extensive dilution (Zoonens et al. 2007; Tribet et al. 2009), or upon flushing of complexes attached to
an SPR chip via a histidine tag carried by the protein (Popot et al. 2003; see Fig. 5.7). Consistent with
these observations, MPs bound to a solid support via a biotinylated APol neither desorb nor become
inactivated upon extensive washing of the chips with surfactant-free buffer (Charvolin et al. 2009;
Basit et al. 2012; Della Pia et al. 2014; Ferrandez et al. 2014; see Chap. 13).
As mentioned in § 5.2.1, APols, whether present as free particles or as a MP-adsorbed layer,
freely mix with detergents, in a nearly ideal manner (Zoonens et al. 2007; Tribet et al. 2009). This
makes it very easy to exchange one type of surfactant for the other (see e.g. Tribet et al. 1997; Zoonens
et al. 2007). The ease and speed (Fig. 5.40) with which detergents can wash APols away from the
surface of MPs may seem contradictory with the strong retention of APols by MPs upon extensive
dilution or flushing with surfactant-free buffers. This apparent paradox is due to the fact that there is
little or no free energy cost to displacing APols from a MP hydrophobic transmembrane surface to a
mixed detergent/APol particle while replacing it with detergent, whereas it is extremely costly to bare
the same surface from any surfactant (Tribet et al. 2009; Giusti et al. 2012).
APols have been used by C.R. Sanders and colleagues to deliver diacylglycerol kinase (DAGK)
(Nagy et al. 2001) and by A.S. Ladokhin to deliver the pore-forming domain of diphtheria toxin
(Kyrychenko et al. 2012) to preformed lipid vesicles, as well as by J.H. Kleinschmidt and colleagues
to deliver two outer membrane β-barrel proteins that had been refolded in A8-35, OmpA from E. coli
and FomA from Fusobacterium nucleatum, to lipid black films (Pocanschi et al. 2006b; Fig. 5.41). In
all cases, the native state of at least some of the proteins inserted was demonstrated by functional tests.
APols have also been used to deliver a synthetic peptide mimicking the single transmembrane helix of
a growth factor receptor to cells in culture. In the hours and days that followed, fluorescence imaging
showed that the peptide (and the APol) was endocytosed (Popot et al. 2011; see Chap. 15, § 15.3).
Fig. 5.40 Displacement of a membrane protein-bound fluorescent amphipol by an excess of detergent.
C 12 E 8 was added to tOmpA/FAPol NBD complexes in a stopped-flow instrument, and the kinetics of
dequenching of the protein’s tryptophan residues followed as a function of time. The final concentrations
of detergent extended from 2Â below the CMC (0.025 gÁL
À1
) to 20Â above it (1 gÁL
À1
). The final protein
and APol concentrations were kept constant at 0.025 and 0.1 gÁL
À1
, respectively. Experimental data (thin
lines) have been normalized to the intensity of tryptophan fluorescence at the first measurable point, taken
as I 330 (t 0 ), and fitted with three exponentials (thick lines) (Reprinted with permission from Zoonens et al.
2007, # 2007 American Chemical Society).
308
5 Formation and Properties of Membrane Protein/Amphipol Complexes
an SPR chip via a histidine tag carried by the protein (Popot et al. 2003; see Fig. 5.7). Consistent with
these observations, MPs bound to a solid support via a biotinylated APol neither desorb nor become
inactivated upon extensive washing of the chips with surfactant-free buffer (Charvolin et al. 2009;
Basit et al. 2012; Della Pia et al. 2014; Ferrandez et al. 2014; see Chap. 13).
As mentioned in § 5.2.1, APols, whether present as free particles or as a MP-adsorbed layer,
freely mix with detergents, in a nearly ideal manner (Zoonens et al. 2007; Tribet et al. 2009). This
makes it very easy to exchange one type of surfactant for the other (see e.g. Tribet et al. 1997; Zoonens
et al. 2007). The ease and speed (Fig. 5.40) with which detergents can wash APols away from the
surface of MPs may seem contradictory with the strong retention of APols by MPs upon extensive
dilution or flushing with surfactant-free buffers. This apparent paradox is due to the fact that there is
little or no free energy cost to displacing APols from a MP hydrophobic transmembrane surface to a
mixed detergent/APol particle while replacing it with detergent, whereas it is extremely costly to bare
the same surface from any surfactant (Tribet et al. 2009; Giusti et al. 2012).
APols have been used by C.R. Sanders and colleagues to deliver diacylglycerol kinase (DAGK)
(Nagy et al. 2001) and by A.S. Ladokhin to deliver the pore-forming domain of diphtheria toxin
(Kyrychenko et al. 2012) to preformed lipid vesicles, as well as by J.H. Kleinschmidt and colleagues
to deliver two outer membrane β-barrel proteins that had been refolded in A8-35, OmpA from E. coli
and FomA from Fusobacterium nucleatum, to lipid black films (Pocanschi et al. 2006b; Fig. 5.41). In
all cases, the native state of at least some of the proteins inserted was demonstrated by functional tests.
APols have also been used to deliver a synthetic peptide mimicking the single transmembrane helix of
a growth factor receptor to cells in culture. In the hours and days that followed, fluorescence imaging
showed that the peptide (and the APol) was endocytosed (Popot et al. 2011; see Chap. 15, § 15.3).
Fig. 5.40 Displacement of a membrane protein-bound fluorescent amphipol by an excess of detergent.
C 12 E 8 was added to tOmpA/FAPol NBD complexes in a stopped-flow instrument, and the kinetics of
dequenching of the protein’s tryptophan residues followed as a function of time. The final concentrations
of detergent extended from 2Â below the CMC (0.025 gÁL
À1
) to 20Â above it (1 gÁL
À1
). The final protein
and APol concentrations were kept constant at 0.025 and 0.1 gÁL
À1
, respectively. Experimental data (thin
lines) have been normalized to the intensity of tryptophan fluorescence at the first measurable point, taken
as I 330 (t 0 ), and fitted with three exponentials (thick lines) (Reprinted with permission from Zoonens et al.
2007, # 2007 American Chemical Society).
308
5 Formation and Properties of Membrane Protein/Amphipol Complexes