Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 325

mTOR becomes detectable by BiFC complex formation of YFP fragments
within 10 min after rapamycin treatment. 80 Given that the signal
continuously increases for at least 8 h, the rapidity of detection does not
necessarily indicate a short half-time of fluorescent complex formation.
Using fragments of Venus, a bright mutant of YFP with fast maturation,
we have been able to monitor growth factor-induced Ras activation
(which generally peaks at approximately 10 min), although this activation
is slowed relative to activation of endogenous Ras. 81 Therefore, this rapid
response might be due partially to the emergence of weak fluorescence
signals emanating from a small fraction of associated fragments of a
fluorescent protein followed by rapid chromophore maturation. Of
course, detection of weak signals can be achieved by combining
highly sensitive fluorescence detection with minimal background in both
imaging systems and objects of observation. However, in general, BiFC
analysis enables rapid detection of complex formation but does not allow
instantaneous visualization of complexes or analysis of the kinetics of
complex formation in real time.
Another limitation of BiFC is that it cannot directly identify novel inter-
acting partners, because fluorescence complementation requires that the
proteins be tagged with a fragment. The relative efficiencies of BiFC com-
plex formation by different combinations of fluorescent protein fragments
do not reflect the equilibriums among alternative interactions. However,
they do reflect the relative efficiencies of complex formation among
alternative interaction partners at the time of fusion protein synthesis.

5. FÖRSTER RESONANCE ENERGY TRANSFER

The abbreviation “FRET” might be better known than its expansion
“F ¨ rster (or fluorescence) resonance energy transfer.” FRET, first theorized
in 1948, 82 is a phenomenon of radiationless excitation energy transfer from a
donor chromophore to an acceptor chromophore. 83 FRET can be observed
only when the donor and acceptor are very close to each other ( 10 nm;

Fig. 8.3A ) and when the following additional conditions are applied: (1) sub-
stantial overlap between donor emission and acceptor excitation spectra
( Fig. 8.3B ) and (2) optimal relative orientation between the donor and the
acceptor ( Fig. 8.3C ). The rate of energy transfer k T (s 1 ) is therefore given by

2

J k l D

n 4 r 6

10 23

k T ¼ 8 : 71 : ½ 8 : 9