Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 329

is negligible. Cells expressing both CFP and YFP are then observed. Reliable
FRET indices are provided by calculation of 91

FRET c ¼ FRET a CFP b

YFP ð ¼ FRET CFP bt YFP bt Þ ½ 8 : 13

and

FRET c
CFP

¼ : ½ :

Emission ratio 8 14

Various methods introducing different observation strategies for FRET
indices and efficiency have been also reported. 92-94
When intermolecular FRET is utilized i n vitro , such arithmetic
processing is no longer necessary to obtain accurate FRET indices. For ex-
ample, if donor- and acceptor-labeled proteins are synthesized in bacteria or
insect cells or via in vitro translation and mixed together at a 1:1 ratio in a
solution in which the proteins can freely diffuse, accurate FRET indices
can be obtained by measuring the emission ratio because the invariable con-
centrations of donor and acceptor keep their bleed-throughs constant. In
addition, their dissociation results in a close-to-infinite distance between
them and guarantees the absence of FRET in the absence of interaction.
Thus, in vitro , intermolecular FRET systems generally produce larger re-
sponses than do intramolecular FRET systems, in which researchers must
use trial and error to optimize the relative orientations among the donor,
acceptor, and host proteins to gain a greater increase or decrease in FRET
in response to interactions or conformational changes (see next section).

5.2. Intramolecular (or unimolecular) FRET
While intermolecular FRET is useful for monitoring protein-protein inter-
actions, intramolecular FRET is well suited for the analysis of conforma-
tional changes. However, intramolecular FRET constructs have a great
advantage over intermolecular FRET in vivo , with respect to the stoichio-
metric issues described above. For example, expression levels of donor- or
acceptor-tagged proteins are influenced by the properties of the host pro-
teins, making it difficult to control relative expression levels of the donor
and the acceptor. In addition, exogenous proteins can bind to endogenous
partners, which dampens an increase in FRET in response to protein inter-
action. In contrast, intramolecular FRET constructs are fairly insensitive to
endogenous molecules and contain the same amounts of donor and acceptor