Biology Reference
In-Depth Information
A
Donor:
Blue or
cyan FP
Acceptor:
Green or
yellow FP
M13
Cam
Excitation:
442 nm
Emission:
486 nm
4 Ca
2
+
0 Ca
2
+
+
FRET
<100 nm
Emission:
530 nm
4 Ca
2
+
Excitation:
442 nm
B
+
Ca
2
+
Ca
2
+
−
530 nm
486 nm
Wavelength
Fig. 4
Cameleon-based F¨ rster resonance energy transfer (FRET). (A) Schematic of the cameleon in
the absence and presence of Ca
2
þ
; note the conformational change in the calmodulin (Cam) and the
Cam-binding domain of myosin light chain kinase (M13) upon binding to Ca
2
þ
that allows for FRET
between the donor and acceptor green fluorescent protein (FP) mutants. (B) The relative emission
intensities at different wavelengths indicate whether or not Ca
2
þ
is present, and hence, FRET occurs.
Since the introduction of cameleons, derivatives have been developed that fuse
Cam with the Cam-binding peptide M13 in a similar fashion as cameleons, but are
based on single GFPs instead of two GFP mutants with di
erent spectral proper-
ties that necessitate Ca
2
þ
imaging by FRET. These derivatives, called pericams,
can therefore be imaged by standard epifluorescence or confocal microscopes, and
present with broad Ca
2
þ
sensitivities and di
V
erent localization sequences that
allow for measurements of a range of Ca
2
þ
concentrations within specific orga-
nelles and intracellular targets (
Kettlewell et al., 2009; Nagai et al., 2001
).
V