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18.3 INTERPRETING RESULTS
It should be noted at this point that several factors affect the amount of FRET
produced. These include the distance between the fluorophores, relative fluorophore
orientation, and the proportion of receptors that are dimeric ( Stryer and Haugland,
1967 ). Therefore, if the FRET produced in different lipid environments changes,
it is important to distinguish between changes in receptor architecture that affect
the orientation of, or distance between, the probes and changes in the equilibrium
of dimerization. If large probes with flexible linkers, such as CFP or YFP, are used,
perturbations in FRET are likely due to changes in the dimerization equilibrium.
However, smaller probes attached to the protein backbone are more likely to be
affected by subtle changes in receptor architecture.
It is possible to quantify the FRET signal to compare different lipid environ-
ments, and this is done by calculation of apparent FRET efficiency ( Harding
et al., 2009; Overton and Blumer, 2002 ). In this case, this is simply the height of
the peak in the FRET spectrum (10) compared to the total amount of YFP in the
same sample (spectrum 7). Apparent FRET efficiency is also affected by the do-
nor-acceptor ratio—we typically use a 1:1 ratio for initial assays, but higher ratios
will give more efficient FRET. In a 1:1 ratio, only half of the acceptor molecules
are in FRET-competent dimers due to the presence of acceptor-acceptor dimers.
As the proportion of donor increases, the likelihood of an acceptor being in an
acceptor-acceptor dimer decreases and FRET efficiency increases.
In Fig. 18.3 , we illustrate typical results seen for the lipid-dependence of NTS1
dimerization. Brain polar lipid (BPL) is a commercially available extract (Avanti
FIGURE 18.3
Lipid dependence of dimerization. Apparent FRET efficiency for NTS1 tagged with eCFP or
eYFP was determined as a measure for dimerization in different lipid mixes (BPL, brain
polar lipid; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphoethanolamine; Ch, cholesterol).
Adapted from Oates et al. (2012) .
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