probably because of a variable degree of coating with SA (Fig. 13.4B). Binding kinetics of α-Bgt-647
were recorded online and data collected at the single-bead level. Their analysis yielded an observed
association constant k obs ¼ 0.018 min
À1 at 10 nM α-Bgt-647, assuming a pseudo-first-order reaction
(Fig.13.4C), which corresponds to k on ¼ (3.0 Æ 0.7) Â 10
4 M
À1
Ás
À1 (SD) for the bimolecular
reaction. This rate is within the range of those reported in the literature for the binding of radiolabeled
α-Bgt to purified Torpedo membranes or detergent-solubilized Torpedo nAChR ((0.8–33) Â 10
4 M
À1
Ás
À1 ; see e.g. Franklin and Potter 1972; Blanchard et al. 1979; Lukas et al. 1981). The binding of α-Bgt647 could be inhibited by carbamylcholine, a small analog of acetylcholine (unpublished data),
opening the way to determining the affinity constant of small, unlabeled ligands.
Study 13.1 thus established the feasibility of using tagged APols to immobilize MPs onto chips
or beads and measure the binding to the immobilized proteins of large (antibodies, ~150 kDa),
medium-sized (α-Bgt, 8 kDa), or small (ACh analogs, a few hundred Da) ligands. In addition to the
features noted above (absence of detergent, biochemical stabilization, absence of MP modification, use
of a single tagged APol for any MP, possibility of mixing tagged and labeled APols), the approach
presents other advantages: (i) unlike in protein-mediated immobilization methods, all extramembrane
domains of the protein are expected to remain free from interactions with the support and to be
comparably exposed to the solution; (ii) at variance with experimental schemes in which the target MP
is embedded in membrane vesicles, all of its extramembrane regions are simultaneously accessible
from the same aqueous compartment.
In most cases, trapping with APols is unlikely to interfere with ligand binding. As discussed in
Chap. 5, § 5.4, it cannot be excluded, however, that, even though APols mostly confine themselves to
the TM surface of trapped MPs, some of their tails or loops may explore MP extramembrane surfaces
and bind to hydrophobic ligand-binding sites. This is not a purely theoretical possibility, because
similar polymers have been shown to interact with serum albumin, a soluble protein, presumably via
the hydrophobic groove that binds fatty acids (Tribet et al. 1998). We have noted in Chap. 5, however,
that no interference of A8-35 is observed with the binding of LTB 4 to the BLT1 or BLT2 receptors
(Dahmane et al. 2009; Catoire et al. 2010), even though, given the hydrophobicity of LTB 4 , its binding
site must be itself quite hydrophobic and could in principle attract APol octyl chains (§ 5.4). In the
frontier region between the TM and extramembrane surfaces of the protein, APols could sterically or
otherwise hinder the binding of macromolecular ligands. Furthermore, polyacrylate-based APols such
as A8-35, being polyanionic, could also bind to strongly cationic pockets or patches: A8-35 has been
shown to aggregate lysozyme (Champeil et al. 2000), and molecular dynamics simulations indicate
that it does interact with extramembrane basic residues of OmpX (Perlmutter et al. 2014; see Chap. 5,
§ 5.3.3). Altogether, however, as noted in Chap. 5, § 5.4, no interference has been noted to date
between APols and ligand binding, with the exception of that between anionic APols and G protein and
arrestin binding to G protein-coupled receptors (see Chap. 5, § 5.4). The latter problem can be avoided
by resorting to non-ionic APols. All things considered, this ensemble of data suggests that the
possibility that APols interfere with ligand binding to APol-immobilized MPs cannot be ignored,
but that, in most cases, it is unlikely to be a serious hindrance.
It is worth noting that, because APols bind to membranes (see Chap. 5, § 5.2.2.2), native
membrane fragments or lipid vesicles could presumably be anchored to solid supports by the same
strategy. This possibility has not yet been exploited.
Biotinylated APols are the only tagged APols that have been used to date in several publications.
BA8-35 has been used to immobilize MPs in Studies 13.2 (Della Pia et al. 2014a; see § 13.2.2) and
13.3 (Ferrandez et al. 2014; see § 13.2.3). A biotinylated phosphorylcholine-based APol has been used
in Study 13.5 to study the interaction between a bacterial outer membrane protein, FhuA, and a protein
from the tail of a bacteriophage (Basit et al. 2012).
13.2 Immobilization of Membrane Proteins Using Biotinylated Amphipols and Its Applications
599
were recorded online and data collected at the single-bead level. Their analysis yielded an observed
association constant k obs ¼ 0.018 min
À1 at 10 nM α-Bgt-647, assuming a pseudo-first-order reaction
(Fig.13.4C), which corresponds to k on ¼ (3.0 Æ 0.7) Â 10
4 M
À1
Ás
À1 (SD) for the bimolecular
reaction. This rate is within the range of those reported in the literature for the binding of radiolabeled
α-Bgt to purified Torpedo membranes or detergent-solubilized Torpedo nAChR ((0.8–33) Â 10
4 M
À1
Ás
À1 ; see e.g. Franklin and Potter 1972; Blanchard et al. 1979; Lukas et al. 1981). The binding of α-Bgt647 could be inhibited by carbamylcholine, a small analog of acetylcholine (unpublished data),
opening the way to determining the affinity constant of small, unlabeled ligands.
Study 13.1 thus established the feasibility of using tagged APols to immobilize MPs onto chips
or beads and measure the binding to the immobilized proteins of large (antibodies, ~150 kDa),
medium-sized (α-Bgt, 8 kDa), or small (ACh analogs, a few hundred Da) ligands. In addition to the
features noted above (absence of detergent, biochemical stabilization, absence of MP modification, use
of a single tagged APol for any MP, possibility of mixing tagged and labeled APols), the approach
presents other advantages: (i) unlike in protein-mediated immobilization methods, all extramembrane
domains of the protein are expected to remain free from interactions with the support and to be
comparably exposed to the solution; (ii) at variance with experimental schemes in which the target MP
is embedded in membrane vesicles, all of its extramembrane regions are simultaneously accessible
from the same aqueous compartment.
In most cases, trapping with APols is unlikely to interfere with ligand binding. As discussed in
Chap. 5, § 5.4, it cannot be excluded, however, that, even though APols mostly confine themselves to
the TM surface of trapped MPs, some of their tails or loops may explore MP extramembrane surfaces
and bind to hydrophobic ligand-binding sites. This is not a purely theoretical possibility, because
similar polymers have been shown to interact with serum albumin, a soluble protein, presumably via
the hydrophobic groove that binds fatty acids (Tribet et al. 1998). We have noted in Chap. 5, however,
that no interference of A8-35 is observed with the binding of LTB 4 to the BLT1 or BLT2 receptors
(Dahmane et al. 2009; Catoire et al. 2010), even though, given the hydrophobicity of LTB 4 , its binding
site must be itself quite hydrophobic and could in principle attract APol octyl chains (§ 5.4). In the
frontier region between the TM and extramembrane surfaces of the protein, APols could sterically or
otherwise hinder the binding of macromolecular ligands. Furthermore, polyacrylate-based APols such
as A8-35, being polyanionic, could also bind to strongly cationic pockets or patches: A8-35 has been
shown to aggregate lysozyme (Champeil et al. 2000), and molecular dynamics simulations indicate
that it does interact with extramembrane basic residues of OmpX (Perlmutter et al. 2014; see Chap. 5,
§ 5.3.3). Altogether, however, as noted in Chap. 5, § 5.4, no interference has been noted to date
between APols and ligand binding, with the exception of that between anionic APols and G protein and
arrestin binding to G protein-coupled receptors (see Chap. 5, § 5.4). The latter problem can be avoided
by resorting to non-ionic APols. All things considered, this ensemble of data suggests that the
possibility that APols interfere with ligand binding to APol-immobilized MPs cannot be ignored,
but that, in most cases, it is unlikely to be a serious hindrance.
It is worth noting that, because APols bind to membranes (see Chap. 5, § 5.2.2.2), native
membrane fragments or lipid vesicles could presumably be anchored to solid supports by the same
strategy. This possibility has not yet been exploited.
Biotinylated APols are the only tagged APols that have been used to date in several publications.
BA8-35 has been used to immobilize MPs in Studies 13.2 (Della Pia et al. 2014a; see § 13.2.2) and
13.3 (Ferrandez et al. 2014; see § 13.2.3). A biotinylated phosphorylcholine-based APol has been used
in Study 13.5 to study the interaction between a bacterial outer membrane protein, FhuA, and a protein
from the tail of a bacteriophage (Basit et al. 2012).
13.2 Immobilization of Membrane Proteins Using Biotinylated Amphipols and Its Applications
599