Biology Reference
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cells, as when calcium increases, endogenous calmodulin becomes activated and
binds to the MLCK calcium-binding domain. This in turn alters the disposition of
the attached GFPs and leads to changes in F
¨
rster resonance energy transfer
(FRET) between the blue and green proteins (
Romoser
et al.
, 1997
).
FRET is the phenomenon on which the cameleon sensor family relies. It occurs
between closely apposed fluorophores that have overlapping emission and excita-
tion spectra (
Jares-Erijman Elizabeth and Jovin Thomas, 2003
). In this example,
the emission spectrum of BFP overlaps with the excitation spectrum of GFP. The
extent of FRET depends on the degree of overlap between the two spectra, the
orientation of the fluorescence dipoles and crucially, the distance between them.
There is a very steep sixth power relationship with distance, so the energy transfer
is very sensitive to distance between fluorophores over the range 1-10 nm (
Jares-
Erijman and Jovin, 2003
). Calmodulin binds to the helical MLCK sequence by
wrapping its two lobes around it (
Ikura
et al.
, 1992
). In FIP-CB
sm
, the steric bulk
of the calmodulin molecule when it binds to the MLCK peptide linker forces the
BFP and RFP further apart and reduces FRET (
Romoser
et al.
, 1997
). FRET can
be measured in a variety of ways (
Jares-Erijman Elizabeth and Jovin Thomas,
2003; Visser
et al.
, 2010
), but conceptually the simplest method is to excite the
donor
fluorophore, here BFP, and measure the emission of both the donor and the
acceptor
, here GFP. FRET takes place by nonradiative energy transfer, so high
levels of FRET transfer energy from BFP to GFP, reducing BFP emission at
around 440 nm and increasing GFP emission at 510 nm. Calmodulin binding
reduces FRET, increasing emission at 440 nm and reducing emission at 510 nm.
These changes can be expressed as a ratio of emission at the two wavelengths, a
value independent of the concentration of the protein. In HEK-239 cells expressing
FIP-CB
sm
, ratio changes (
F
510
/
F
440
) of around three- to fourfold could be observed
after raising free intracellular calcium concentration with the calcium ionophore
ionomycin (
Romoser
et al.
, 1997
).
FIP-CB
sm
relied on endogenous calmodulin to generate a calcium-sensitive
FRET signal between GFPs. Tsien's construct concatenated
Xenopus laevis
cal-
modulin and an MLCK calmodulin-binding peptide, M13 (
Ikura
et al.
, 1992
),
together between BFP and GFP and also in an analogous construct between two
other GFP variants, enhanced cyan fluorescent protein (ECFP) and enhanced
yellow fluorescent protein (EYFP). In this concatenated configuration, binding
of calcium to calmodulin causes it to loop back toward the M13 peptide (the
cameleon's tongue) as it binds, reducing the distance between the two GFP
variants and enhancing FRET (
Miyawaki
et al.
, 1997
). This study beautifully
exemplifies the power of the cameleon concept linked to selective mutagenesis:
the original BFP/GFP construct (cameleon-1) worked well
in vitro
, but did not
express su
ciently in mammalian cells; the enhanced variant with mammalian
codon usage (EBFP/EGFP—cameleon-2) showed much improved expression, but
the best expression, brightness, and signal-to-noise data were seen with enhanced
cyan and yellow variants of GFP (ECFP/EYFP—yellow cameleon-2). These ben-
efits came, however, at the expense of a lower FRET change between calcium
Y
