and 12, § 12.3.3). The origin of this phenomenon is uncertain, and it has not been observed in
all EM studies (see Liao et al. 2013, 2014; for discussions, see Huynh et al. 2014; Zoonens
and Popot 2014, and Chap. 12, § 12.3.4.1). If real, it may conceivably interfere with
crystallization. It would be of interest to examine whether mixed APol/detergent belts exhibit
or not the same feature.
Whereas crystals of ternary complexes were obtained, the few of them that could be tested
diffracted only to ~20-Å resolution. It is currently not known whether this represents an intrinsic limit
or is simply due to the small number of crystals that could be examined and/or to difficulties in
handling and/or freezing them. Nevertheless, the mere fact that 3D crystals of APol-trapped MPs could
be obtained is an encouragement to pursue such attempts. Those could be carried out using either
A8-35 or more recently developed APols with different chemical structures, e.g. those described in
Diab et al. (2007a, b), Bazzacco et al. (2012), and Sharma et al. (2012), which can be used under a
globally neutral or totally uncharged form (see Chap. 4). Of particular interest is the coming of age of
non-ionic APols, whose latest versions have shown an excellent ability to trap and stabilize MPs
(Bazzacco et al. 2012; Sharma et al. 2012).
Finally, one may note two other ways in which APols may help to obtain the crystallographic
structure of MPs. First, it is now established that APol/detergent mixtures can (i) stabilize MPs as
compared to pure detergent (Champeil et al. 2000) (see Chap. 5, § 5.6.1) and (ii) yield 3D crystals
(Charvolin et al. 2014). APols therefore could possibly be used as additives, to stabilize MPs whose
instability in pure detergent solution prevents crystals from growing to a sufficient size. Second, APols
have proven an excellent medium in which to fold MPs to their native state (Chap. 6), as well as to
synthesize them in vitro (Chap. 7). They could therefore be used as mere tools to produce the folded
protein, whereas crystallization would be attempted after transferring the latter either to a detergent, a
bicelle, or a lipidic mesophase (Fig. 11.17). The latter approach, which is described in the next section,
has permitted to obtain crystals diffracting to <2 Å of BR transferred from A8-35 or SMA to a lipid
mesophase (Polovinkin et al. 2014; Broecker et al. 2017).
11.3.2 Using Amphipols as a Vehicle to Crystallize Membrane Proteins in Meso
One of the best investigated examples of MP trapping and stabilization by APols is that of BR. Of
relevance to the present section are the following observations, which have been described in the
preceding chapters:
• BR can be transferred in its native state from BR/lipid/detergent complexes to various APols,
an environment where it is functional (Tribet et al. 1996, 1997; Gohon et al. 2008; Bazzacco
et al. 2012; Sharma et al. 2012; Dahmane et al. 2013) (Chap. 5).
• It can be renatured by transfer from sodium dodecyl sulfate to either A8-35 or non-ionic
APols either in the presence or absence of lipids (Pocanschi et al. 2006; Bazzacco et al. 2012;
Dahmane et al. 2013) (Chap. 6) or synthesized in vitro in the presence of non-ionic APols
(Bazzacco et al. 2012) (Chap. 7).
• In the course of these works, it was noted that archaebacterial lipids displaced by detergents
rebind to BR upon its transfer to APols, with functional consequences on the photocycle
(Gohon et al. 2008; Dahmane et al. 2013).
516
11 Amphipols and Membrane Protein Crystallization
all EM studies (see Liao et al. 2013, 2014; for discussions, see Huynh et al. 2014; Zoonens
and Popot 2014, and Chap. 12, § 12.3.4.1). If real, it may conceivably interfere with
crystallization. It would be of interest to examine whether mixed APol/detergent belts exhibit
or not the same feature.
Whereas crystals of ternary complexes were obtained, the few of them that could be tested
diffracted only to ~20-Å resolution. It is currently not known whether this represents an intrinsic limit
or is simply due to the small number of crystals that could be examined and/or to difficulties in
handling and/or freezing them. Nevertheless, the mere fact that 3D crystals of APol-trapped MPs could
be obtained is an encouragement to pursue such attempts. Those could be carried out using either
A8-35 or more recently developed APols with different chemical structures, e.g. those described in
Diab et al. (2007a, b), Bazzacco et al. (2012), and Sharma et al. (2012), which can be used under a
globally neutral or totally uncharged form (see Chap. 4). Of particular interest is the coming of age of
non-ionic APols, whose latest versions have shown an excellent ability to trap and stabilize MPs
(Bazzacco et al. 2012; Sharma et al. 2012).
Finally, one may note two other ways in which APols may help to obtain the crystallographic
structure of MPs. First, it is now established that APol/detergent mixtures can (i) stabilize MPs as
compared to pure detergent (Champeil et al. 2000) (see Chap. 5, § 5.6.1) and (ii) yield 3D crystals
(Charvolin et al. 2014). APols therefore could possibly be used as additives, to stabilize MPs whose
instability in pure detergent solution prevents crystals from growing to a sufficient size. Second, APols
have proven an excellent medium in which to fold MPs to their native state (Chap. 6), as well as to
synthesize them in vitro (Chap. 7). They could therefore be used as mere tools to produce the folded
protein, whereas crystallization would be attempted after transferring the latter either to a detergent, a
bicelle, or a lipidic mesophase (Fig. 11.17). The latter approach, which is described in the next section,
has permitted to obtain crystals diffracting to <2 Å of BR transferred from A8-35 or SMA to a lipid
mesophase (Polovinkin et al. 2014; Broecker et al. 2017).
11.3.2 Using Amphipols as a Vehicle to Crystallize Membrane Proteins in Meso
One of the best investigated examples of MP trapping and stabilization by APols is that of BR. Of
relevance to the present section are the following observations, which have been described in the
preceding chapters:
• BR can be transferred in its native state from BR/lipid/detergent complexes to various APols,
an environment where it is functional (Tribet et al. 1996, 1997; Gohon et al. 2008; Bazzacco
et al. 2012; Sharma et al. 2012; Dahmane et al. 2013) (Chap. 5).
• It can be renatured by transfer from sodium dodecyl sulfate to either A8-35 or non-ionic
APols either in the presence or absence of lipids (Pocanschi et al. 2006; Bazzacco et al. 2012;
Dahmane et al. 2013) (Chap. 6) or synthesized in vitro in the presence of non-ionic APols
(Bazzacco et al. 2012) (Chap. 7).
• In the course of these works, it was noted that archaebacterial lipids displaced by detergents
rebind to BR upon its transfer to APols, with functional consequences on the photocycle
(Gohon et al. 2008; Dahmane et al. 2013).
516
11 Amphipols and Membrane Protein Crystallization