Biology Reference
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bound and unbound forms (1.5 vs. 1.8 when expressed as a ratio of emission
wavelengths) and a greater pH sensitivity. Mutagenesis can also be applied to the
calcium-binding a
nity of the calmodulin moiety: calmodulin has two classes of
calcium-binding sites and site-directed mutations in either high-(
K
0
d
70 nM) or low
(
K
0
d
11
m
M) a
Y
Y
Y
nity sites are
suppressed to give a monotonic binding curve (
K
0
d
4.4
m
M: cameleon-3) or low-
a
nity sites give rise to constructs in which high-a
nity sites are altered to give an enhanced range over four orders of magnitude of
calcium concentration (
K
0
d
s of 83 nM and 700
m
M: cameleon-4). The third dimen-
sion of modification adds signal tags to the constructs. Nuclear localization tags
gave cameleon-2nu and ER localization tags produced yellow cameleon-3er (
K
0
d
4.4
m
M) and cameleon-4er (
K
0
d
s of 83 nM and 700
m
M).
Tsien's seminal paper also exemplifies some challenges in the approach: on the
one hand, the complexities of permutation and combination of mutant variants
and their concomitant properties and on the other hand, the relatively low magni-
tude of FRET modulation by calcium over a very wide range of concentrations.
The subsequent proliferation of family members results from attempts to improve
brightness and dynamic range, but at the expense of adding to the combinatorial
complexity.
Persechini's second sensor design also concatenated GFPs, MLCK peptide, and
calmodulin, though in di
Y
erent order. A calmodulin whose EF hand calcium-
binding sites had been reversed in order (CN-CaM) was added to the FIP-CB
sm
C-terminal to BFP to make FIP-CA (
Persechini
et al.
, 1997
). This produced a
sensor with a monotonic FRET response and a
K
0
d
of 100 nM. Variants with lower
a
V
nities for calcium were obtained by mutating the MLCK calmodulin-binding
peptide sequence, rather than the calmodulin calcium-binding sites. As with FIP-
CB
sm
, calmodulin binding reduced FRET, the ratio (now expressed as
F
440
/
F
510
)
increasing approximately 1.7-fold over the calcium dynamic range. The interaction
was markedly pH sensitive in the range 6.5-7.5. This configuration of calmodulin
and calmodulin-binding peptide did not lead to later variants and appears to have
been an evolutionary dead end.
The cameleon family of calcium sensors is shown in
Fig. 1
.
Y
2. Evolution
The EYFP in yellow cameleon-2 and-3 shows an apparent p
K
a
of 6.9, so
contains a significant proportion of the protonated species at physiological pH
(
Miyawaki
et al.
, 1999
). The protonated species does not participate in FRET
(
Habuchi
et al.
, 2002
). As pH can vary by several tenths of a pH unit when cells are
stimulated; changes in pH would be read as changes in calcium ion concentration.
Two adjacent point mutations in EYFP (V68L and Q69k) lower the p
K
a
to 6.1,
markedly reducing the pH sensitivity in the physiological range (
Miyawaki
et al.
,
1999
). Replacing EYFP with EYFP-V68L/Q69K abolished pH sensitivity above
pH 6.9 (
Miyawaki
et al.
, 1999
). This substitution produces yellow cameleon-2.1
(YC2.1;
K
0
d
s for calcium: 100 nM and 4.3
m
M) and yellow cameleon-3.1 (YC3.1;
