From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 163

different colors, 29 enabling scientists to consider dynamic localization of
several proteins of interest in living cells. 1
The properties of these FPs reveal that they are excellent candidates for
FRET-based biological applications (see Table 5.1 ). The choice of a particular
FP as a donor or an acceptor is very important and is mainly based on the analysis
of its respective excitation and emission spectra. FPs must meet certain criteria to
form a FRET pair: (i) an effective overlap between the emission spectrum of the
fluorophore donor and the excitation spectrum of the acceptor; (ii) a large ex-
tinction coefficient at the region of excitation; (iii) a high quantum yield (ratio
of photons emitted/photons absorbed); (iv) a separation between the excitation
and emission spectra of the donor andof the acceptor; (v) good photostability; (vi)
high brightness; (vii) minimal perturbation to the environment by FPs (toxicity);
(viii) minimum sensitivity to the cellular environment (pH, chloride); and (ix)
inability or at least limited capacity to dimerize and/or oligomerize. The last re-
quirement is very important because the use of oligomerizing FPs may compro-
mise the interpretation of the FRET signal. In 2006, Dunn et al. 43 elegantly
demonstrated that the use of monomeric FPs significantly increased the FRET
efficiency of aKAR. However, the FPs' expression levelmust be sufficiently high
to provide enough signal, but not too high, it should become cytotoxic. In con-
trast, some tetrameric FPs can be toxic to bacteria when produced in large quan-
tities, but this is not the case withmonomeric FPs. All these requirements need to
be considered and therefore a compromise in the choice of a particular FP in a
specific context is suggested. Fortunately, FPs are continually being subjected to
molecular engineering to improve their intrinsic properties and to increase
the number of variants. 44,45 Generally, for biosensing approaches, FRET
can be evaluated by ratiometric methods by measuring the fluorescence
emitted by the acceptor in response to the excitation of the donor
fluorophore. However, when considering protein interactions studies by
FRET measurement, FLIM is a preferred method. Thus, other approaches
have emerged in recent years with fluorescent protein engineering that rely
on donor FLIM for biosensing studies.
4. FRET MEASUREMENTS: METHODS AND
INSTRUMENTATION
As previously described, FRET induces modification of several prop-
erties of the emitted fluorescence. Different techniques thus arise from these
modified measurements, 46,47 such as monitoring the fluorescence emission