Biology Reference
In-Depth Information
nity
K
0
d
of 40 nM, compared to the two
K
0
d
sof70nMand
11
m
M of cameleon-2 (
Miyawaki
et al.
, 1997
). YC4.6 has
K
0
d
sof58nMand
14.4
m
M, compared to
K
0
d
s of 83 nM and 700
m
M in cameleon-4. The YCX.60
series of cameleons show the rapid maturation and low pH and chloride sensitiv-
ities of their Venus forbears, YCX.12, and are the best performing native M13-
based cameleons to date.
Both citrine and the circularly permutated Venus (cpv) of the YCX.60 series were
used as alternative acceptors in a further series of cameleons based on Cameleon
D1ER (
Palmer
et al.
, 2006
). Computational design of novel M13 and CaM-based
binding pairs led to Cameleons D2, D3, and D4 and D2cpv, D3cpv, and D4cpv,
o
has a single high-a
Y
nities, good sensor dynamic range (the cpv series
comparable to YC3.60), and insensitivity to endogenous calmodulin. This cameleon
set showed good performance in reporting cytoplasmic and mitochondrial calcium
concentrations in HeLa cells and peri-plasmalemmal calcium concentrations in
hippocampal neurones when localized with the appropriate targeting sequences.
ECFP/EYFP-based cameleons require excitation at near-UV wavelengths.
It would be convenient to have FRET-based calcium sensors that can be excited
at visible wavelengths. One possibly solution is to use a FRET couple in which
GFP is paired with a red fluorescent protein. GFP-like red fluorescent proteins are
found in corals (
Baird
et al.
, 2000; Miyawaki
et al.
, 2003b
). However, they are less
tractable than GFP and its variants as they oligomerize, mature very slowly via a
green-emitting intermediate and in general, show low extinction coe
V
ering a wide range of calcium a
Y
cients and
quantum yield (
Miyawaki
et al.
, 2003b
). A GFP/RFP cameleon has been devel-
oped using a DsRed variant—a tandem dimer mutant (
Yang
et al.
, 2005
). The
maturation rate is tens of hours and the emission ration change is less than 1.2-fold
when cells expressing the sensor are challenged with ionomycin (
Yang
et al.
, 2005
).
Y
3. Changing the Sensor Mechanism 1
Solution NMR showed that the calmodulin-binding peptide of calmodulin-
dependent kinase kinase (CKKp) has a di
erent relation to the two lobes of
calmodulin than M13 peptide (
Truong
et al.
, 2001
). The structural modeling
suggested that the peptide might be concatenated in a recombinant construct
between
the N- and C-terminal lobes of calmodulin. Calculations suggested that
if ECFP and EYFP-V68L/Q69K were attached to the N- and C-terminals of the
split calmodulin, then the distance between the fluorophores when calcium was
bound and the calmodulin interacting with its binding peptide might be less than
40
˚
, rather than the 50-60
˚
in M13-based YC2.1. Given the sixth power depen-
dency of FRET on distance between fluorescent dipoles (
Jares-Erijman and Jovin,
2003
), this approach promised an improvement of the dynamic range of the ratio
of fluorescence emission. The splitting of the N- and C-domains of calmodulin in
this construct (termed YC6.1) led to a monotonic calcium-binding curve with a
K
0
d
of 110 nM, in some respects more suited to measurement of smaller changes in
intracellular free calcium concentration. While in the event, YC6.1 showed a more
V
