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emission light; the emitted light is more isotropically distributed. Therefore, quan-
tification of the fluorescence anisotropy can be used to determine homo-FRET.
The homo-FRET method can be calibrated using reference measurements on
protein clusters of known size. Subsequently, EGFR cluster sizes can be determined
directly frommeasured fluorescence anisotropy values. As a tool for the referencemea-
surements, we have used FKBP-mGFP, which dimerizes via its ligand AP20187. Sim-
ilarly, 2xFKBP-mGFP can form oligomers by the addition of AP20187. Clusters made
with the FKBP constructs can therefore be either monomeric in the absence of their
ligand or dimers or oligomers in the presence of ligand. The level of anisotropy of
monomers, dimers, or oligomers can be used as a reference for cluster size measure-
ments. This homo-FRET method can be employed to study the clustering behavior of
EGFR for fundamental research and anticancer drug research. For example, it has been
shown that EGFR can also form inactive dimers in the absence of EGF; these predimers
are formed independently of the C-terminal tail. In contrast, EGF-dependent oligomer-
ization depends on tyrosine kinase activity and more particular on the nine tyrosine
residues in the C-terminal tail of the receptor (
Hofman et al., 2010
).
16.1
THEORY HOMO-FRET QUANTIFICATION
16.1.1
Steady-state fluorescence anisotropy imaging
In homo-FRET experiments, a linearly polarized excitation laser beam is used to ex-
cite the fluorophores. Next, the intensities of the parallel and perpendicular polarized
fluorescence are detected. Here, parallel and perpendicular are defined with respect
to the polarization direction of the excitation laser. From these intensities of the fluo-
rescence, the anisotropy can be calculated using Eq.
(16.1)
. Upon homo-FRET be-
tween fluorophores, the anisotropy decreases because the energy transfer between
two fluorophores results in emission by a molecule with a different orientation than
the molecule directly excited by the laser (
Fig. 16.1
)(
Bader et al., 2009; Lakowicz,
2006; Runnels & Scarlata, 1995
):
I
par
I
per
I
par
rt
ðÞ¼
(16.1)
2
I
per
Besides homo-FRET, other processes can affect the fluorescence anisotropy, such as
rotational diffusion of fluorophores. If the excited fluorophore changes its orientation
in the time period between excitation and emission, the anisotropy will be lowered.
However, in case of a large, slowly rotating molecule like a green fluorescent protein
(GFP), the depolarization due to rotations is negligible (
Bader, Hofman, van Bergen
en Henegouwen, & Gerritsen, 2007
). The rotation correlation time of fluorescent
proteins is typically in the order of 20 ns, while the fluorescence lifetime is usually
<
2.5 ns. The fluorophores in the sample can be separated into two groups, fluoro-
phores that are directly excited by the polarized excitation light and fluorophores that
are indirectly excited by the energy transfer involved in homo-FRET.
Runnels and
