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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
 
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