Biology Reference
In-Depth Information
become a powerful method to assess such interactions at the molecular level. The
method is based on the principle of F¨rster resonance energy transfer, which postu-
lates that the efficacy of energy transfer between a donor and an acceptor is inversely
proportional to the sixth power of the distance between them.
When studying receptor-receptor interactions, one protomer is fused to the donor
(Rluc) and the other protomer to the acceptor (fluorescent protein). If the two fusion
protomers interact and the distance between the energy donor and acceptor is
<
10 nm, a resonance energy transfer occurs and the emission signal from the accep-
tor protein can be detected. However, the energy transfer process not only depends on
the distance between donor and acceptor but also relies on the overlap of the emission
spectrum of the donor with the excitation spectrum of the acceptor and the relative
spatial orientation of donor and acceptor. Therefore, the absence of BRET signal be-
tween two receptors does not necessarily mean that these receptors do not interact
with each other. Thus, all such factors must be taken into account. On the other hand,
increasing the acceptor/donor ratio may lead to a detectable but nonspecific BRET
signal. This can be illustrated in BRET saturation assays, where a fixed amount of
donor is coexpressed with increasing amount of the acceptor. Nonspecific BRET sig-
nals tend to increase linearly with increasing acceptor concentrations. In contrast, a
progressive increase of the BRET signal in a hyperbolic manner represents the com-
plete saturation of all donors with acceptor molecules and a specific BRET signal
(
Marullo & Bouvier, 2007; Pfleger & Eidne, 2006
).
Over the last years, different generations of BRET (BRET
1
, BRET
2
, eBRET
2
,
BRET
3
, and QD-BRET) have been developed, depending on the type of enzyme sub-
strate and the nature of donor/acceptor pairs (
Kocan, See, Seeber, Eidne, & Pfleger,
2008; Pfleger et al., 2006; Xing, So, Koh, Sinclair, & Rao, 2008
). As a result, the
nomenclature for describing each of the BRET forms has not followed a unique rig-
orous pattern. The original BRET method using coelenterazine h (benzyl-
coelenterazine) as substrate is nowadays called BRET
1
(Marullo & Bouvier, 2007).
In BRET
1
, the maximal emission of Rluc is observed at 480 nm, a wavelength that
is appropriate for excitation of a yellow fluorescent protein (enhanced YFP: EYFP),
which subsequently reemits light at 530 nm. Several other variants of the YFP with
identical excitatory properties are YFP topaz, YFP citrine, YFP venus, and YPet
(
Bacart, Corbel, Jockers, Bach, & Couturier, 2008
). BRET
1
is characterized by
strong signals and long lifetime, making it one of the most suitable approaches
for BRET saturation assays. Changes in the Rluc substrate used resulted in the second
generation of BRET methods, the BRET
2
.IntheBRET
2
, the bisdeoxycoelenterazine
(DeepBlueC
TM
) or didehydrocoelenterazine (coelenterazine-400a) is used as Rluc
substrate, resulting in different donor emission spectra that shift the maximal light
emission of Rluc to 395 nm. Appropriate acceptors are GFP2 and the GFP10 with
excitation and emission maxima of 400 and 510 nm, respectively. BRET
2
in compar-
ison to BRET
1
has an improved separation of donor and acceptor emission peaks.
This makes this form a better choice for screening assays where high signal-to-noise
ratios are required. However, a clear limitation of BRET
2
is the low light emission
(DeepBlueC
TM
as a substrate leads to up to 300 times less light emission) and the
