From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 159

proteins), segmental motions of the donor and acceptor tend to randomize
the orientations and

2

2/3 is classically used. 6,20

k ¼ Computational

2

simulations showed that k converges to 2/3 in a FRET sensor where

the D/A pair is presumed to be freely mobile. 21 Finally, the evaluation of
errors in the distance ( r ) due to approximation on k

2 has been reported,

but they are no more than 10%. 20

3.5. Energy transfer efficiency
The efficiency of energy transfer ( E ) can be defined as the ratio of the relaxation
rate due to energy transfer to the sum of all relaxation rates.

K T r ðÞ

E ¼ ½ 5 : 21

t 1

ðÞ

þ K T r

D

The rate of energy transfer is often defined as a function of inverse sixth
power of the distance between the two molecules.

R 0
R 0 þ

E ¼ ½ :

5 22

r 6

The first factor that affects the FRET signal is the distance ( r ) between the
fluorophores. The most sensitive range of r is 0.7-1.4 R 0 , corresponding to
90-10% FRET efficiency ( Fig. 5.9 ). R 0 usually ranges from 4 to 7 nm; hence
protein conformational change in this range is ideal for the largest FRET
dynamic in biosensors.
The transfer efficiency is typically measured using the relative fluorescence
intensity of the donor in the absence ( F D )orpresence( F DA ) of acceptor.

ð I DA t

ðÞ d t

F DA
F D ¼

ð I D t

E ¼ 1 1 ½ 5 : 23

ðÞ d t

where F DA is the intensity of donor fluorescence emission in presence of ac-
ceptor, F D is the intensity of donor fluorescence in absence of acceptor, and
I D and I DA are, respectively, the intensity decays of the donor alone and the
donor in the presence of the acceptor.
The transfer efficiency can also be calculated from the lifetimes under
these respective conditions ( t D and t DA ):