Biology Reference
In-Depth Information
Emmision of
mGFP after energy
transfer
Excitation with
polarized light
Emmision of directly
excited mGFP
par
par
per
per
mGFP
Homo-FRET
FIGURE 16.1
Schematic representation of homo-FRET in a dimer of monomeric GFP (mGFP), causing
depolarization of the polarized excitation light.
Scarlata (1995)
introduced a simple model to calculate cluster sizes from the steady-
state anisotropy (
r
ss
) (Eq.
16.2
):
1
þ ot
r
et
N
ð
1
Þot
r
ss
¼
r
mono
þ Not
þ
(16.2)
1
1
þ Not
Here,
r
mono
is the anisotropy of monomers,
r
et
is the anisotropy after energy transfer,
N
is the number of molecules in the cluster, and
o
is defined as
6
o ¼
ð
R
0
=
R
Þ
=t
where
R
0
is the F¨rster distance,
R
is the distance between two fluorophore, and
t
the
fluorescence lifetime. The homo-FRET rate
can be determined from time-resolved
anisotropy experiments. In steady-state anisotropy experiments, the rate is either es-
timated or assumed to be much faster than the rate of fluorescence. In the latter case,
ot!1
o
(under these conditions
E
¼
1) and Eq.
(16.2)
reduces to Eq.
(16.3)
:
r
mono
1
r
et
N
1
r
ss
¼
N
þ
(16.3)
N
In the original work by Runnels and Scarlatta, they presented an example where an-
isotropy after a single energy transfer step is about zero. This further simplifies
Eq.
(16.2)
to
