Biology Reference
In-Depth Information
affinity chromatography, cleaved with TeV, and then active receptors are purified
using an NT-affinity column. This process is described in detail elsewhere
(
Attrill et al., 2009
).
18.1.2
Labeling strategies for FRET
FRET studies require the labeling of each of the dimerization partners with donor or
acceptor fluorophores that have spectral overlap between their emission and absorp-
tion spectrum, respectively. When choosing donor and acceptor pairs, one must try to
optimize this spectral overlap while minimizing spectral overlap of the donor and
acceptor absorption spectra, and of their emission spectra, as that would lead to
artifacts such as crosstalk and bleedthrough, respectively (this will be discussed in
a later section).
The most common labeling strategy is the use of autofluorescent FPs, where a
variant of the green fluorescent protein (GFP) is fused to the GPCR of interest
through standard genetic engineering (for more detailed experimental procedure,
see
Harding et al. (2009)
). Frequently used FRET-compatible pairs are CFP and
YFP
(cyan and yellow fluorescent protein), but vectors of many GFP variants
with different absorption properties to suit experimental requirements are commer-
cially available (e.g., Living Colors
™
, BD Biosciences, or Vivid Colors
™
, Life
Technologies). These FPs not only are typically fused to the N- or C-terminus of
the target protein to minimize problems with folding and retain GPCR functionality
but also can be inserted into loop regions. Often, a linker region is introduced
between the FP and the target protein to allow some flexibility between the two
domains to minimize steric hindrance. Different linkers have been reported,
varying in both length and content. Typically, these linkers have a high glycine
and/or serine content, as these flexible and hydrophilic residues are thought to
form a random coil structure and are thus unlikely to interact with the protein
domains or to interfere with their folding (
Evers, van Dongen, Faesen, Meijer, &
Merkx, 2006
).
The use of FPs has the benefit that all receptors are directly labeled during ex-
pression, and no cofactors or additional labeling and purification steps are required.
FPs are relatively large (
27 kDa), however, and can impair protein function when
inserted into functionally important domains (
Hoffmann et al., 2005
). The tendency
of GFP to self-dimerize, leading to false-positive results, has also been raised as a
concern (
Tsien, 1998
), but new “enhanced” variants of GFP, such as eCFP, do
not exhibit this propensity to the same extent (
Espagne et al., 2011
).
Cell-free expression of GPCRs also allows fluorescently tagging receptors prior
to purification, for example, by incorporation of nonnatural amino acids or by
conjugation of labels to the C-terminus (
Jackson, Boutell, Cooley, & He, 2004
).
The applicability hereof will however be dependent on the possibility for
in vitro
expression of the GPCR of interest.
Another popular technique is posttranslational site-directed labeling using
small organic dyes, such as Alexa Fluor dyes (e.g., Alexa488/Alexa647) (
Panchuk-
