A genetically encoded sensor for H2O2 with expanded dynamic range.
ABSTRACT Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator's dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.
A genetically encoded sensor for H2O2with expanded dynamic range
Kseniya N. Markvichevaa, Dmitry S. Bilana, Natalia M. Mishinaa, Andrey Yu. Gorokhovatskyb,
Leonid M. Vinokurovb, Sergey Lukyanova, Vsevolod V. Belousova,*
aShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia
bShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, RAS, Pushchino, Russia
a r t i c l ei n f o
Received 19 May 2010
Revised 30 June 2010
Accepted 7 July 2010
Available online 5 August 2010
a b s t r a c t
Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by
selective oxidation of redox active thiolates in proteins. Changes in intracellular [H2O2] can be tracked
in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive
and selective for H2O2due to the properties of its sensing domain derived from the Escherichia coli OxyR
protein, many applications may benefit from an improvement of the indicator’s dynamic range. We here
report HyPer-2, a probe that fills this demand. Upon saturating [H2O2] exposure, HyPer-2 undergoes an
up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated
by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was pre-
viously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the
A233V mutation stabilizes the dimer and expands the dynamic range of the probe.
? 2010 Elsevier Ltd. All rights reserved.
Aerobic organisms reduce molecular oxygen to produce energy.
Most of the oxygen consumed undergoes a four-electron reduction
to water. However, a fraction of the oxygen within cells receives
only one electron. This process gives birth to a superoxide anion
radical, a highly reactive molecule serving as a precursor of other
reactive oxygen species (ROS).1,2Under a variety of pathological
conditions, ROS production in the cells increases enormously lead-
ing to oxidative stress and nonspecific oxidation of lipids, DNA, and
proteins.1At the same time, ROS participate in normal physiology
modifying cellular signaling cascades activated in response to
external stimuli such as growth factors and cytokines.3Among
ROS, H2O2is best studied as a second messenger. It mainly acts
in the cells by modifying critical thiol residues of proteins thereby
regulating their catalytic activities or other functions. Due to the
reducing conditions within the cell, H2O2is able to react with only
those Cys residues that have low pKa(below 6.5) and, therefore,
tend to be deprotonated at physiological pH.2A well-documented
example of such regulation is an oxidation of protein tyrosine
phosphatases (PTPs). Activation of receptor tyrosine kinases (RTKs)
leads to production of H2O2 by NADPH oxidase (Nox) family
enzymes.4This H2O2 inhibits PTPs by oxidizing their catalytic
thiolates thus allowing an extended propagation of the phosphor-
For many decades since the discovery of biological ROS, their
detection inside cells was difficult due to the absence of adequate
measurement technique. Despite their high dynamic range, small
molecule chemical probes for ROS detection have one or several
of the following disadvantages: low specificity, poor to zero cell
permeability, photodynamic ROS production leading to signal
self-amplification. We overcame these drawbacks by development
of the first genetically encoded sensor for H2O2, HyPer.7HyPer can
be expressed in apparently any compartment of the cell by trans-
fecting cells with DNA encoding the HyPer fusion with a subcellu-
lar localization tag or a protein of interest. HyPer has two
excitation peaks corresponding to the protonated (420 nm) and
charged (500 nm) forms of Tyr residue of the YFP chromophore.
Both forms can be easily visualized by laser excitation of a confocal
system or with widefield fluorescent microscopy. Ratiometric
readout avoids artifacts associated with cell movement or differ-
ences in the sensor expression level between cells. However, for
cells that do not move significantly and do not change the shape
in the course of the experiment, single wavelength monitoring is
possible. The properties of the H2O2-sensing domain of HyPer, de-
rived from the bacterial OxyR protein,8,9dictate high selectivity of
the probe, high sensitivity and, importantly, good reversibility in
the intracellular environment.7
Wild-type OxyR consists of two domains, a DNA-binding and a
regulatory (OxyR-RD) domain.9The latter contains two cysteine
residues, C199 and C208, critical for the protein function as an
H2O2sensor. C199 resides in a hydrophobic pocket and therefore
is inaccessible for charged oxidants such as superoxide anion
0968-0896/$ - see front matter ? 2010 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +7 495 4298020; fax: +7 495 3307056.
E-mail address: email@example.com (V.V. Belousov).
Bioorganic & Medicinal Chemistry 19 (2011) 1079–1084
Contents lists available at ScienceDirect
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radical.8However, amphiphilic H2O2molecules are able to pene-
trate the pocket and oxidize C199 to a sulphenic acid intermediate
that is repelled by its hydrophobic surrounding due to its charge.
Initially separated, C199 and C208 now get sufficiently close to
form the disulphide.10This reaction changes the overall conforma-
tion of the OxyR-RD permitting OxyR binding to a specific region
on DNA.11OxyR was shown to function as either dimer or tetramer
(dimer of dimers)12and therefore contains one or two oligomeriza-
tion interfaces. Crystallization of OxyR-RD reveals a dimer inter-
face that consists of a.a. 226–233 positioned C-terminally behind
the ‘redox loop’ that hosts C199 and C208.8
The dynamic range of HyPer F500/F420 ratio is 3. This is satis-
factory for most of the experimental conditions. However, expan-
sion of the dynamic range could be beneficial for applications in
amounts of H2O2produced for cell signaling. Here we report Hy-
Per-2, the probe with the twice-expanded dynamic range com-
pared to HyPer.
2.1. Investigating the dynamic range of the HyPer-N353S-
During HyPer subcloning to introduce nuclear export signal se-
quences (NES), we accidentally produced two random PCR-derived
mutations leading to amino acid substitutions. The first mutation
affected the sequence of the fluorescent protein (N353S corre-
sponding to N121S in GFP). The other mutation was A406V corre-
sponding to A233V in wtOxyR (Fig. 1a). When expressed in HeLa
cells, this probe demonstrated expanded dynamic range compared
to regular HyPer. To carefully evaluate the amplitude of the hydro-
peroxide response, we designed a simple assay to measure imme-
diately [H2O2] changes in cells. Usually, addition of compounds to
the glass-bottom cell culture dish mounted on a microscope table
takes several seconds. To ensure proper mixing, the solution
should be added in a large volume, not less than1=4of the volume
of the media in the dish. Adding this carefully to avoid focus shifts
requires up to 10 s. Giving the large volume of the media in the
dish, diffusion of the compound may also take time. In case of
H2O2addition imaged with HyPer, the oxidation profile may vary
between dishes. We therefore used l-slides to culture, transfect
and image cells exposed to H2O2. The scheme of the experiment
is shown in Figure 1b. The total volume of l-slide-VI channel with
its two reservoirs is 120 ll. Prior to imaging, we removed 90 ll of
the medium via one of the reservoirs of the channel. The remaining
30 ll of the media were left inside the channel. We chose cells, ad-
justed the focus and started a time series. After acquiring the first
several frames (Fig. 1c), we added 90–100ll of H2O2-containing
medium as a drop into the front reservoir of the channel. In this
setting, the H2O2medium immediately replaced the medium in-
side the channel.
Upon addition of saturating amounts of H2O2(100 lM) to the
cells expressing HyPer-N353S-A406V-NES, the F500/F420 ratio of
the probe changed 6- to 7-fold (Fig. 1c and d).
2.2. HyPer-2 dynamics in living cells
The reason for the dynamic range expansion could be the NES
attached to the C-terminus of HyPer, or one of two amino acid
Figure 1. HyPer with N353S and A406V substitution demonstrates a high dynamic range in its ratio change upon addition of H2O2. (a) HyPer consists of cpYFP (green)
inserted between residues 205 and 206 of OxyR-RD (blue). It contains two critical Cys residues, C121 and C381 (199 and 208 in wtOxyR). N353 corresponds to N121 in wtGFP.
A406 is A233 in wtOxyR. (b) Scheme of the experiment with cells cultured and transfected in l-slides. (c) Images of ratio F500/F420 change in cells transfected with HyPer-
N353S-A406V exposed to 100 lM H2O2. Numbers indicate timing in seconds. H2O2was added between the 2nd and 3rd frames shown. Scale bar 40 lm. (d) Timing of ratio
change in cells shown on panel c (black lines) compared to HyPer expressing cells (red lines).
K. N. Markvicheva et al./Bioorg. Med. Chem. 19 (2011) 1079–1084
substitutions. Addition of C-terminal NES to HyPer without muta-
tions had no effect on the dynamic range of the probe (data not
shown). We therefore introduced each mutation separately into
HyPer and expressed the resulting proteins in HeLa cells. Whereas
the N353S substitution had no effect on HyPer’s spectral proper-
ties, the A406V mutant appeared to exhibit an elevated dynamic
range (Fig. 2a and b). The A406V substitution was therefore the sole
cause of the property change. We named this probe HyPer-2.
We next compared the properties of intracellular HyPer-2 ver-
sus HyPer response to externally added H2O2. For this, we mea-
sured the half-oxidation and the half-reduction time of the
probes. HyPer demonstrated faster oxidation and reduction than
HyPer-2 (Fig. 2c and d). Both half-oxidation and half-reduction
times of HyPer-2 were doubled compared to HyPer. This might
indicate the reduced availability of the OxyR disulphide in
HyPer-2 to reducing enzymes. It may also indicate increased sensi-
tivity of HyPer-2 to H2O2with higher probe-H2O2reaction rates
allowing a higher HyPer-2 oxidation rate in the presence of pro-
gressively decreasing concentrations of H2O2.
2.3. Oligomerization state of HyPer-2 versus HyPer
A233V mutation of OxyR corresponding to A406V in HyPer had
been previously characterized as one destabilizing wtOxyR dimer
interface.8,12,13We proposed that destabilization of the dimer
interface leads to increased mobility of the ‘redox loop’, the region
between a.a. 205 and 225 (wtOxyR nomenclature).8a-Helix host-
ing A233 is (i) packed into the dimer interface and (ii) flanks C-ter-
minally the ‘redox loop’ into which the cpYFP is incorporated in
HyPer. We performed gel-filtration chromatography to evaluate
oligomerization states of HyPer and HyPer-2 (Fig. 3). Surprisingly,
whereas HyPer appeared to be eluted as a mixture of dimers and
monomers in concentrated samples or to be monomeric when
diluted, HyPer-2 showed strong dimerization independently on
the concentration of the protein. It suggests either that HyPer
and HyPer-2 have a different structure of the interface compared
to wtOxyR or that the substitution of A233 to the more hydropho-
bic Val stabilizes the HyPer interface via hydrophobic interaction
with I110 and L124 (wtOxyR numbers).8
Notably, the ex497/ex420 ratio is higher for the dimers com-
pared to the monomers (Fig. 3b). Therefore, it could be suggested
that HyPer and HyPer-2 may change spectral properties upon
dimerization. Our observations (unpublished data) show that
ex497/ex420 ratio of reduced HyPer within the living cells resem-
bles the ratio of the monomeric form of HyPer shown in Figure 3b
suggesting HyPer exists in the monomeric form in the cell. Good
performance of HyPer in fusions also supports its monomeric nat-
ure. However, for HyPer-2 the overall amplitude of its response to
H2O2may theoretically reflect two sequential processes, oxidation
and oxidation-induced dimerization, which both may increase the
ratio. This model could also explain slow response of HyPer-2 be-
cause its dimerization after oxidation may take time.
2.4. Detection of endogenous cellular H2O2with HyPer-2
HyPer has been previously shown as a suitable probe for mon-
itoring low amounts of H2O2produced upon activation of growth
factor receptors.7,14,15The dynamic range of HyPer-2 could be ben-
eficial for signaling H2O2studies because the oxidant is produced
in subsaturating concentrations. We therefore stimulated NIH-
3T3 fibroblasts expressing HyPer-2-NES or HyPer-Cyto with
10 ng/ml PDGF. All the cells responded by a progressive increase
in H2O2(Fig. 4). However, cells that express HyPer-2-NES demon-
strated a higher amplitude of fluorescence response than those
expressing regular HyPer. Therefore, HyPer-2 is more suitable for
monitoring low concentrations of endogenous H2O2than HyPer.
Figure 2. HyPer-2 versus HyPer response on H2O2. (a) Changes in ratio F500/F420 of HyPer-2 (upper raw) versus HyPer (lower raw). Numbers indicate time in seconds and
show timing of both upper and lower rows. Scale bar 40 lm. (b) Typical profiles of the ratio F500/F420 change in individual cells expressing HyPer-2 (black line), HyPer-
N353S (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.
K. N. Markvicheva et al./Bioorg. Med. Chem. 19 (2011) 1079–1084
We introduced HyPer-2, the improved version of HyPer, a
genetically encoded sensor for H2O2. The dynamic range of
F500/F420 ratio change is 6, which is twice as high as in HyPer.
This improvement was a result of a single point mutation chang-
ing Ala 406 for a Val residue. HyPer consists of two domains: a
circularly permuted fluorescent protein (cpYFP) inserted into a
regulatory domain of OxyR (OxyR-RD). The A406V substitution
corresponds to A233V of wtOxyR. This mutation is one of several
well-studied mutations of OxyR.8,12,13OxyR A233V is a constitu-
tively active mutant probably due to reduced dimerization.12,13
In case of full-length OxyR, activity means binding of OxyR to a
specific DNA region and transcription from the corresponding
promoter. Therefore OxyRA233V binds DNA irrespectively of the
redox state of its dicysteine pair. Alanine 233 is one of the key
residues of the interface between OxyR monomers in the dimer.8
Substitution to the bigger Val residue has been shown to destabi-
lize this interface resulting in a conformational shift in OxyR that
improves DNA binding.8,12,13In case of HyPer, the same mutation
led to the stabilization of the dimer interface leading to changes
in mobility of the ‘redox loop’, the region flanked by position
205 (wtOxyR nomenclature) and a short alpha-helix participating
in the dimer interface and containing the residue 233 (Fig. 5).
cpYFP is inserted between a.a. 205 and 206. Therefore conforma-
tional changes in this region are critical for shifts in the cpYFP
In our previous attempts to improve HyPer we mutated cpYFP
and the linkers between the fluorescent protein and OxyR-RD. It
seemed logically that the ‘naturally designed’ sensing part should
be kept intact to preserve activity. Our results presented here show
that the mutagenesis of this part is a promising strategy of the sen-
sor improvement. In fact, this strategy was successfully used for
the rational design of calcium probes. Tian and co-authors modi-
fied EF-hands in the Ca2+-sensing domain to increase the affinity
of the sensor to Ca2+to produce GCaMP3.16
For most genetically encoded sensors, the limiting step in prop-
erty improvement is the number of clones that can be manually
screened. In case of simple fluorescent protein, any easy to detect
parameter such as brightness or maturation speed can serve as a
readout for mutagenesis. Fluorescent sensor candidates need to
be visualized twice—before and after the change in the measured
signal. The absence of high throughput strategies for sensor
improvement restricts the potential applicability of the probes be-
cause most of the sensors published have a low dynamic range
which prevents more general applications.
HyPer, and now HyPer-2, have been used to monitor the gener-
ation of H2O2produced by cells activated by various growth fac-
tors, namely, NGF,7EGF,14PDGF, and insulin.15Detection of H2O2,
therefore, can be used as readout in high throughput/high content
screening of chemicals inhibiting RTK pathways and in the search
for isoform-specific NADPH oxidases (Nox) inhibitors. HyPer or Hy-
Per-2 need to be expressed in a proper cell line expressing the
receptor of interest or a specific Nox isoform. Then, after addition
of compounds and the agonist, cells expressing HyPer or HyPer-2
can be fixed with up to 4% paraformaldehyde and the HyPer emis-
sion ratio is acquired. Apart from the for above-mentioned recep-
tors, many other growth factor and cytokines receptors and even
some GPCRs that activate NADPH oxidases4may be assayed using
HyPer/HyPer-2 in the future. We believe that HyPer-2 will be use-
ful in novel screening platforms replacing the often used multistep
screens based on phospho-MAPKs immunostaining.17
Final words should be said about influence of luck on experi-
mental results. Three years of site-directed and random mutagen-
esis, screening of hundreds of clones in search for better mutant of
HyPer did not give us an improved sensor. In fact, most variants
performed worse then the original one. The first real improvement
of the sensor properties came by chance as a side effect of the sub-
cloning procedure. There is a phrase attributed to a famous bio-
chemist Isaac Asimov: ‘The most exciting phrase to hear in
science, the one that heralds new discoveries, is not ‘Eureka!’
(I found it!) but ‘That’s funny ...’
Figure 3. Gel-filtration elution profiles and excitation spectra of HyPer and HyPer-2. (a) Whereas HyPer (two upper plots) is eluted as a dynamic mixture of monomers and
dimers, HyPer-2 (two lower plots) is a dimer independently on the protein concentration. (b) Excitation spectra of monomers and dimers of HyPer and HyPer-2.
K. N. Markvicheva et al./Bioorg. Med. Chem. 19 (2011) 1079–1084
H2O2and molecular weight markers for gel-filtration were from
Sigma. Hanks balanced salts solution (HBSS) was from PanEko
(Russia), DMEM, MEM, Opti-MEM, FCS and FuGene6 transfection
reagent were from Invitrogen. l-Slides-IV were from IBIDI, glass
bottom dishes were from MatTec. HeLa and NIH-3T3 cells were
from ATCC. HyPer expression vectors were from Evrogen.
4.2. Cell culture and transfection
HeLa-Kyoto and NIH-3T3 cells were cultured in DMEM supple-
mented with 10% FCS at 37 ?C in atmosphere containing 95% air
and 5% CO2. Cells were splitted every 2nd day and seeded to the
l-slides-IV. After 12–24 h cells were transfected by the mixture
of vector DNA and FuGene6 transfection reagent according to the
liposomes manufacturer recommendations with the following
optimization: because of a small volume of the l-slide-IV, ready
liposome-DNA solution was mixed with 10? volume of cell culture
medium and used to replace the culture medium inside the chan-
nels of l-slide-IV. Detailed protocols of media changing in l-slides
can be found at www.ibidi.com.
For experiments with growth factors, NIH-3T3 cells were seeded
to glass bottom dishes. Twenty-four hours later cells were transfec-
ted by the mixture of vector DNA and FuGene6 transfection reagent
according to the liposomes manufacturer recommendations.
4.3. Stimulation of the cells and imaging
Twenty-four hours after transfection the culture medium was
changed to either HBSS (for experiments with externally added
H2O2) or MEM without phenol red and serum (for PDGF stimula-
Figure 4. HyPer-2 versus HyPer response on H2O2generated intracellularly upon addition of PDGF to NIH 3T3 cells. (a) Confocal images of a HyPer-2-NES expressing cell after
stimulation with 10 ng/ml PDGF. Scale bar 20 lm. (b) Confocal images of a HyPer-Cyto expressing cell after stimulation with 10 ng/ml PDGF. Scale bar 20 lm. (c) Profiles of
F500/F420 ratio change in the cells shown on the panel a (black line) or panel b (red line). PDGF is added at time zero.
K. N. Markvicheva et al./Bioorg. Med. Chem. 19 (2011) 1079–1084
tion). l-Slides-IV with HeLa cells were transferred onto the micro-
scopic stage of the widefield (Leica 6000) microscope equipped
with an HCX PL APO lbd.BL 63x 1.4NA oil objective. FRET filter cube
together with external CFP and YFP excitation filters were used to
sequentially excite two excitation peaks of HyPer detecting emis-
sion using external YFP filter. Laser scanning confocal inverted
microscopes Carl Zeiss LSM 510-META equipped with an HCX PL
APO lbd.BL 63x 1.4NA oil objective and environmental chamber
was used for growth factor stimulation experiments. Laser lines
(405–488 nm) were used to sequentially excite two excitation
peaks of HyPer detecting emission at 500–550 nm wavelength
range. After 6 h (NIH-3T3) of incubation in serum-free medium,
glass bottom dishes were transferred onto the microscopic stage.
NIH-3T3 cells were stimulated by adding 10 ng/ml PDGF-BB. HeLa
cells were stimulated by 100 lM H2O2.
4.4. Time series processing
Time series were analyzed using ImageJ free software down-
loaded from EMBL ALMF website (http://www.embl.de/almf/).
Stacks of 420–500 nm (correspond to 2 HyPer excitation peaks)
were Gaussian filtered with sigma radius 2 (skipped for widefield
images) and background was subtracted. Images were converted
to 32 bit and 420 nm stack was thresholded to remove pixel values
from background (Not-a-Number function). Five hundred nanome-
ters stack was divided to 420 nm stack frame-by-frame. The re-
sulted stack was pseudocolored using ‘Ratio’ lookup table. Time
course of HyPer fluorescence was calculated for regions of interest
(ROIs) inside the imaged cell.
4.5. HyPer oligomerisation state estimation
Gel filtration chromatography was performed using a Superdex
200 10/300 GL column (Amersham Biosciences), equilibrated with
40 mM Tris–HCl (pH 7.5), 150 mM NaCl buffer at a flow rate of
0.4 ml/min. Elution profiles were monitored by absorbance at
280, 420, and 497 nm using a Varian ProStar 335 diode array detec-
tor and by fluorescence using in-line Varian ProStar 363 fluores-
cence detector. To obtain a fluorescence signal within a dynamic
range of the detector, excitation/emission wavelengths were 457/
566 nm for 1 mg/ml samples or 477/536 nm for 0.02 mg/ml sam-
ples. Apparent molecular masses were calculated by interpolating
an elution volume versus log (molecular mass) calibration curve
using ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa),
BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa),
and ribonuclease A (13.7 kDa).
The work is supported by Federal Education Agency (project
no. P256), grant of the President of Russian Federation (MR-
3567.2009.4), the Russian Academy of Sciences Program in
Molecular and Cell Biology, the Russian Foundation for Basic
Research (09-04-12235, 10-04-01561) and the Howard Hughes
Medical Institute (55005618). K.N.M. received FEBS short-term
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.014. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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Figure 5. Image of the reduced OxyR-RD structure (PDB ID 1I69). C199 (upper) and
C208 (lower) are shown in red. The flexible region of OxyR from a.a. 205 to 225 is
yellow; the a-helix facing the dimer interface and hosting A233 (magenta) is shown
in cyan. The second monomer of the dimer is dark blue. The image was obtained
using PyMOL software.
K. N. Markvicheva et al./Bioorg. Med. Chem. 19 (2011) 1079–1084