discussed in more detail in Chap. 5 (§ 5.4), that this effect may not apply to MPs whose function
does not involve large conformational changes at the interface between the protein and the
membrane. Thus, (i) the nicotinic acetylcholine receptor undergoes, following trapping with
A8-35, ligand-induced conformational changes with kinetics similar to that in its native membrane
environment (Martinez et al. 2002); (ii) BR recovers, following transfer from detergent to A8-35, a
photocycle very similar to that observed in its native purple membrane (Gohon et al. 2008;
Dahmane et al. 2013); and (iii) TRPV1 was imaged in different conformational states after adding
agonists and antagonists to the APol-trapped channel (Cao et al. 2013b). Yet, some MPs, such as the
sarcoplasmic calcium pump (Champeil et al. 2000; Picard et al. 2006) or the F 1 F O ATP synthase
(Wilkens 2000), are reversibly inhibited by APols, which could be related to APols slowing down or
preventing transconformations required by the enzymatic cycle (see Chap. 5, § 5.6). In such cases, it
could be desirable either to leave the system enough time to relax to the state induced by one or the
other ligand before freezing it or to trap it after the ligand has been added.
As with detergents, the question arises whether the APol-trapped state is identical to that – or to
one of those – adopted by the protein in its native membrane environment. An interesting
observation concerns BR, whose TM region looks, according to solution-state NMR, very similar
in DDM, A8-35, or nanodiscs, whereas the conformation of the loops resembles more that in the
membrane following APol trapping than in DDM solution (Etzkorn et al. 2013). This cannot be
due to the thickness of the polar head region, which is closest to that in the membrane in DDM
than in A8-35. It suggests, rather, a change in dynamics (see Chap. 5, § 5.6). This is a very
interesting issue that deserves to be studied in more depth.
(iii) A point that remains to be clarified is that of the nature and origin of the “bumps” of the APol belt
observed at medium resolution (19 Å) with the respirasome/A8-35 complex (Fig. 12.19A; Althoff
et al. 2011), but not at higher resolution (~4 Å) in subsequent cryo-EM studies of TRP channels
(Figs. 12.23 and 12.25) (Liao et al. 2014; Paulsen et al. 2015). One possibility that may perhaps
deserve to be kept in mind is that the formation of bumps may result from the frustration created
by the adsorption of the APol onto the nearly flat surface of the respirasome, which forces the
APol/water interface away from its natural curvature. This situation is not reproduced, or not to
the same extent, with the much smaller TRP channels.
12.3.4.2 Imaging
The reported effects of APol-trapping vs. detergent solubilization on imaging fall in three categories:
the contrast, the dispersity of the particles, and their orientation.
(i) Contrast. In the case of the ryanodine receptor, it was noted that the contrast was significantly
better with the A8-35-trapped complex than with that kept soluble by CHAPS (Baker et al. 2015).
Although this is not discussed by other authors, one should expect that this effect can be significant
with high-CMC detergents such as CHAPS or CHAPSO, which need to be present at relatively
high concentration (>8 mM) to keep the protein from aggregating, probably much less so with
low-CMC detergents such as Triton X-100, DDM, digitonin, or the MNG detergents.
(ii) Dispersity. It has been noted in several studies (e.g. Flötenmeyer et al. 2007; Baker et al. 2015;
Jeong et al. 2016) that particles that, in detergent samples, tend to crowd at the rim of the holes in
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12 The Use of Amphipols for Electron Microscopy
does not involve large conformational changes at the interface between the protein and the
membrane. Thus, (i) the nicotinic acetylcholine receptor undergoes, following trapping with
A8-35, ligand-induced conformational changes with kinetics similar to that in its native membrane
environment (Martinez et al. 2002); (ii) BR recovers, following transfer from detergent to A8-35, a
photocycle very similar to that observed in its native purple membrane (Gohon et al. 2008;
Dahmane et al. 2013); and (iii) TRPV1 was imaged in different conformational states after adding
agonists and antagonists to the APol-trapped channel (Cao et al. 2013b). Yet, some MPs, such as the
sarcoplasmic calcium pump (Champeil et al. 2000; Picard et al. 2006) or the F 1 F O ATP synthase
(Wilkens 2000), are reversibly inhibited by APols, which could be related to APols slowing down or
preventing transconformations required by the enzymatic cycle (see Chap. 5, § 5.6). In such cases, it
could be desirable either to leave the system enough time to relax to the state induced by one or the
other ligand before freezing it or to trap it after the ligand has been added.
As with detergents, the question arises whether the APol-trapped state is identical to that – or to
one of those – adopted by the protein in its native membrane environment. An interesting
observation concerns BR, whose TM region looks, according to solution-state NMR, very similar
in DDM, A8-35, or nanodiscs, whereas the conformation of the loops resembles more that in the
membrane following APol trapping than in DDM solution (Etzkorn et al. 2013). This cannot be
due to the thickness of the polar head region, which is closest to that in the membrane in DDM
than in A8-35. It suggests, rather, a change in dynamics (see Chap. 5, § 5.6). This is a very
interesting issue that deserves to be studied in more depth.
(iii) A point that remains to be clarified is that of the nature and origin of the “bumps” of the APol belt
observed at medium resolution (19 Å) with the respirasome/A8-35 complex (Fig. 12.19A; Althoff
et al. 2011), but not at higher resolution (~4 Å) in subsequent cryo-EM studies of TRP channels
(Figs. 12.23 and 12.25) (Liao et al. 2014; Paulsen et al. 2015). One possibility that may perhaps
deserve to be kept in mind is that the formation of bumps may result from the frustration created
by the adsorption of the APol onto the nearly flat surface of the respirasome, which forces the
APol/water interface away from its natural curvature. This situation is not reproduced, or not to
the same extent, with the much smaller TRP channels.
12.3.4.2 Imaging
The reported effects of APol-trapping vs. detergent solubilization on imaging fall in three categories:
the contrast, the dispersity of the particles, and their orientation.
(i) Contrast. In the case of the ryanodine receptor, it was noted that the contrast was significantly
better with the A8-35-trapped complex than with that kept soluble by CHAPS (Baker et al. 2015).
Although this is not discussed by other authors, one should expect that this effect can be significant
with high-CMC detergents such as CHAPS or CHAPSO, which need to be present at relatively
high concentration (>8 mM) to keep the protein from aggregating, probably much less so with
low-CMC detergents such as Triton X-100, DDM, digitonin, or the MNG detergents.
(ii) Dispersity. It has been noted in several studies (e.g. Flötenmeyer et al. 2007; Baker et al. 2015;
Jeong et al. 2016) that particles that, in detergent samples, tend to crowd at the rim of the holes in
574
12 The Use of Amphipols for Electron Microscopy