Biology Reference
In-Depth Information
VI. F ¨ rster Resonance Energy Transfer Microscopy
Confocal microscopy coupled with FRET imaging has experienced something
of a surge of interest recently fromCa
2
þ
researchers, because of the development of
Ca
2
þ
-sensitive cameleons. The FRET process is based upon nonradiative energy
transfer from a fluorophore in an excited state (''donor'') to another chromophore
(''acceptor'') that usually is, but does not have to be, a di
erent fluorescent
molecule within a range of 10-100 Angstroms (1-10 nm) (fluorophore) (
Jares-
Erijman and Jovin, 2003
). Thus, when FRET occurs, it is not only the acceptor
emission that will be recordable, but the measurable emission from the donor will
also be greatly reduced because of the energy transfer. Also, the intensity of the two
emission bands will depend on the distance between the two donor and acceptor
fluorescent proteins or molecules even within the FRET distance. The closer the
distance between the donor and acceptor, the greater the longer-wavelength emis-
sion (from the acceptor) and the less the shorter-wavelength emission (from the
donor). The fluorescence emission from the acceptor is discernible from the donor
emission due to the spectral redshift, such that the instrumental requirement is that
the two emission bands must be separately recordable. The presence of the longer-
wavelength acceptor emission will confirm that the acceptor molecule is within
the FRET distance of the donor, that is, within a distance of
V
10 nm. Likewise,
the absence of it suggests that the physical distance between the donor and
acceptor is larger than that. Traditionally, FRET imaging has been used to study
protein-protein interactions by tagging di
erent proteins with fluorescent probes
(e.g., yellow or green fluorescent proteins (YFP or GFP)) and thereby study
whether they exist within or outwith the FRET distance.
However, it is the recent development of cameleons that has moved FRET
imaging to also become a sought-after technique with respect to studies of Ca
2
þ
.
Cameleons are genetically encoded fluorescent proteins that are sensitive to Ca
2
þ
,
in which a blue- or cyan-mutant of GFP (serving as the donor), calmodulin that
can bind to Ca
2
þ
, a calmodulin-binding peptide such as the calmodulin-binding
domain of skeletal muscle myosin light chain kinase (the M13 peptide), and a
long-wavelength yellow mutant or normal GFP (serving as the acceptor) are
tandemly fused (
Miyawaki et al., 1997
). This configuration forms a stable and
compact complex that remains intact once transfected into the target cell, and
subsequent development has also improved the spectral properties and rendered
the cameleons less susceptible to changes in pH (enhanced GFPs). In the absence
of Ca
2
þ
, the cameleon remains in its linear tandem configuration, whereby the
two GFP mutants (donor and acceptor) at the two flanks of the tandem are too
far apart to create FRET. However, when the concentration of free Ca
2
þ
increases, calmodulin binds to Ca
2
þ
and undergoes a conformational change
that also leads it to bind and wrap around the M13 peptide, which creates a
compact configuration of the cameleon and therefore brings the donor and
acceptor GFP mutants to within a distance where energy transfer may occur
V
