Biology Reference
In-Depth Information
FKBP-mGFP
2xFKBP-mGFP
Pentamer
Tetramer
Trimer
Dimer
Dimer
Monomer
Monomer
AP20187
-
+
-
+
FIGURE 16.2
Native PAGE analysis of dimerization constructs. Cells expressing FKBP-mGFP or 2xFKBP-
mGFP, either incubated with AP20187 or mock-treated, were lysed under nonreducing
and nondenaturing conditions. After size separation on a native PAGE gel and blotting to
PVDF membrane, proteins were detected with anti-GFP antibodies.
Adapted from
Bader et al. (2009)
.
r
mono
1
N
r
ss
¼
(16.4)
However, our experimental work indicated that the assumptions mentioned earlier
are not correct for our applications. Often, the energy transfer efficiency is not high
enough to ignore and the anisotropy after energy transfer is not equal or close to zero.
Therefore, Eq.
(16.3) and (16.4)
are not generally applicable. Determination of
r
et
is
not trivial; therefore, we opted for using reference measurements to relate anisotropy
with cluster sizes. This also takes into account effects due to an energy transfer that is
not equal or close to 100%. We employ reference constructs to produce clusters of
known size. To this end, FKBP-mGFP and 2xFKBP-mGFP constructs were used;
they form monomers in the absence of AP20187 but dimers and oligomers, respec-
tively, when AP20187 is added (
Fig. 16.2
).
Relative steady-state depolarization values due to clustering,
r
/
r
mono
, of the
reference constructs were determined (
Fig. 16.3
)(
Bader et al., 2009
). The value
of
r
/
r
mono
for dimers was 0.86 and for oligomers 0.78. Using these reference values,
steady-state anisotropy can be converted into an average cluster size on a scale of
N
¼
1,
N
¼
2, and
N
3.
16.1.2
Time-resolved fluorescence anisotropy imaging
In time-resolved fluorescence anisotropy (imaging), the anisotropy decay is
measured. This method provides more detailed information about the homo-FRET
process than steady-state anisotropy measurements. For large molecules or proteins,
the influence of rotation can be neglected and the decay due to homo-FRET can be
described by Eq.
(16.5)
(
Gautier et al., 2001; Tanaka & Mataga, 1979
):
