Biology Reference
In-Depth Information
dynamic range below pH 6.8 and little e
V
ect in the physiological range pH 6.8-7.3.
Calcium o
rates were similar to or slightly faster than that of YC2.3 (
Heim and
Griesbeck, 2004
). The TN-L15 sensor was targeted to the plasma membrane using
GAP43, Ras, or Synaptobrevin. In direct comparison with YC2.1 and YC 3.3, it
showed markedly greater sensitivity and no diminution of dynamic range.
Mutations to EF hands III and IV and substitution of citrine with a circularly
permutated variant, Citrine-cp174 produced a TnC-based sensor that showed no
magnesium dependence, a fourfold dynamic range and a
K
0
d
of 2.5
m
M—TN-XL.
TN-XL has a very fast o
V
rate with a dominant component with a time constant of
142 ms (
Mank
et al.
,2006
). Expressed in
Drosophila
under a UAS/Gal4 neuronal
promoter, it showed response times to calcium signals at the neuromuscular junction
significantly faster than other sensors—YC2.0, YC3.3, Inverse Pericam, G-CaMP1.3,
andG-CaMP1.6. Further mutagenesis and rearrangement of the TnC domain gave a
higher a
V
nity variant, modestly named TN-XXL, that was capable of long-term
monitoring of individual neuronal responses in flies and mice (
Mank
et al.
,2008
).
Y
B. Camgaroos
1. Circular Permutation of EYFP
Remarkably perhaps, the beta-can that surrounds the cyclized and oxidized
fluorophore is amenable to circular permutation, by which is meant the insertion
of a peptide linker between N- and C-terminals of the protein and the creation of a
newN- and C-terminal pair elsewhere in the sequence, in the loops that connect the
component beta-sheets and in the beta sheets themselves (
Baird
et al.
, 1999
). As we
have seen, circular permutation of Venus led to YC2.60 and YC3.60, the two
cameleons with the largest emission ratio dynamic range (
Nagai
et al.
, 2004
). The
discovery that N- and C-terminals of EYFP could be rearranged prompted the
discovery that a calcium sensor could be fashioned by insertion of calmodulin
within EYFP itself.
Xenopus
calmodulin was inserted between residues 144 and 146
of each of ECFP, EYFP, and EGFP. Each of these constructs was a calcium
sensor, with the EYFP insertion giving the largest calcium response. In calcium-
free conditions, the construct absorbs predominantly at 400 nm, while in calcium-
saturating conditions, the dominant absorption peak is at 490 nm. The 400-nm
absorption is due to the protonated form of EYFP and the 490-nm absorption to
the unprotonated form. As discussed in
Section II.A.2
, in EYFP, the protonated
species is not fluorescent (
Habuchi
et al.
, 2002
), so the excitation spectrum shows a
single peak at 490 nm and both the excitation and emission spectra are strongly
dependent on calcium concentration, with around an eightfold increase in emission
intensity at saturating calcium concentrations. Calcium binding was monotonic
with an apparent dissociation constant of 7
m
M. Calcium binding clearly shifts the
proportion of protonated and unprotonated forms at constant pH, so the p
K
a
's for
the two forms are di
erent: 10.1 and 8.9, respectively. Continuing the whimsical
tradition, this calcium sensor is termed Camgaroo-1, because it is yellowish, carries
V
