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sensors ( Nagai et al. , 2001 ). Circular permutation of EYFP-V68L/Q69K to give an
EYFP with Y145 as the N-terminal and N144 as the C-terminal (cpEYFP)
produced an EYFP variant that could be concatenated with M13 and calmodulin
(bearing the E104Q mutation that conferred a monophasic calcium-binding curve;
Miyawaki et al. , 1997 ). The construct with calmodulin at the N-terminal (CaM-
cpEYFP-M13) showed no calcium-dependent properties, confirming the finding
reported for a cpGFP variant ( Nakai et al. ,2001 ), but the opposite concatenation
(M13-cpEYFP-CaM) gave a construct that showed threefold brighter 520 nm
fluorescence in high calcium media compared to calcium-free media when excited
at 485 nm. This construct was given the name pericam (from a circularly permuted
YFP and CaM —calmodulin). Pericam was the prototype from which three peri-
cams with enhanced features were developed. Flash pericam has three additional
point mutations that confer an eightfold increase in 520-nm fluorescence on
calcium binding. Flash Pericam is a single wavelength, nonratiometric indicator
with a K 0 d of 0.7 m M . Knowing that substitution of phenylalanine at residue 203 in
YFP conferred fluorescence on the protonated form, this mutation was introduced
into Flash Pericam. The result, Ratiometric Pericam, was a sensor whose emission
ratio at 520 nm when excited at 494 nm or 415 nm changes 10-fold between
calcium-free and calcium-saturating conditions with a K 0 d of 1.7 m M; this excita-
tion ratio sensor is functionally analogous to fura-2 ( Grinkiewicz et al ., 1985 ).
Further semirandommutagenesis of Ratiometric Pericam gave a single wavelength
construct whose fluorescence intensity at 513-515 nm decreased on calcium bind-
ing—Inverse Pericam ( K 0 d ; 0.2 m M). Two advantages of Inverse Pericam are that it
is bright and has excitation/emission characteristics similar to fluorescein; the latter
advantage it shares with Flash Pericam: these two YFP-based indicators are
functionally equivalent to the Fluo-3 and Fluo-4 single wavelength calcium sensors
( Gee et al ., 2000; Kao et al ., 1989; Minta et al. , 1989 ). Expression in HeLa cells
showed that Ratiometric Pericam and Inverse Pericam expressed significantly
better at 37 C than did Flash Pericam. Ratiometric Pericam gave a 2.5-fold
increase in excitation ratio emission after addition of histamine, while Flash and
Inverse Pericams o
100% increase and decrease in signal, respectively, with
the same agonist. As might be expected from our earlier discussion of the camgar-
oos, the calcium-free and calcium-bound forms of all three pericams showed
di
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er a
erent p K a 's and all three have pH sensitive emissions in the physiological pH
range. Miyawaki showed a proof of principle that the excitation ratio-based
Ratiometric Pericam can be used in the context of confocal imaging ( Shimozono
et al. , 2002 ); recent confocal microscopes based on acousto-optical filters o
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er
turnkey solutions to excitation ratiometric imaging.
2. GCaMPs
Single wavelength nonratiometric sensors that use the same sensor strategy
as pericams but are based on circularly permutated GFP rather than EYFP
were developed at almost the same time as the pericams, their development
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