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increased brightness than the conventional CFP (see Table 5.1 ). mCitrine
shows better tolerance to pH variations and quenching by chloride ions,
while Venus is characterized by a quicker maturation within cells.
Along these lines, a “sticky” FRET pair, known as CyPET (optimized
eCFP for FRET) and YPet (optimized YFP for FRET), has been optimized
from a cyan-yellow FRET pair by Daugherty's lab in 2005, using a quan-
titative evolutionary strategy. 132 The resulting FRET pairs showed a 20-fold
ratiometric FRET signal change instead of the 3-fold for the parental pair, in
a context where fluorophores were separated by a caspase 3 cleavable sub-
strate. While this FRET pair provides substantial improvement in sensitivity
and dynamic range for some biosensors, 132 the poor folding and expression
of CyPET at 37 C limits its usefulness in living cell studies. 31 Nevertheless,
YPet remains one of the brightest YFPs and displays superior FRET effi-
ciency when paired with a cyan donor fluorophore such as eCFP, alleviating
any ill effects stemming from CyPET. 127,133
Lately, circularly permuted (cp) fluorophores have appeared as a goodmeans
to enhance the dynamic range of biosensors. These cp fluorophores have
flourished in scientific reports as a solid solution for biosensor optimization. 41,71
Technically, they consist of fluorescent proteins in which the N- and C-termini
are fused together through a flexible linker. This protein engineering has allowed
the emergence of other types of biosensors resting solely on the fluctuation of
fluorescence properties upon binding of the analyte to a molecular switch
positioned in the linker region. 134 Note that such fluorophores are more
sensitive to their photochemical environment such as pH and temperature in
this configuration/protein structure. The first biosensor successfully improved
using this strategy is the Ca 2 þ indicator (yellow cameleon), exhibiting nearly
sixfold increase in dynamic range of FRET efficiency upon Ca 2 þ binding to
the biosensor. 135 Since then, cpFPs, in particular cpVenus, have been
implemented in other biosensors, including AKAR3, 41 AKAR4, 71 and
recently T Epac VV. 73 ThesimpleswappingoftheCFPfortheCeruleanas
donor between AKAR3 and AKAR4 in combination with the cpVenus
improves the dynamic range of the AKAR series. Indeed, AKAR4 shows a
slight average difference in fluorescence lifetime in control experiments when
compared to AKAR3 ( Fig. 5.21 ).
6.4.2 Green/red FRET pairs
Since 2004, many other variants of fluorescent proteins have emerged with
the discovery and characterization of a new red fluorescent protein (RFP)
named Discosoma sp . red fluorescent protein (DsRed) isolated from the coral
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