Biology Reference
In-Depth Information
amino acids (
Tsien, 1998
). Fluorescence develops relatively slowly when the pro-
tein is expressed in cells, the process of what is known as maturation taking tens of
minutes to hours; maturation is also temperature dependent, oxidation to form the
fluorophore being the rate-limiting step. Another potential di
Y
culty with FRET-
based probes using the CFP/YFP partners is that maturation of YFP is substan-
tially slower than that of CFP, particularly at mammalian body temperatures
(
Miyawaki
et al.
, 1999
), a very important consideration especially for expression
in transgenic mammals. If the YFP partner of the FRET couple matures more
slowly than the CFP partner, then the sensors dynamic range is compromised, as
mature CFP in a sensor that contains immature YFP will contribute to the 476 nm
emission in the absence of 528 nm emission from the same construct, so that the
overall population 528/476 emission ratio will be depressed as a function of the
proportion of disparately matured sensor constructs (as illustrated by the behavior
of YC6.1 discussed below;
Evanko and Haydon, 2005
). The F46L mutation in
YFP greatly accelerates oxidation to the mature fluorophore and four additional
point mutations contributed to create a construct that matured two orders of
magnitude faster than EYFP from a urea-denatured state (
Nagai
et al.
, 2002;
Rekas
et al.
, 2002
); because of its resulting brightness, this YFP construct was
given the name Venus. Venus also has a low p
K
a
(6.0) and low sensitivity to
chloride, comparable to citrine in these respects (
Griesbeck
et al.
, 2001
), though
it lacks citrine's improved resistance to photobleaching. Substitution of Venus for
EYFP-V68L/Q69K resulted in a new rapidly maturing yellow cameleon (YC2.12).
Bright YC2.12 fluorescence was seen to develop rapidly after ballistic transfection
of Purkinje cells in cerebellar slices, though the fold ratio change after depolariza-
tion suggests that its dynamic range was not much altered from earlier family
members (
Nagai
et al.
, 2002
).
The challenge of improving dynamic range was addressed systematically by
altering the orientation of the YFP fluorescence dipole relative to the CFP dipole
(
Jares-Erijman and Jovin, 2003
) to maximize FRET (
Nagai
et al.
, 2004
). Changes
in orientation were achieved by circular permutation (see below,
Section II.B.1
)of
the Venus construct. The YC3.12-based construct with EYFP-V68L/Q69K sub-
stituted by circularly permutated Venus with a new N-terminal at Asp-173 (termed
YC3.60) showed the largest increase in fluorescence emission ratio dynamic range
between calcium free and calcium-bound forms
in vitro
: around 6.6-fold compared
to 2.1-fold for YC3.12. This large improvement in dynamic range was verified by
expression of each the two sensors in HeLa cells and challenge with ATP to raise
cytoplasmic free calcium levels (
Nagai
et al.
, 2004
). This study also illustrates the
important point that altering the properties and conformation of the FRET
partners at the N- and C-terminals of the sensor can also alter the apparent calcium
activation characteristics of the calmodulin-M13 inner pair as measured by FRET.
YC3.60 showed a monotonic increase with calcium concentration, as would be
expected from a construct based on the monotonically increasing cameleon-3
(
Miyawaki
et al.
, 1997
), but the apparent dissociation constant for YC3.60 is
0.25
m
M, compared to 4.4
m
M for cameleon-3. YC2.60, based on cameleon-2,
