Biology Reference
In-Depth Information
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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