no aplica

1. DOI: 10.1002/cbic.200600048
Application of Bioluminescence Resonance
Energy Transfer (BRET) for Biomolecular
Interaction Studies
Anke Prinz, Mandy Diskar, and Friedrich W. Herberg*[a]
1. Introduction
It is widely appreciated that the investigation of components
in protein networks and signalling cascades requires multiple
approaches and that analysis of protein interactions in living
cells to complement in vitro studies is indispensable. This field
was revolutionised with the molecular cloning of the green flu-
orescent protein (GFP, MW =27 kDa) from the jellyfish Aequorea
victoria by Prasher and co-workers.[1]
Since then, GFP and its
derivatives[2]
have been applied as genetically encoded fluores-
cent reporters for protein localisation studies, to visualise traf-
ficking of proteins in real time, and to investigate protein–pro-
tein interactions through resonance energy transfer (RET[3]
).
This review focuses on bioluminescence resonance energy
transfer (BRET) in the study of subunit interaction of the cAMP-
dependent protein kinase (PKA) in intact cells. As depicted in
Figure 1, BRET is based on the nonradiative transfer of energy
between a bioluminescent donor protein (e.g., firefly (Photinus
pyralis)[4]
or Renilla reniformis luciferase;[5]
for a review on bio-
chemical properties and use of bioluminescent proteins, see
ref. [6]) and a fluorescent acceptor protein, most often a var-
iant of GFP. A change in fluorescence/luminescence ratio
allows protein–protein interactions to be quantitatively ana-
lysed. RET efficiency is inversely proportional to the distance
between donor and acceptor molecules, varying with the sixth
power of the distance.[3]
Besides the orientation of the donor
and acceptor proteins towards each other, the occurrence of
BRET is dependent on molecular proximity (<10 nm), which
makes this homogenous assay suitable for monitoring molecu-
lar interactions with the aid of genetically encoded mono- or
bimolecular sensors.[7–9]
2. Protein–Protein Interaction Studies Based
on BRET
BRET was pioneered in 1999, being used to investigate the di-
merisation of cyanobacterial circadian clock proteins in bacteri-
al culture.[10]
The originally developed assay was based on Re-
nilla reniformis luciferase (Rluc, MW =35 kDa) as the energy
donor. Upon oxidation of its cell-permeable substrate coelen-
terazine h, this enzyme emits luminescent light at 480 nm,
with the GFP variant eYFP as the acceptor fluorophore (excita-
tion maximum at 513 nm, emission maximum at 527 nm). ACHTUNGTRENNUNGThis
first BRET assay (BRET1
) yielded a spectral separation of
~50 nm.[9]
Rluc, however, generates a broad emission peak,
substantially overlapping the GFP emission, which contributed
to a relatively low signal to background ratio. A new genera-
[a] Dr. A. Prinz, Dipl.-Biol. M. Diskar, Prof. Dr. F. W. Herberg
Kassel University, Department of Biochemistry
Heinrich-Plett-Strasse 40, 34132 Kassel (Germany)
Fax: (+49)561-804-4466
E-mail: herberg@uni-kassel.de
Figure 1. Schematic illustration of a BRET sensor based on the PKA holoen-
zyme. A) Rluc and GFP2
are fused to the R and C subACHTUNGTRENNUNGunits, respectively. After
addition of the luciferase substrate DeepBlueCB (DBC), BRET occurs between
the energy donor (Rluc) and the acceptor (GFP2
) provided that the two re-
porters are kept in close proximity (1–10 nm) by PKA–subACHTUNGTRENNUNGunit interaction.
B) Binding of two cAMP molecules (red circles) to each regulatory (R, black)
subACHTUNGTRENNUNGunit monomer causes a conformational rearrangement followed by the
release of the C subACHTUNGTRENNUNGunits and a subsequent decrease in BRET signal.
ChemBioChem 2006, 7, 1007 – 1012 C 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1007

2. tion BRET (BRET2
) was developed, utilising the proprietary sub-
strate coelenterazine DeepBlueCB (Perkin–Elmer) with an emis-
sion maximum between 390–400 nm. GFP2
, tailored to this
emission wavelength, re-emits light at 505–508 nm, which in
turn increased the spectral separation to about 100 nm.[9]
Fur-
thermore, the reporter protein genes in BRET2
were codon-op-
timised for expression in human cell lines. Commercially avail-
able vectors allow amino-terminal (N-terminal) and carboxy-ter-
minal (C-terminal) cloning of the interaction partners of inter-
est.[11,12]
Other genetically encoded BRET systems with different
donor/acceptor pairs (e.g., firefly luciferase/DsRed[13]
(from Dis-
cosoma sp.) and Vargula hilgendorfii luciferase/enhanced
yellow fluorescent protein (eYFP)[14]
) have been used sporadi-
cally.
The Rluc-based BRET1
/BRET2
systems have been applied to
characterise G protein-coupled receptor (GPCR) dimer and/or
oligomer formation, as well as regulation of GPCRs by arrestins
and G proteins (reviewed in refs. [8,9,15]; see Figure 2). Angers
et al.[16]
used BRET to demonstrate that human b2- adrenergic
receptors form constitutive homodimers in the plasma mem-
branes of HEK-293 cells. Upon treatment with the b-adrenergic
receptor agonist isoproterenol, the BRET signal increased, indi-
cating that the agonist interacts with receptor dimers at the
cell surface. Since then, quantitative BRET1
/BRET2
assays of con-
stitutive or agonist-promoted receptor–receptor interactions
have been extended to a multitude of GPCRs and other recep-
tors of the plasma membrane, such as melatonin recep-
tors,[17,18]
the thyrotropin-releasing hormone receptors,[19–21]
chemokine receptors,[22–25]
opioid receptors,[12,26,27]
the chole-
cystokinin receptor,[28]
insulin receptor,[29,30]
or the leptin recep-
tor.[31]
More recently, BRET was used to study the oligomerisation
of transcription factor complexes, belonging to the zinc-finger
protein family, in the nuclear compartment.[32]
Other applica-
tions of BRET cover specific protease activity, in which a se-
quence containing a consensus cleavage site for the protease
of interest is fused between the luminescent donor and GFP,
and protease activity results in a sharp reduction in the BRET
signal.[14,33]
A post-translational protein modification—ubiquiti-
nation—has also been investigated with the BRET technolo-
gy.[34]
3. Considerations for Developing a BRET-Based
Sensor
As noted in the first paragraph, RET efficiency is dependent on
the relative distance between the donor/acceptor molecules
(within 10 nm of each other) and a sufficient, but not too
large, spectral overlap of donor protein emission and acceptor
protein excitation. Also, the relative orientation of the donor
and acceptor dipoles with respect to each other is critical, so
donor and acceptor fluorophores usually require significant
freedom of movement to allow an orientation suitable for RET
to occur. In some cases, RET efficiency can be improved by in-
troducing linkers between the reporter proteins and the pro-
teins of interest. A circular permutation approach was success-
fully used to optimise the dynamic range of a “cameleon” con-
struct, sensing changes in intracellular Ca2+
.[35]
However, sever-
al FRET-based PKA type Ia sensors, varying in their linker
ACHTUNGTRENNUNGsequence, failed (M. Zaccolo, personal communication). Inter-
estingly, BRET with Renilla luciferase as the donor protein was
seemingly less influenced by perturbative effects than FRET
based on GFP donor proteins, as four out of six BRET combina-
tions in the PKA system yielded significant BRET, albeit with dif-
ferent efficiencies (see Figure 3).
BRET was shown to be particularly well suited to quantifying
resonance transfer because BRET—in contrast to FRET—does
not require optical excitation. As a consequence, all the light
emitted by GFP must be the result of the resonance energy
transfer; this eliminates background fluorescence, sample pho-
tobleaching, photoconversion of GFP and associated prob-
lems.[36]
A more elaborate comparison of BRET and FRET tech-
nologies has been published elsewhere and has therefore
been omitted here.[7,8]
It should be noted that steric hindrance by the rather large
reporter proteins fused to the proteins of interest could
hamper protein interaction. This problem could possibly be
overcome by using smaller reporter proteins (e.g., Gaussia
princeps luciferase,[37,38]
MW =20 kDa). Alternate labelling ap-
proaches include the tetracysteine-biarsenical system,[39,40]
fluo-
rescent nanoparticles[41,42]
or quantum dots.[43]
3.1. Techniques for detecting BRET
BRET was first measured by scanning spectroscopy.[10]
Higher
throughput can be achieved by using a plate reader capable
of detecting one wavelength window for the donor and for
the acceptor emission, respectively. Recently, De and Gambhir
Figure 2. Components of the GPCR–PKA signalling cascade. Ligand binding
to a G protein-coupled receptor (GPCR) activates a trimeric G protein, which
activates an adenylyl cyclase. Subsequently, cAMP is generated, and binds
to the regulatory subACHTUNGTRENNUNGunit dimer (black) of the inactive PKA type I or type II
holoACHTUNGTRENNUNGenzyme, causing the release of the catalytic subACHTUNGTRENNUNGunits (C, grey) phos-
phorylating targets in the cytosol and in the nucleus. Type II holoenzyme is
predominantly anchored to subcellular compartments via A-kinase anchor-
ing proteins (AKAPs).
1008 www.chembiochem.org C 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2006, 7, 1007 – 1012
F. W. Herberg et al.

3. demonstrated BRET in living mice. HEK-293 cells, transfected ex
vivo with a BRET2
sensor, were implanted and RET signals were
determined by means of a cooled charge-coupled device
(CCD) camera capable of sequential integration of filtered light
signals.[44]
Because of the low quantum yield of the luciferase/
coelenterazine system, microscopy of (single) cells is rarely
done with BRET.[18]
For this application, FRET has superior spa-
tial and temporal resolution. In combination with automated
liquid handling systems, however, BRET signal determination
can be performed in real time.[8,45,46]
The limiting factor of the
timespan of the assay is the short half-life of the coelentera-
zine substrate (25 min in aqueous solution). By employing
BRET1
, this problem can be overcome by using EnduRenB, a
cell-permeable coelenterazine h derivative (Promega Inc.), with
a half-life of about 24 h. It is metabolised to the free substrate
by intracellular esterases.[47]
These technical improvements
make BRET a suitable technology for medium–high-throughput
pharmacological compound screening and evaluation
assays.[48,49]
4. BRET and the Protein Kinase A Model
System
We have engineered BRET sensors downstream of the G pro-
tein-coupled receptor complex (Figure 2), using the PKA cata-
lytic and the regulatory subACHTUNGTRENNUNGunit as reporters to quantify
changes in intracellular cAMP concentration.[50]
PKA is a key
regulator of many metabolic processes and modulates various
intracellular signalling processes. Malfunction of the cAMP sig-
nalling cascade has implications in many diseases—such as
diabetes mellitus, cardiovascular disease, memory disorders
(e.g., Alzheimer’s disease) and certain cancers (e.g., Carney
complex)[51–53]
—so PKA is an interesting drug target, and this
interest has resulted in the development of several FRET-based
reporters.[54–56]
The novel BRET sensors were created to investi-
gate subACHTUNGTRENNUNGunit dynamics of the two major PKA isoforms (type Ia
and type IIa) side by side.[50]
Both BRET sensors respond to
changes in the intracellular cAMP concentration following the
dissociation of PKA holoenzyme, composed of two catalytic (C)
subACHTUNGTRENNUNGunits and a regulatory (R) subACHTUNGTRENNUNGunit dimer (Figure 1A). Each R
subACHTUNGTRENNUNGunit monomer subsequently binds two cAMP molecules,
followed by the release of the C subACHTUNGTRENNUNGunits[57]
(Figure 1B); this is
reflected in a decrease in the BRET signal.
4.1. Selection of BRET pairs based on PKA type Ia
To determine the most efficient type Ia BRET pair, several C-ter-
minal and N-terminal fusion proteins with Rluc and GFP2
were
generated and tested for BRET (Figure 3). The combination of
R-Rluc/GFP2
-C yielded the strongest BRET signals, which were
about two to three times the background level (here the
amount of luminescent light detected with the filter for GFP2
fluorescence, determined for each experiment by use of cells
expressing distinct Rluc and GFP2
proteins encoded by inde-
pendent expression plasmids (see also Figure 4)). In general, re-
porters fused to the C terminus of the human PKA RI
a subACHTUNGTRENNUNGunit
Figure 3. Selection of BRET2
pairs for monitoring of PKA type Ia holoenzyme
formation. Several N- and C-terminal fusions were tested to select the most
efficient BRET pair. The plasmids for expression of the PKA R and C subACHTUNGTRENNUNGunit
chimeras were constructed on the basis of the BRET2
system as described in
ref.[50]. BRET assays were performed as detailed in the legend to Figure 4,
with use of equal amounts (0.5 mg) of donor and acceptor DNA per micro-
plate well. BRET is given as the ratio between luminescence at 410 nm and
fluorescence at 515 nm (bg=background). Data are meansS.E.s of n=3–
10 inACHTUNGTRENNUNGdependent experiments.
Figure 4. Reproducibility of the BRET assay. A) To test signal stability of the
light emission of Rluc and GFP2
and the calculated BRET2
signals (B), the
BRET2
positive control vector [direct fusion of Rluc and GFP2
(Rluc GFP2
)],
as well as the combination of both vectors expressing either Rluc or GFP2
(Rluc+GFP2
), were tested. For the BRET assay, 2N104
COS-7 cells were seed-
ACHTUNGTRENNUNGed in each well of a 96-well microplate and cultivated at 378C, 7.5% CO2.
After 24 h, cells were transfected by lipofection with plasmid DNA (1 mg per
well) and transfection. Nontransfected cells are shown on the right-hand
side of plot A (COS-7 cells). BRET measurements were performed two days
after transfection by addition of DeepBlueCB luciferase substrate (5 mm) in
glucose containing phosphate-buffered saline (D-PBS). Light emitted by Rluc
(410 nm80 nm) and GFP2
(515 nm30 nm) was sequentially detected for
each well by use of a multi-label reader (Fusion a-FP, Perkin–Elmer) and is
given as counts per second (CPS). B) BRET signals for the BRET2
positive
ACHTUNGTRENNUNGcontrol vector (Rluc-GFP2
) as well as the individual reporters co-expressed
(Rluc+GFP2
; bg=background) were calculated from the data in A as fol-
lows: [emission515 nm (not transfected) COS-7 cells515 nm]/[emission410 nm (not
transfected) COS-7 cells410 nm]. Data shown are meansS.E.s, n=12.
ChemBioChem 2006, 7, 1007 – 1012 C 2006 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim www.chembiochem.org 1009
BRET for Biomolecular Interaction Studies

4. produced a higher BRET ratio and thus a better signal-to-noise
ratio than N-terminal fusion constructs.
4.2. Optimisation of assay conditions
The ratiometric nature of BRET signal determination suggests
that the sensor performance is independent of the concentra-
tion of the sensor inside the cell. This is supported by the fact
that enrichment of GFP-expressing cells by flow cytometry
prior to BRET analysis does not result in significantly different
BRET values.[58]
In GPCR expression studies, nonspecific BRET
was reported to occur only at high receptor concentrations
(above ~45 pmol per mg protein).[59]
Still, it is important to op-
timise the assay conditions with respect to the transfection
conditions to avoid nonspecific RET due to molecular crowd-
ing.[18,58]
Creating a stable cell line might be worthwhile for
cases in which a cell is difficult to transfect, a protein is cyto-
toxic at higher expression levels, or a high degree of standardi-
sation is needed. Once assay conditions have been established,
BRET provides a robust reporter assay with good reproducibili-
ty between individual experiments (Figure 4). Raw data as re-
ported in Figure 4A translate into a BRET ratio with high dy-
namic range when comparing directly fused reporter proteins
with the co-expressed, individual reporters (Figure 4B; see
figure legend for details). A 1:1 ratio of donor/acceptor DNA at
0.5 mg DNA per microplate well was found to result in maxi-
mum BRET signals for PKA type Ia BRET sensor (R-Rluc/GFP2
-C;
see Figure 5). The concentration of donor protein DNA was
kept constant at 0.5 mg, and the acceptor fluorophore DNA
was varied from 0.25 to 3 mg per well (Figure 5A). The experi-
ment was repeated the other way round, with variation of the
donor DNA concentration (Figure 5B). In the case of a specific
interaction, such titration curves should result in a saturation
curve.[59]
In our hands, both with the PKA type Ia (Figure 5A)
and with the type IIa sensor (not shown), the expression of
high levels either of GFP2
-C or of R-Rluc resulted in a reduction
in BRET signal, possibly due to a cytotoxic effect of DNA
(Figure 5). The protein expression levels for GFP2
-C and RI-Rluc
proteins were about two to three times higher than the en-
dogenous protein concentrations, as estimated by Western
blot analyses with use of specific antibodies raised against the
catalytic subACHTUNGTRENNUNGunit (Ca, PKA-anti-Cat, Santa Cruz Biotechnolo-
gy, Inc.) and the regulatory subACHTUNGTRENNUNGunit (hRI
b, Eurogentec) of PKA
(not shown).
4.3. Pharmacological compound characterisation
BRET assays result in robust signal-to-noise ratios for the PKA
sensors expressed in COS-7 cells. Addition of the cell-permea-
ble cAMP analogue 8-bromoadenosine-3’, 5’-cyclic monophos-
phate (8-Br-cAMP; 10 mm) results in a reduction of the BRET
signal of about 30% when the type Ia sensor is investigated in
intact cells. In contrast, the signal is reduced to background
values after cell lysis and subsequent incubation with 8-Br-
cAMP (1 mm; Figure 6), indicating complete holoenzyme disso-
ciation. Type I holoenzyme does not completely dissociate
even at maximally elevated cAMP levels, because of its high in-
Figure 5. Optimisation of plasmid DNA concentration. A) COS-7 cells were
transfected with a constant amount (0.5 mg) of GFP2
-C DNA (acceptor) in the
presence of increasing amounts of R-Rluc DNA (donor) as indicated in the
plot. A 1:1 ratio of donor/acceptor DNA resulted in the highest BRET values.
The titration is strongly dependent on the acceptor DNA concentration; the
reduction in BRET signal is probably due to cytotoxic effects of high DNA
concentrations. B) Complementary experiment with transfection of cells with
R-Rluc DNA (donor, 0.5 mg fixed) and increasing amounts of GFP2
-C (acACHTUNGTRENNUNGcep-
ACHTUNGTRENNUNGtor), yielding similar saturation effects as in A. Data are means (S.E.s) of
ACHTUNGTRENNUNGindividual experiments, each performed three times with n=4 (bg=back-
ground).
Figure 6. BRET works in intact cells and in cell lysate. The sensitivity of the
sensor towards agonist treatment was tested in COS-7 cells transiently ex-
pressing the sensor. Intact cells were treated with 8-Br-cAMP (10 mm) for
30 min or were lysed with Passive lysis bufferB (Promega) and subsequently
treated with 8-Br-cAMP (1 mm). Normalised data (meansS.E.s, n=6) are
shown after background subtraction.
1010 www.chembiochem.org C 2006 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim ChemBioChem 2006, 7, 1007 – 1012
F. W. Herberg et al.

5. tracellular concentration.[60]
However, once the holoenzyme
complex is diluted in the lysis buffer, it may dissociate com-
pletely, reflected in a decrease in the BRET signal to back-
ground level.
Intracellular cAMP concentrations can also be increased by
stimulation of the endogenous adenylyl cyclases’[61]
activity
with forskolin and by simultaneous inhibition of phosphodi-
ACHTUNGTRENNUNGesterases[62]
with 3-isobutyl-1-methylxanthine (IBMX). Figure 7
shows the decrease in BRET signal after forskolin treatment in
the presence of IBMX in a dose-dependent manner, demon-
strating that the sensor provides a sufficient dynamic range for
quantitative description of holoenzyme dissociation in re-
sponse to intracellular cAMP. The corresponding EC50 value of
about 2 mm is in good agreement with previous results ob-
tained in living cells.[63,64]
5. Future Perspectives
BRET is a powerful tool for quantitative monitoring of protein–
protein interactions in live cells and will probably be applied
to a multitude of candidate proteins in the near future. Besides
the characterisation of GPCR receptors located at plasma mem-
branes, proteins of the particulate and soluble compartment of
the cell are likely to be investigated.
For the first time, a PKA type Ia-specific RET-based biosensor
has been engineered, allowing comparative studies of type Ia
and IIa isoenzymes. The sensors are currently used to monitor
isoform-specific and membrane localisation-dependent[51]
regu-
lation of enzyme activity. The cAMP signal propagation can be
influenced by cellular compartmentalisation mediated by A-
kinase anchoring proteins (AKAPs). Through displacement of
the PKA type IIa from AKAPs with peptide-anchoring disrup-
tors, the holoenzyme becomes insensitive to a local, b-adrener-
gic receptor-mediated rise in intracellular cAMP concentra-
tion.[50,65]
BRET does not require an external light source and so it has
a superior signal-to-noise ratio; this enables its use for non-
ACHTUNGTRENNUNGinvasive monitoring of disease progression or targeted drug
action in animal models. In a proof-of-principle approach, BRET
from a Rluc/GFP-based sensor was detected in mice, albeit
only in superficial tissue, as described above.[44]
Luciferase pro-
teins with higher photon yields and luciferase substrates with
improved pharmacological properties are therefore needed for
protein–protein interaction assays in live animals (e.g., split-lu-
ciferase fragment complementation[66–68]
). To overcome the lim-
itations—due to light absorption, scattering and attenuation—
of blue/green light-emitting probes, novel donor/acceptor
pairs shifted to longer wavelengths are being developed. Im-
proved in vivo molecular imaging methods based on biolumi-
nescent[69]
and fluorescent[70,71]
proteins are thus evolving rap-
idly, generating valuable tools for the analysis of novel diag-
nostics and therapeutics, as well as in genomics and functional
proteomics.[68,72]
Acknowledgements
We would like to thank A. Erlbruch for the construction of several
BRET vectors, A. Wattrodt for technical assistance and Dr. Silke
Hutschenreiter for critical reading of the manuscript. The work
was supported by grants from the European Commission (EU-
RTD-QLK3-CT-2002-02149), the Deutsche Forschungsgemeinschaft
(DFG, He1818/4) and the German Ministry for Education and
ACHTUNGTRENNUNGResearch (BMBF 01GR0441) to F.W.H. M.D. is supported by the
Otto-Braun-Fonds of the University of Kassel.
Keywords: bioluminescence resonance energy transfer ·
biosensors · fluorescent probes · luminescence · protein
kinase A
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Received: February 2, 2006
Published online on June 6, 2006
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