Biology Reference
In-Depth Information
FIGURE 14.2
Principles of FRET. The technique of F
¨
rster resonance energy transfer permits
determination of the approach between two molecules within several nanometers, a distance
sufficiently close for molecular interaction to occur. It is a strategy for detection of protein-
protein interaction tagged with fluorescent proteins. For more detailed information, see Ref.
Lakowicz (2006)
.
average molecular distance is far above
R
0
and FRET could be only seen if interac-
tion occurs. More important, FRET allows us to investigate protein-protein interac-
tion, which occurs far below spatial resolution of optical microscopy.
14.1.2
Fluorescent proteins and FRET
To study protein-protein interaction by FRET, the first step is to tag proteins of in-
terest with either donor or acceptor fluorophore. Several labeling strategies are avail-
able for FRET signal detection (
Lam et al., 2012
). Genetic labeling with fluorescent
proteins (FPs) for live-cell FRET imaging represents one of the central labeling strat-
egies. At present, there is a large availability of green fluorescent protein (GFP) var-
iants as possible donor-acceptor pairs that can be used to study quantitative FRET in
living cells. Cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)
derivatives are widely used in fluorescence microscopy studies as acute FRET pairs
(
Piston & Kremers, 2007
). For protein-protein interaction, it is important to ensure
that the FPs per se do not interact with each other. It is known that original GFP
showed intrinsic tendency to dimerize to some extent; hence, special effort was taken
