Biology Reference
In-Depth Information
modest fold emission ratio change than predicted (2.1 vs. 1.4 for YC2.1 in parallel
experiments), the twofold change was expressed over a narrower range of calcium
concentrations (0.05-1
m
M) in the physiologically relevant cytoplasmic range.
YC6.1 of course su
V
ers from the pH and chloride sensitivity and the slow
maturation of its EYFP-V68L/Q69K fluorophore that we discussed above. Repla-
cing EYFP-V68L/Q69K with Venus (
Evanko and Haydon, 2005
) gives the sensor
VC6.1 (Venus cameleon 6.1: the nomenclature is confusing and unhelpful, given
that the Venus CaM-M13 cameleons are known as YC2.12 and YC3.12). VC6.1
shows a emission ratio change of around 2.1-fold between zero and saturating
calcium concentrations. Thus, as with substitution with Venus for EYFP-V68L/
Q69K to produce YC2.12 from YC2.1, dynamic range is not much altered, while
improvements in maturation and pH and chloride sensitivity are obtained. It
would be logical to develop a YC6 sensor that contains the circularly permutated
Venus used in YC2.6 and YC3.6 (
Nagai
et al.
, 2004
); this would be predicted to
much improve the ratio dynamic range.
Small improvements in dynamic range for YC6.1 and VC6.1 can be obtained by
excluding from analysis cells that express a low resting YFP/CFP ratio (
Evanko
and Haydon, 2005
): the authors very reasonably suggest that this screens out cells
in which the YFP partner is less-mature relative to its CFP pair.
4. Changing the Sensor Mechanism 2
One potential disadvantage of calmodulin-based sensors is that calmodulin is a
near-ubiquitous protein with many binding partners. It is possible that calmodulin-
based sensors may su
er interference from binding partners when expressed in the
cytoplasm or other cellular compartments. While there is no direct evidence to
support this conjecture, it is nonetheless true that performance
in vivo
does not
always mirror the sensor properties demonstrated
in vitro
(
Hasan
et al.
, 2004; Heim
and Griesbeck, 2004
). With this potential pitfall in mind, a sensor has been devel-
oped based on troponin C, a calcium-binding protein and close homologue of
calmodulin that is, however, expressed only in muscle. The approach was to
concatenate TnC with CFP and citrine (
Heim and Griesbeck, 2004
). While devel-
oping these CFP-TnC-citrine sensors, a variant strategy was pursued to concate-
nate TNI, a TnC-binding partner, alongside TnC by analogy with the M13 binding
partner of calmodulin in the classical cameleons; this was unsuccessful. The con-
structs showing the greatest change in FRET between calcium-free and calcium-
bound forms contained a chicken skeletal muscle TnC with an N-terminal 14
residue truncation, TN-L15, and a human cardiac TnC, TN-humTnC. TN-L15
showed a 140% change and TN-humTnC a 120% change, measured in the absence
of magnesium ion. At physiological (1 mM) magnesium concentrations, the
dynamic ranges were 100% and 70%, respectively. Apparent dissociation constants
were 470 nM for TN-L15 and 1.2
m
M for TN-humTnC. The TnCEF hand calcium-
binding sites in TN-L15 were mutated to give
K
0
d
s of 300 nM and 29
m
M. The pH
sensitivities were similar to the other CFP/citrine-based sensors, with a reduction in
V
