Biology Reference
In-Depth Information
K
0
d
for calcium: 1.5
m
M) with around a twofold di
erence in 528/476 nm emission
ratios in calcium-free and calcium-saturating conditions. Recalling that the calci-
um-dependent signal from FIP-CB
sm
relied on binding of endogenous calmodulin,
an obvious concern would be that YCs would be perturbed by such interactions
and also perhaps themselves perturb downstream calcium-signaling pathways.
In fact, EC
50
s for YC2.1 and YC3.1 stimulation of calmodulin-dependent phos-
phodiesterase were two to three orders of magnitude greater than for calmodulin
and the sensors were unperturbed by addition of 3
m
M calmodulin. Of course, the
YC constructs will bu
V
er calcium inside cells. This was tested by studying the
calcium oscillations induced in HeLa cells induced by addition of histamine. At a
YC3.1 concentration of 150
m
M, calcium oscillations were evident whereas at
concentrations greater than 300
m
M, oscillations were not seen, though the overall
magnitude of the response was little altered. The loss of oscillations suggests
calcium bu
V
ering. Below around 20
m
M, the fluorescent signal was too faint to
give acceptable signal-to-noise ratios (
Miyawaki
et al.
, 1999
). Thus, working YC
concentrations in the range 40-150
m
M do not substantially perturb calcium-
dependent signaling mechanisms.
Yellow fluorescent proteins, besides being sensitive to pH, are more prone than
GFP to photobleaching and to quenching by biological anions such as chloride.
Because YFPs show such utility as one of the partners in the CFP/YFP FRET
couple, this defect is worth fixing. Mutagenesis by error-prone PCR and expression
in
Escherichia coli
uncovered a mutation to methionine in residue 69 that was much
more resistant to chloride quenching than EYFP-V68L/Q69K, twice as resistant to
photobleaching, with a p
K
a
of 5.7 rather than 6.1 and of comparable spectral
properties including brightness (
Griesbeck
et al.
, 2001
). This YFP is known as
citrine, and substituted for EYFP-V68L/Q69K as the FRET acceptor produced
the cameleons YC2.3 and YC3.3. These two cameleons express well at 37
C, show
a ratio change of around 1.5 to calcium over their dynamic range and are pH
insensitive down to around pH 6.5. To demonstrate the utility of YC3.3 in an
acidic compartment, it was targeted to the Golgi using an 81 residue N-terminal
construct from human galactosyl transferase type II. The sensor was saturated
when expressed in the Golgi, suggesting high resting levels of free calcium concen-
tration in this cellular compartment (
Griesbeck
et al.
, 2001
).
The CFP/citrine couple was also used in an ER-targeted sensor, Cameleon
D1ER. Here, the rationale was to design a sensor based on the M13/CaM-biding
pair that would be insensitive to interaction with endogenous calmodulin (
Palmer
et al.
, 2004
), as had been reported (
Hasan
et al.
, 2004; Heim and Griesbeck, 2004
).
The M13 and CaM were co-mutated to provide a binding pair that would not
interact strongly with endogenous calmodulin. Cameleon D1ER has a very wide
range of calcium sensitivity with
K
0
d
s of 0.81 and 60
m
M, appropriate for ER
calcium sensing, and was successfully used in HeLa cells to monitor cytoplasmic
and ER calcium simultaneously in conjuction with Fura2 (
Palmer
et al.
, 2004
).
The GFP family of proteins is remarkable in possessing a visible wavelength
fluorophore that is formed through an oxidation reaction involving adjacent
V
