Principles of Fluorescence Spectroscopy

458 ENERGY TRANSFER
Figure 13.21. RET indicator for estrogens using the ligand-binding domain of estrogen receptor. The color scale shows the intensity at 480 nm divided
by the intensity at 535 nm. Revised and reprinted with permission from [45]. Copyright © 2004, American Chemical Society.
other mutants there was almost no RET. By examination of
the six mutant proteins the orientation of MBP on the surface
of the a QD could be determined (Figure 13.20, lower
panel). The center of the QD is shown as a pink dot, but the
QD itself is not shown.
13.5. RET SENSORS
Resonance energy transfer has been used to develop a number
of sensors. The use of donor-to-acceptor intensity ratios
is valuable because the measurements become mostly independent
of the overall intensity. This independence is especially
important in fluorescence microscopy, where it is
usually not possible to know or control the local fluorophore
concentration.
13.5.1. Intracellular RET Indicator for Estrogens
RET has been used with green fluorescent proteins (GFPs)
and their variants to develop sensors for a variety of analytes.
One example is an intracellular indicator for estrogens.
45 In this case cyan (CFP) and yellow (YFP) fluorescent
proteins form the RET pair (Figure 13.21). These pro-
teins are linked by a peptide containing both the ligandbinding
domain of estrogen receptor α (ER) and an estrogen-dependent
ER-interaction site. The latter domain is
comprised of the sequence LXXLL, where L is leucine and
X is any other amino acid. Upon binding the ER against 17β
estradiol, the ER ligand-binding domain becomes competent
to interact with the LXXLL motif. The resulting conformational
change brings the tethered CFP and YFP moieties
into close proximity, thus increasing the efficiency of
energy transfer.
An advantage of using a GFP and its mutants is the
potential to express the indicator in the cell rather than
injecting or loading the indicator into the cell. A plasmid
expression vector encoding the RET indicator was transfected
into CHO cells, which then synthesized the indicator.
The labeled cells were exposed to 17-βE and imaged at the
emission wavelength of CFP (480 nm) and YFP (535 nm).
The ratio of intensities was used to create a false color
image of the cells (Figure 13.21). After exposure to 17-βE
the intensity at 535 nm increased, while the intensity at 480
nm decreased. This approach allows cell-based assays for
estrogen potency or the blocking effects of estrogen antagonists.