such as pathogen defense and programmed cell death.

The unique chemical reactivity of the sulfur atom in thiol groups made the cysteine residue of proteins the target for studies of redox biology because the redox potential of the cell drives thiol groups to multiple oxidation states that affect protein conformation, enzyme activity, effector binding, protein degradation, and the capacity for protein–protein and protein–DNA interactions (5) (Fig. 1). In this context, two outstanding features of FROG/B contribute to estimate the intracellular redox potential: 1) the cysteine pair adequately inserted into GFP, and 2) the dual emission peaks sensitive to the intracellular redox state, i.e., green and blue emission under oxidized and reduced conditions, respectively. Given that thiolates are considerably better nucleophiles than their protonated counterparts, the acid dissociation constant of cysteine residues also influences the biochemical activity. Although cysteine is not a predominant amino acid in proteins, a large body of biochemical studies have shown that the sulfur atom in this residue adopts numerous oxidation states. When the thiol moiety (oxidation state: −2) is oxidized, it proceeds 1) reversibly toward two different oxidation states (sulfenic acid [oxidation state: 0], intra- or intersubunit disulfide bonds [oxidation state: −1]); or 2) irreversibly toward to sulfinic (oxidation state: +2) and sulfonic (oxidation state: +4) acids. In the nearly three decades since the description of a thiol–disulfide cascade in many organisms the concept of redox signaling has pervaded physiology, genetics, and biochemistry, embracing many molecular mechanisms involved in adaptations to obnoxious metabolites.

Fig. 1.

Modification of the oxidation state of protein thiols. The sulfur atom in reduced protein thiols proceeds through various oxidation states in the cell. Reversible modifications: Deprotonation of thiol (Prot-SH) generates the reactive thiolate anion (Prot-S⁻) that reversibly can adopt two oxidation states: 1) via thiol-disulfide exchange, the formation of an intramolecular disulfide bridge in the protein or intermolecular disulfide with another protein or low-molecular-weight compounds (e.g., glutathionylation) (oxidation state: −1); or 2) via oxidation with H₂O₂, the formation of sulfenic acid (oxidation state: 0). Glutaredoxins and thioredoxins bring back the thiolate/thiol in these reversible redox modifications. Irreversible modifications: Reactive sulfenic acids can be further oxidized to sulfinic acid (oxidation state: +2) or even to sulfonic acids (oxidation state: +4). However, the concerted action of sulfiredoxins and 2-Cys-peroxiredoxins may reduce the sulfinic acid. G-S-S-G, oxidized glutathione.

On these bases, many redox-sensitive GFPs allow the visualization of the oxidation state in real time (6, 7). The most remarkable feature of GFP is the posttranslational modification of native protein that creates the chromophore out of specific amino acids (Ser65-Tyr66-Gly67). Anchored covalently to the protein and via an H-bonding network, the GFP core chromophore brings two ends close, including 1) the hydroxyl of Ser65, 2) the hydroxyl of Tyr66, 3) the hydroxyl of Ser205, 4) a water molecule, and 5) the carboxylate of Glu222. The excited-state intramolecular proton transfer (ESIPT) takes place via the proton relay of the amino acids and water molecules to the remote residue Glu222, resulting in an intense fluorescence. Sugiura et al. (2) address the rational design of the genetically encoded redox biosensor FROG/B by perturbation of the ESIPT mechanism (8). Consequently, the authors mutated specific amino acids into the A. victoria GFP gene: 1) two residues (T203V/S205V), 2) 10 amino acids (K26R/S30R/Y39N/F64L/N105T/M153T/V163A/I171V/S175G/A206K/H231L) to improve the thermal stability, 3) a cysteine pair at positions 149 and 202 to function as redox switch, 4) an amino acid (V150D) to act as a new terminal, and 5) a final amino acid (Q69A) to improve the weak emission in Escherichia coli. The final protein, FROG/B, exhibits a single absorption peak around 400 nm but the fluorescence emission is green and blue under oxidized and reduced conditions, respectively. Germane to monitoring the intracellular redox states, the midpoint redox potential (E_m) of FROG/B at pH 7.0 (E_m = −293 mV) is higher than that in cytoplasm at identical pH (E_m = −320 mV).

Complementary measurements of FROG/B sensor in phototrophic cyanobacteria uncover an excellent tool for examining redox homeostasis in organisms where the autofluorescence complicates measurements. The filamentous cyanobacterium Anabaena sp. PCC 7120 has proven to be an excellent model for the study of various aspects not only of photosynthesis but also of heterocyst development (9). When nitrogen sources are scarce, the cyanobacterium differentiates some vegetative cells into nitrogen-fixing cells called heterocysts. Fluctuations of light give rise to rapid changes in the intracellular redox status of phototrophs through the photosynthetic electron-transport chain. In this context, the expression of FROG/B sensor in Anabaena sp. PCC 7120 quantitatively estimates changes in intracellular redox potentials when dark-adapted cyanobacterial cells are illuminated (2).

Notably, the fluorescence signal of FROG/B can be observed even in the presence of the autofluorescence originating from the light-harvesting pigments of cyanobacterial cells. Following the emission fluorescence of the sensor expressed in cyanobacteria not only evaluates the in vivo glutathione redox balance but also provides an additional benefit. The observation of in vivo redox balance under the confocal microscope is the advantage relative to mass spectrometry or NMR measurements because the latter procedures lack the capacity to discriminate signals from heterocysts in the presence of vegetative cells. In Anabaena sp. PCC 7120, both vegetative cells and heterocysts appeared to be the oxidized form under dark conditions. The midpoint redox potential of FROG/B (E_m = −293 mV) is too low to measure precisely the redox dynamics in Anabaena 7120 cells under light conditions, but Sugiura et al. (2) estimate that the proportion of reduced FROG/B is 61% and 46% in vegetative cells and in heterocysts, respectively.

From early attempts (10) to the expression of Grx1-roGFP2 (11), GFP-based sensors are the strategy to study cell redox events. Given that Sugiura et al. (2) demonstrate clearly that FROG/B measures redox changes in phototrophic cyanobacteria, it will be interesting to see whether the sensor becomes a tool for measuring changes during photosynthetic activity in land plants. Hopefully, FROG/B may fill in this gap.