Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 327

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relative orientation of the two chromophores ( is assumed to be equal to

2/3), the efficiency of FRET ( E ) can be given by

k

R 0 6
R 0 6

E ¼

r 6 ;

½ 8 : 10

þ

where the F¨rster distance R 0 is the distance between chromophores that pro-
vides the half-maximal FRET efficiency ( E /2) ( Fig. 8.3A ). Initially, this tech-
nique was used by a limited number of biologists; with the explosion of
fluorescent protein-based techniques since the cloning of GFP in 1992 2,85
and the discovery of GFP-like proteins from a variety of animals, 6,10,13,86,87
use of FRET has increased. In most GFP-based FRET experiments, CFP
and YFP are used as the donor and the acceptor, respectively.

5.1. Intermolecular (or bimolecular) FRET
As described above, the GFP chromophore is fixed in the -barreled struc-

ture, and the mobility of fluorescent proteins is restricted because of fusion to
partner proteins. Hence, the efficiency of GFP-based FRET reporters
largely depends on the orientation factor because of the time of rotation
of such a large structure, which is longer than the excited-state lifetime
(2-5 ns). Thus, GFP-based FRET cannot be a simple spectroscopic molec-
ular ruler, and the efficiency of FRET must be determined by alternative
methods. The FRET efficiency is also defined as the probability of energy
transfer per donor excitation event. E can therefore be obtained by measur-
ing the intensity of donor emission in the absence ( F d ) and presence ( F d 0 )of
an acceptor 88,89 :

b

F d 0
F d :

E ¼ 1 ½ 8 : 11

Although the actual measurements are far more complex, for intensity-
based measurements of intermolecular FRET, in which the donor and ac-
ceptor are not linked covalently, their dissociations guarantee disappearance
of FRET. In this case, the fluorescence intensity of the donor is measured to
obtain FRET efficiency. A donor fluorescent protein (CFP) and an acceptor
fluorescent protein (YFP) are fused to host proteins A and B, respectively,
which can associate with each other ( Fig. 8.3D ). By exciting at 440 nm to a
certain extent, F d 0 can be obtained (here, it is assumed to be 50). Next, YFP
is specifically photobleached by a strong excitation at 510 nm, and F d (100) is
measured with the same amount (as that for obtaining F d 0 ) of excitation at
440 nm. Here, E is given by