Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 324

leucine zippers 74 and subsequently utilized in mammalian cells to investigate
interactions between basic leucine zipper (bZIP) and the nuclear factor-

B
(NF- k B)/Rel family of transcription factors under physiological conditions. 66
Since then, BiFC has been successfully used in various model organisms,
including mammalian cell lines, plants, and microorganisms. 75-77

k

4.1. Advantages of BiFC
As described above, one important advantage of BiFC is the intrinsic fluo-
rescence of the formed complex, which makes it possible to visualize
protein-protein interactions without other reagents that are required in
complementation methods using other protein fragments. In addition, mul-
tiple protein interactions can be simultaneously observed in a single cell
using multicolor BiFC analysis. 78 In this approach, different interaction part-
ners are fused to fragments of each different fluorescent protein, the binding
of which therefore produces fluorescent complexes with distinct colors. In
addition, thanks to the structural similarities among fluorescent proteins,
some fluorescent protein fragments can associate with other different com-
plementary fragments and produce BiFC complexes with distinct spectra
( Fig. 8.2B ). 78 This method allows visualization of competition for molecular
binding among alternative interaction partners, 79 wherein the amino termi-
nal fragments determine the spectra emitted by the formed fluorescent com-
plexes. It is worth repeating that the interrupted positions for circular
permutation are located in the carboxyl terminal halves of fluorescent pro-
teins (see Section 2.3 ); therefore, the chromophores are present in the amino
terminal fragments ( Fig. 8.2B ).

4.2. Disadvantages of BiFC
The intensity of fluorescence emanating from resulting BiFC complexes does
not correspond to the kinetics of interactions between the fused proteins.
First, the time required both for fluorescent protein fragment association
and the chemical reactions that produce and mature the chromophore results
in a delay between the beginning of the interaction and the beginning of fluo-
rescence emission. 66,72 The extent of the delay is likely to depend on the
kinetics of chromophore formation; therefore, BiFC complex fluorescence
can often be detected much faster than expected (within minutes after
fusion protein interaction) when quickly maturing fluorescent proteins are
used. For instance, rapamycin-induced binding between FK506 binding
protein (FKBP) and the FKBP-rapamycin binding (FRB) domain of