Molecular Biology Assignment

Genetically Encoded Fluorescent Redox Probes

S. Pavithra

14MBT0040

1st year- M Tech Biotechnology

VIT University

Abstract:

Redox reactions are one of the most important reactions that take place in the major cellular

functions. Thus imaging the redox process is emerging to be of great interest. Genetically

encoded fluorescent probes are the imaging tools that provide excellent opportunity in

observing the redox reactions in live cell. HyPer, rxYFP and roGFPs, are few of those

genetically encoded fluorescent probes that have a range of applications, from cultured cells

to transgenic animals. High specificity, possibility of transgenesis and subcellular targeting

are the major advantages of these GEFIs. For proper selection of a redox sensor for a

particular model, it is important to understand that HyPer and roGFP2-Orp1 are the probes

for H2O2, while, roGFP1/2, rxYFP and roGFP2-Grx1 are the probes for GSH/GSSG redox

state. Possible pH changes should be carefully controlled in experiments with HyPer and

rxYFP. The advent of genetically encoded fluorescent probes has allowed the real time

imaging of live cells and tissues for the reactive oxygen species and thiol redox state. In

future, FP- based redox probes shall be expanded to red and far- red parts of the spectrum.

And these can be used to quantify other critical species like Nitric oxide, oxygen and

superoxide.

1. Introduction:

ORP, redox potential or Oxidation reduction potential is the measure of the reduction or

oxidation potential of a solution. In biological reactions, the measurement of redox potential

would help in understanding different processes like signalling (1).

1.1. Limitations of conventional probes

Measurement of the redox potential of certain biological compound like glutathione,

ascorbate, NADPH, and thioredoxin (Trx), are performed by use of enzymatic assays, HPLC,

or gel mobility. One major drawback of these convention methods of measuring the redox

potential is that they lead to cell disruption. Thus these lack the resolution up to cellular

compartments.

Second drawback is that, few methods involving fluorescent dyes like in thiol- reactive dyes

intended to estimate the glutathione in living cells, provides data only about the content and

not the redox potential of the couple (2). Most of the probes are characterised by partial non

specific behaviour, irreversibility, and lack of compartment specificity.

1.2. Need for a new redox probe:

The drawback of conventional redox sensors are that, they consider the redox processes in the

biological reactions to be a single entity. In reality different oxidation and reduction processes

taking place within a biological system are being kinetically controlled and are far from not

much in equilibrium with each other. They are stearically, kinetically and compartmentally

separated.

All the reactions in the biological systems are controlled by kinetic catalysts the enzymes.

Thus each redox reaction is selective and is seldom uniform within and between cellular

compartments. Individual redox reactions have specific distinct functions and are

independently regulated. Hence the need for a looking into the reactions specifically with

specific functional role is necessary.

Redox probes should be designed in such a way that they are specific to a reaction or a redox

pairs. Since these probes are to be used in biological system, they have to be easy to use in

measurement in live cells, tissues and organisms, without disturbing the behaviour of the

cells. Probes for superoxide (O2˙¯), Hydrogen peroxide (H2O2), nitric oxide (NO˙),

glutathione (reduced form: GSH; oxidized form: GSSG), and ascorbate (reduced form: AA;

oxidized form: dehydroascorbate, DHA)(3).

Advanced probes – genetically encoded fluorescent redox probes - have thus been developed,

in order to overcome these limitations of the conventional probes.

1.3. Genetically encoded fluorescent redox probes

Green fluorescent protein (GFP) and other homologous fluorescent proteins (FPs) of different

colours from other marine creatures fluoresce due to their unique chromophore groups. Wild

GFP is a protein with 27 kDa and was isolated from the jellyfish Aequorea Victoria.

Its structure is made up of 238 amino acid long polypeptide which forms an 11-stranded b-

barrel shielding an internal a-helix (4). The chromophore of GFP is formed through

intramolecular cyclization of the three amino acids S65=Y66=G67.

Oxidation leads to formation of a conjugated system of p-electrons capable of absorbing and

emitting visible light. Fluorescent proteins (FP) have several applications, like activity of

target gene promoters can be monitored by placing FP under their control. This can be used

to reveal the spatial and temporal patterns of gene activities. Intracellular distribution and

dynamics of proteins of interest can be visualised by encoding FP in frame with a target

protein. Fluorescence (Forster) resonance energy transfer (FRET) between FPs of appropriate

colours (e.g., cyan and yellow, or green and red) can be used to monitor the protein–protein

interactions (5). Finally, in construction of genetically encoded fluorescent indicators (GEFIs)

for ions, small molecules, and enzymatic activities.

The first genetically encoded redox probes were Green fluorescent protein (GFP) that

allowed the quantification of dithiol–disulfide equilibrium in living cells. GFP and its

variants posse resistance to common proteases and are found to be stable under physiological

pH conditions. These properties provide an excellent scaffold for the development of

biosensors.

GFP-based redox-sensitive probes were developed by the groups of Jakob Winther and James

Remington by placing artificial disulfides onto fluorescent protein (FP) scaffolds. Redox-

sensitive GFP (roGFP) and Redox sensitive yellow fluorescent protein (rxYFP) both allow

non-invasive quantitative imaging of the associated dithiol–disulfide equilibrium. The set of

redox-sensitive FPs was recently supplemented with a circularly-permuted YFP variant

(cpYFP), which appears to be responsive to O2 ˙¯.

2. Genetically encoded redox probes - Principles:

2.1. Single FP based sensors

In the simplest GEFIs the FP chromophore is sensitivity to small ions. This used to trace the

changes in concentration of the ions. The sensors for pH and chloride are common examples.

The chromophores of most of the yellow fluorescent variants of avGFP are sensitive to Cl−

and the wild type GFP from Aequorea victoria (avGFP) is sensitive to pH (6).

Figure 1: Green Fluorescent Protein (GFP)

Wild-type GFP has two excitation peaks, the first excitation peak with a maximum at 395nm

corresponds to the protonated neutral form of the chromophore (A-band) and the second peak

with a maximum at 475nm corresponds to the de-protonated, anionic form (B-band). It

posses only a single emission peak with a maximum at 509 nm.

Chromophore of FP is buried deep inside the β-barrel structure and thus is not accessible to

large molecules. The number of substances that can be measured is limited to a few small

ions. Hence the structure is modified by random mutagenesis in search for mutants with

enhanced H+ or Cl− sensitivity.

Figure 2: The simplest GEFIs sensitive to changes in concentration of small ions such as H+ or

Cl− that are reflected in spectral shifts.

2.2. FRET based sensors:

Fluorescence resonance energy transfer between two fluorescent proteins is used in these

sensors. The basic principle is the energy transfer from the donor, fluorophore (light excited)

to a longer wavelength acceptor (chromophore) whose absorption spectrum overlaps with

that of the emission spectrum of donor which are placed at a distance of less than 5nm and

whose planes are at optimal angle (7).

The principle behind using these genetically encoded FRET based sensors is to fuse the two

FPs into a single polypeptide chain with sensing protein domains. These domains have the

ability to change the conformation on interaction with a substance of interest. The

subsequent change in the angle or distance between the donor and acceptor will change the

FRET.

Figure 3: Conformation changes in a linker flanked by two FPs comprising a FRET pair.

Structural rearrangements of the domains comprising the linker lead to changes in FRET

efficiency

The advantage in these FRET bases sensors is that they produce a ratiometric signal (i.e.) the

radio between the two wavelengths. Unlike the singe wavelength signal, this radiometric

signal will not be affected by the concentration of the sensor in cell or its compartment,

changes in the shape or cytoplasmic condensation.

The disadvantage of FRET based sensors are their low dynamic range with a large molecular

weight. This makes them difficult in subcellular targeting and using for broad part of the

spectrum. Most of the FRET sensors are sensitive pH. Thus, two different chromophores

may have complex pH driven responses.

2.3. Circularly permuted FP based sensors:

The β-barrel of the GFP is a rigid structure and is therefore unlikely to change conformation

n responses to the forces applied to its termini. A method called circular permutation is

performed where a small polypeptide (6 – 10 a.a.) is attached between the N and C terminal

of the β-barrel. This circularly permutated FPs becomes sensitive to the conformational

changes made in the sensing domain attached to the termini. Thus any change in the structure

of the sensing domain will induce change in the FP chromophore (8).

Figure 4: Sensors based on circularly permuted proteins consisting of cpFP fused to the domain

that undergoes conformation changes upon interaction with an environment.

2.4. Translocation based sensors:

Translocation based sensors are usually designed by fusing the FP with protein domains that

change position within a cell due to a change in any specific parameter. Measurement of

phosphoinositol is being done by one such sensor. Phosphorylation and Dephosphorylation

takes place at the inositol ring of these compounds. The phosphorylated phosphoinositol acts

as a messenger for certain domains which are fused to the GFP. This enables the imaging of

the translocation form or to the plasma membrane (9).

3. Genetically encoded fluorescent probes:

Efficient methods for measurement of ROS (Reactive Oxygen Species) are gaining

importance. Although convention probes like fluorogenic, luminescent and colorimetric are

accurate, but are poor in intracellular imaging.

Live cells have the capacity to sense the parameters in and around the cell in order to control

the transcriptions or enzyme responses. For example, An E.coli cell can measure a superoxide

radical by clusters of protein, SoxR and hydrogen per oxide can be measured by redox active

cysteines of OxyR (10). This quality of the cells can be used for designing a specific probe.

3.1. HyPer: Hydrogen peroxide sensor:

OxyR is a transcription regulator protein of E.coli that selectively senses tiny variations in the

H2O2 concentrations by its regulatory domain. OxyR-RD. Two of the several cys residues in

the protein are critical in sensing H2O2. Cys199 has a low pKa due to adjacent Arg residue

which allows it to react with H2O2 with a rate constant of 105 - 107 M-1 s-1.

This residue is located in a hydrophobic surrounding, which restricts its penetration only to

amphiphilic H2O2 and not to superoxide anion radical. After being oxidised by the H2O2, the

Cys199 with sulfenic acid gets close to the Cys208, where a disulphide bond is formed. This

formation of a disulphide bond leads to a conformational change in the OxyR-RD fold (11).

Thus integration of OxyR to cpYPF will results in a Hydrogen Peroxide sensor, HyPer.

The advantage of HyPer is its reversibility. It can be oxidised by cellular thiol reducing

systems. The midpoint potential of redox active Cys pair of OxyR and thus, of HyPer is

-185mV. Hence, HyPer can be used only in the relatively reducing environment of the

nucleocytoplasmic compartment, mitochondria and peroxisomes, but not outside the cell or in

the lumen of endosomes and the endoplasmic reticulum. Also since OxyR-RD is bacterial, it

has no interaction partner within the mammalian cells, and less subject to post translational

modification (12).

The wild chromophore of GFP and all the other GFP like proteins contains a tyrosine residue

that can be either protonated or deprotonated. In a protein that’s pH sensitive the

chromophore environment is organised in such a way that allows the proton transfer from

chromophore environment to the media. Since HyPer has a modified barrel, its pH sensitivity

is also high.

Its 420 nm and 500 nm excitation peaks correspond to protonated and anionic forms of the

chromophore, respectively. Acidification of the environment leads to an increase in the

protonated form (ex.420 nm) and a decrease in the deprotonated form (ex. 500 nm), therefore

mimicking reduction of the probe. In contrast, alkalization, will correspond to oxidation of

HyPer. This problem is overcome by using a pH control, SypHer, which is used to track pH

changes in the cytoplasm.

3.2. Probes with disulfide bonds engineered into the GFP b-barrel:

Glutathione (GSH) is an important antioxidant in plants, animals, fungi and some bacteria

and archaea, preventing damage to important cellular components caused by reactive oxygen

species such as free radicals and peroxides. It is a tripeptide with a gamma peptide linkage

between the carboxyl group of the glutamate side-chain and the amine group of cysteine.

Glutathione reduces disulfide bonds formed within cytoplasmic proteins to cysteines by

serving as an electron donor. In the process, glutathione is converted to its oxidized form,

glutathione disulfide (GSSG). Once oxidized, glutathione can be reduced back by glutathione

reductase, using NADPH as an electron donor.

Introducing cysteines near the FP chromophore could result in proteins that can change their

spectra on oxidation or reduction. Mutated forms of YF was developed by modifying two

surface amino acids, positioned in the β- strands and are close to the chromophore- N149C

and S202C. Formation of disulphide bonds in these positions will result in changes in

adjacent residues, H148 and Y203 and results in spectral changes (13).

The absorption spectrum changes upon oxidation. The 404 nm decreases while the 512 nm

decreases. However, the 404 nm peak is not fluorescent and only 512 can be imaged. The

midpoint potential of rxYPF is -261mV. The protein equilibrates very slowly with

glutathione in different redox states, when purified. Addition of recombinant glutaredoxin

significantly accelerates the reaction. Thus a fusion of rxYFP-Grx1p would provide an

efficient sensor for glutathione redox buffer.

3.2.1. roGFPs

Introduction of Cys residues in place of S147 and Q204, close to positions 148 and 203

facing the chromophore, and mutation C48S, resulted in roGFP1. The introduction of the

S65T mutation to roGFP1 resulted in roGFP2. Practically, roGFP2 appears to be more useful

than roGFP1for several reasons. First, roGFP2 is brighter and has a higher dynamic range

when excited with conventional laser wavelengths (405 nm and 488 nm). Second, in roGFP2

the anionic form of the chromophore (ex. 490 nm) dominates over the protonated form (ex.

400 nm), while the opposite is true for roGFP1. Also, with roGFP2, the strong anionic form

decreases and the weak protonated form increases making imaging easier.

Unlike roGFP1, roGFP2 is pH sensitive with a pKa of ~6. However, pH shifts the thiol-

disulfide equilibrium thereby affecting the roGFP2 response. However, the roGFP2

ratiometric readout can be considered to be more-or-less pH-insensitive since the pKa for

most of the cysteines in the cell, including roGFPs, is close to 9, which is far greater than

physiological pH values (14).

3.2.2. Grx1-roGFP2

The problem of the slow equilibration with external thiols described for rxYFP persists in

roGFPs as well. Glutaredoxin catalyses the reaction of thiol-disulfide exchange between

glutathione and roGFPs. Availability of glutaredoxin is therefore a rate-limiting factor in

roGFPs equilibration with intracellular thiols. As mentioned earlier, this issue has been

solved for rxYFP by equipping each rxYFP molecule with Grx via fusing the two proteins.

The fused protein, Grx1-roGFP2, was reactive to either GSH or GSSG in a time scale of

minutes, whereas GFP2 alone dint not respond (15).

Also, Grx1-roGFP2 was sensitive to trace amounts of GSSG and senses redox potential

changes between −240 mV and −320 mV. But this protein was not sensitive to H2O2.

Although, addition of GSH led to H2O2-induced oxidation of the sensor, indicating that H2O2

influences the sensor only via oxidation of GSH.

3.2.3. Orp1-roGFP2

If peroxidase was fused with GFP2, it can use H2O2 as one substrate and roGFP2 as a

reducing substrate. Thus fusing of roGFP2 and yeast peroxidase, Orp1 results in a H2O2

probe. This probe has the spectral properties of roGFP2- ratiometric pH- stable readout

reporting submicromolecular H2O2 (16). Orp1-roGFP2 with HyPer revealed similar profiles

of response to H2O2 in the cytoplasm of mammalian cells. HyPer showed faster oxidation in

with the different mechanisms of probe oxidation: direct reaction of HyPer and peroxidase-

mediated reaction of Orp1-roGFP2.

3.3. FRET-based sensors

FRET based sensor was created by connecting ECFP-EYFP FRET pair by a linker having

two Cys residues able to form disulfide upon oxidation (17).

This probe was suggested to be suitable for redox imaging in oxidized compartments, like

ER. The probe was used to measure EGSH in the ER lumen. The midpoint potential of the

probe is more than 80 mV less negative than that of roGFP1-iX (−229 mV) making it a

roGFP1 version with improved performance under oxidizing conditions.

Two novel FRET probes were developed, OxyFRET, and PerFRET. OxyFRET contains two

cysteine-reach domains (CRD) of Yap1 (a component of the Orp1–Yap1 redox relay)

separating cyan and yellow FPs. Oxidation in the CRDs results in FRET change. PerFRET

consists of one CRD domain and Orp1 peroxidase flanked by the FRET pair. Both sensors

have similar performance in different cell culture models. The dynamic range of the probes is

rather small that they were successfully used to monitor NADPH oxidase activation.

3.4. NAD+/NADH GEFIs

NADP+/NADPH and NAD+/NADH are two redox pairs that provide reducing equivalents to

all downstream redox systems. NAD+/NADH couple is mainly used in fuel oxidation

reactions, while, NADP+/NADPH provides electrons for anabolic processes. Both the redox

couples are involved in production/decomposition of ROS. In cytoplasm, electrons from

NADPH are taken up by NADPH oxidases to produce superoxide and, to support antioxidant

systems. In mitochondria, electrons from NADH can leak from the electron transport chain to

oxygen to produce superoxide. NADP exists in the cell mostly in its reduced form, while,

NAD is predominantly oxidized (18).

Two Genetically Encoded Fluorescent proteins for NAD+/NADH named Peredox and Frex

were developed with bacterial Rex domains capable of binding adenine nucleotides. Rex

domains are evolved to selectively bind NADH in presence of excess of NAD+. Peredox and

Frex sensors explore changes in the Rex dimerization state upon NADH binding. cpFP

incorporated into an engineered linker between two Rex subunits. This allows a spectral

change upon NADH binding. Perdox has an extreme sensitivity to NADH, which makes it

useful in mitochondrial compartments, where NAD+/NADH ratio is low. Since Frex sensors

are based on cpYFP, they need pH control.

4. Usage of GEFI:

ROS are measured indirectly through the thiol-disulfide exchange between the probe and its

environment. Both HyPer and roGFP-based probes have been used to reveal the spatial and

temporal patterns of H2O2 production in the form of glutathione redox state, in a quantitative

way (19).

HyPer ratio values can be transformed into H2O2 values similar to roGFP2 → EGSH

transformation. Since HyPer competes with powerful antioxidant systems such as, Prx, the

amount of H2O2 measured by HyPer can be used as an estimate rather than absolute numbers.

Application of GEFI could be seen in several levels of complex biological systems. Real time

imaging of living cells are possible, which were once not possible due to non specificity of

the intracellular dyes. GEFIs made it possible to observe processes at the single-cell level for

the first time. By fusing GEFI to proteins that localize to specific micro domains

(mitochondria, nucleus, cytoplasm, ER and others) within the organelle it is possible to track

the redox processes at the level of the diffraction limit of fluorescent microscopy.

4.1. HyPer in organelles

HyPer and its derivatives have been successfully used in a number of experimental systems

that report H2O2 production in response to EGF (Epidermal Growth Factor), PDGF (Platelet-

derived growth factor), NGF (Nerve Growth Factor), high frequency electrical stimulation of

neurons, insulin and other stimuli (20).

Since HyPer is composed of protein domains that have no evolved partners in eukaryotic

cells, it diffuses very rapidly averaging the signal (21). As mentioned earlier, a pH controller

has to be used along HyPer. BCECF-AM was used as a pH control in experiments with NGF.

4.2. Imaging redox microdomains

HyPer has been used for visualizing the sites of ROS production within the compartments.

Diffusion of the probe inside the cell is a major limitation in imaging of localized H 2O2. This

can be overcome by immobilizing the sensor inside the cell (22). Thus, it can be fused with

different proteins localized on the cytoplasmic face of the plasma membrane, endosomes and

the ER membrane. This has allowed imaging of H2O2 microdomain formation upon

activation of receptor tyrosine kinases such as EGFR and PDGFR.

4.3. In vivo imaging of H2O2 using HyPer

On cutting the tail fins of 3 dpf larvae of Zebra fish Danio rerio, a Hydrogen peroxide

gradient was observed in response to the wound. The gradient acts to attract the neutrophils to

the site of wounding and represents the earliest event in the inflammatory response. HyPer

expression was achieved by injecting mRNA into the oocyte. The neutrophils arriving at the

wound dissipate the H2O2 gradient by means of myeloperoxidase, an enzyme of antibacterial

defence machinery. At the tissue level, myeloperoxidase acted as an H2O2-scavenging

enzyme, efficiently consuming H2O2 in the surrounding tissue. The cell- scale, H2O2 gradients

in the neutrophils migrating to wound was visualised by generating a line of transgenic fish

expressing HyPer (23).

4.4. In vivo roGFP2 imaging

Age and condition-related alterations of GSH/GSSG and H2O2 in living larval and fixed

adult Drosophila melanogaster was studied by fusing, roGFP2 to Grx1 and Orp1. The Orp1-

roGFP2 and roGFP2-Grx1were targeted to the cytoplasm or mitochondrial matrix and were

expressed in transgenic animals. GSH/GSSG level in cytoplasm appeared to be stable in

different tissues and in adult flies of different ages (1 to 14 days), while, mitochondrial

GSH/GSSG was more heterogeneous: more oxidized in Malpighian tubules and fat tissue and

more reduced in muscles. Midgut entherocytes were found to be a major source of aging-

associated H2O2 production. Higher H2O2 in the gut was correlated to an increase in the

lifespan. Also, the important finding was the absence of an antioxidant effect of N-acetyl-

cysteine in vivo. The study clearly showed that differences in [H2O2] does not correlate with

differences in GSH/GSSG and that the two parameters should be considered and measured,

separately (24).

4.5. roGFP2-Grx1 in yeasts

Approximately 100:1 GSH:GSSG ratio corresponding to a glutathione redox potential of

−240 mV was resulted in estimating cytoplasmic GSH/GSSG redox state using whole-cell

extracts. Quantitative measurement of cytosolic EGSH with rxYFP and roGFP2-Grx1

showed a much more reduced GSH/GSSG state (−320 mV, 50,000:1 GSH:GSSG ratio). It

was showed that under oxidative challenge or glutathione reductase Glr1 deficiency highly

reducing glutathione state in yeast cells is maintained by rapid active export of GSSG from

the cell by ABC-C transporter Ycf1 (25). A mammalian homolog of Ycf1, MRP1, had earlier

been shown to transport GSSG from the cells under oxidative stress conditions. This

mechanism seems to be conserved in eukaryotic species.

5. Conclusion

Redox GEFIs has enabled the real-time monitoring of GSH/GSSG, H2O2 and NAD+/NADH

redox state. Since the current GEFIs emit light in the green part of the visible spectrum, they

cannot be used for simultaneous imaging with other green-emitting probes. Therefore, red

fluorescent sensors are in high demand. Far-red probes have potential utility because the

“optical window” of tissues allows a penetration of light in a near-IR range. Most of them

have large stocks shift: they emit in the near-far-red and are excited by an orange light.

Therefore, excitation light penetration in tissues can still be a limiting factor.

Several reactive oxygen intermediates are still to be visualized. The utility of cpYFP as a

superoxide probe is still under discussion, other ways to make superoxide sensors are

possible. Other molecules like nitric oxide and oxygen have remained hidden from a direct

observation for several decades. Cells have evolved protein domains that sense all the factors

critical for their existence. This gives a hope that these cells’ domains could be used in

designing probes for NO, O2 and other critical redox related molecules.

6. Reference:

(1) American Public Health Association, American Water Works Association, Water

Environment Federation (2005) Standard Methods for the Examination of Water &

Wastewater, 21st ed.; Washington, DC. Bühler, H., Baumann, R., (1982) Redox

Measurement, Principles and Problems; Zürich

(2) Schmidt B, Ho L, and Hogg PJ. Allosteric disulfide bonds. Biochem. 45: 7429–7433,

2006.

(3) Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4:

181–189, 2004.

(4) Shimomura O, Johnson FH, and Saiga Y. Extraction, purification and properties of

aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell

Comp Physiol 59: 223–239, 1962

(5) F.S. Wouters, P.I. Bastiaens, Imaging protein–protein interactions by fluorescence

resonance energy transfer (FRET) microscopy, Chapter 5, Curr. Protoc. Neurosci.

(2006), (Unit 5 22).

(6) M.F. Abad, G. Di Benedetto, P.J. Magalhaes, L. Filippin, T. Pozzan, Mitochondrial pH

monitored by a new engineered green fluorescent protein mutant, J. Biol. Chem. 279

(2004) 11521–11529.

(7) E.B. van Munster, T.W. Gadella, Fluorescence lifetime imaging microscopy (FLIM),

Adv. Biochem. Eng. Biotechnol. 95 (2005) 143–175.

(8) H.J. Carlson, R.E. Campbell, Genetically encoded FRET-based biosensors for

multiparameter fluorescence imaging, Curr. Opin. Biotechnol. 20 (2009) 19–27.

(9) P. Varnai, T. Balla, Live cell imaging of phosphoinositide dynamics with fluorescent

protein domains, Biochim. Biophys. Acta 1761 (2006) 957–967.

(10)C. Rota, Y.C. Fann, R.P. Mason, Phenoxyl free radical formation during the oxidation

of the fluorescent dye 2′,7′-dichlorofluorescein by horseradish peroxidase. Possible

consequences for oxidative stress measurements, J. Biol. Chem. 274 (1999) 28161–

28168.

(11)V.V. Belousov, A.F. Fradkov, K.A. Lukyanov, D.B. Staroverov, K.S. Shakhbazov,

A.V. Terskikh, S. Lukyanov, Genetically encoded fluorescent indicator for intracellular

hydrogen peroxide, Nat. Methods 3 (2006) 281–286.

(12)K.N. Markvicheva, D.S. Bilan, N.M. Mishina, A.Y. Gorokhovatsky, L.M. Vinokurov,

S. Lukyanov, V.V. Belousov, A genetically encoded sensor for H2O2 with expanded

dynamic range, Bioorg. Med. Chem. 19 (2011) 1079–1084.

(13)V.V. Belousov, A.F. Fradkov, K.A. Lukyanov, D.B. Staroverov, K.S. Shakhbazov,

A.V. Terskikh, S. Lukyanov, Genetically encoded fluorescent indicator for intracellular

hydrogen peroxide, Nat. Methods 3 (2006) 281–286.

(14)A.J. Meyer, T.P. Dick, Fluorescent protein-based redox probes, Antioxid. Redox

Signal. 13 (2010) 621–650.

(15)M. Gutscher, A.L. Pauleau, L. Marty, T. Brach, G.H. Wabnitz, Y. Samstag, A.J. Meyer,

T.P. Dick, Real-time imaging of the intracellular glutathione redox potential, Nat.

Methods 5 (2008) 553–559.

(16)M. Gutscher, M.C. Sobotta, G.H. Wabnitz, S. Ballikaya, A.J. Meyer, Y. Samstag, T.P.

Dick, Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases, J.

Biol. Chem. 284 (2009) 31532–31540.

(17)V.L. Kolossov, B.Q. Spring, A. Sokolowski, J.E. Conour, R.M. Clegg, P.J. Kenis, H.R.

Gaskins, Engineering redox-sensitive linkers for genetically encoded FRET-based

biosensors, Exp. Biol. Med. (Maywood) 233 (2008) 238–248.

(18)E.A. Sickmier, D. Brekasis, S. Paranawithana, J.B. Bonanno, M.S. Paget, S.K. Burley,

C.L. Kielkopf, X-ray structure of a Rex-family repressor/NADH complex insights into

the mechanism of redox sensing, Structure 13 (2005) 43–54.

(19)Konstantin A. Lukyanov, Vsevolod V. Belousov, Genetically encoded fluorescent

redox sensors,Biochimica et Biophysica Acta 1840 (2014) 745–756.

(20)M. Malinouski, Y. Zhou, V.V. Belousov, D.L. Hatfield, V.N. Gladyshev, Hydrogen

peroxide probes directed to different cellular compartments, PLoS One 6 (2011).

(21)A. Espinosa, A. Garcia, S. Hartel, C. Hidalgo, E. Jaimovich, NADPH oxidase and

hydrogen peroxide mediate insulin-induced calcium increase in skeletal muscle cells, J.

Biol. Chem. 284 (2009) 2568–2575.

(22)Y.M. Go, D.M. Duong, J. Peng, D.P. Jones, Protein cysteines map to functional

networks according to steady-state level of oxidation, J. Proteomics Bioinform. 4

(2012) 196–209.

(23)L. Pase, J.E. Layton, C. Wittmann, F. Ellett, C.J. Nowell, C.C. Reyes-Aldasoro, S.

Varma, K.L. Rogers, C.J. Hall, M.C. Keightley, P.S. Crosier, C. Grabher, J.K. Heath,

S.A. Renshaw, G.J. Lieschke, Neutrophil-delivered myeloperoxidase dampens the

hydrogen peroxide burst after tissue wounding in zebrafish, Curr. Biol. 22 (2012)

1818–1824.

(24)S.C. Albrecht, A.G. Barata, J. Grosshans, A.A. Teleman, T.P. Dick, In vivo mapping of

hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity

of redox homeostasis, Cell Metab. 14 (2012) 819–829.

(25)B. Morgan, D. Ezerina, T.N. Amoako, J. Riemer, M. Seedorf, T.P. Dick, Multiple

glutathione disulfide removal pathways mediate cytosolic redox homeostasis, Nat.

Chem. Biol. 9 (2013) 119–125.