5.2.3
Folding Membrane Proteins in Amphipols
As shown in Table 5.1, a dozen MPs have been transferred and folded in APols starting from a
denatured state. The denaturing medium is typically sodium dodecyl sulfate (SDS) for α-helical
proteins (BR and seven G protein-coupled receptors (GPCRs)) and urea for β-barrel ones (four
bacterial outer membrane proteins). The dodecyl sulfate is typically removed by precipitating it as
its potassium salt, PDS, whereas the urea is diluted. The procedure has been validated for A8-35
(Pocanschi et al. 2006b; for a recent review, see Le Bon et al. 2018), SAPols (Dahmane et al. 2009),
NAPols (Bazzacco et al. 2012), and amphipathic poly-γ-glutamic acid (APG) (Han et al. 2014). A few
synthetic peptides have been transferred to A8-35 from either urea or trifluoroethanol (Gohon 1996;
Duarte et al. 2008; Stangl et al. 2014). In comparative studies, it was shown that, provided lipids have
not been removed, the functional properties of BR refolded in A8-35 are exactly the same as those of
native BR trapped in the same APols starting from a solution in non-denaturing detergent (Dahmane
et al. 2013). Similarly, GPCRs folded in APols from SDS-solubilized inclusion bodies exhibit the same
pharmacological properties as native receptors (Dahmane et al. 2009; Banères et al. 2011; Bazzacco
et al. 2012; Mary et al. 2014; Logez et al. 2016). Folding MPs to their native form is one of the most
promising uses of APols and will be discussed in detail in Chap. 6.
Amphipol-assisted folding of membrane proteins
(# 2018 by Francis Haraux)
Alternatively, MPs can be synthesized in vitro using APols as the accepting medium. However,
polyanionic APols such as A8-35, SAPols, and SMA have all been found to inhibit cell-free synthesis
of MPs, possibly by interacting with cationic sites at the surface of the translation apparatus (Park et al.
2011; Periasamy et al. 2013). Only glucose-based NAPols (Bazzacco et al. 2012) and NVoy (Guild
et al. 2011; Klammt et al. 2011), both of which are non-ionic, have been validated to date for use in MP
cell-free expression. Using APols for this application will be discussed in Chap. 7.
5.2 Forming Membrane Protein/Amphipol Complexes
265
Folding Membrane Proteins in Amphipols
As shown in Table 5.1, a dozen MPs have been transferred and folded in APols starting from a
denatured state. The denaturing medium is typically sodium dodecyl sulfate (SDS) for α-helical
proteins (BR and seven G protein-coupled receptors (GPCRs)) and urea for β-barrel ones (four
bacterial outer membrane proteins). The dodecyl sulfate is typically removed by precipitating it as
its potassium salt, PDS, whereas the urea is diluted. The procedure has been validated for A8-35
(Pocanschi et al. 2006b; for a recent review, see Le Bon et al. 2018), SAPols (Dahmane et al. 2009),
NAPols (Bazzacco et al. 2012), and amphipathic poly-γ-glutamic acid (APG) (Han et al. 2014). A few
synthetic peptides have been transferred to A8-35 from either urea or trifluoroethanol (Gohon 1996;
Duarte et al. 2008; Stangl et al. 2014). In comparative studies, it was shown that, provided lipids have
not been removed, the functional properties of BR refolded in A8-35 are exactly the same as those of
native BR trapped in the same APols starting from a solution in non-denaturing detergent (Dahmane
et al. 2013). Similarly, GPCRs folded in APols from SDS-solubilized inclusion bodies exhibit the same
pharmacological properties as native receptors (Dahmane et al. 2009; Banères et al. 2011; Bazzacco
et al. 2012; Mary et al. 2014; Logez et al. 2016). Folding MPs to their native form is one of the most
promising uses of APols and will be discussed in detail in Chap. 6.
Amphipol-assisted folding of membrane proteins
(# 2018 by Francis Haraux)
Alternatively, MPs can be synthesized in vitro using APols as the accepting medium. However,
polyanionic APols such as A8-35, SAPols, and SMA have all been found to inhibit cell-free synthesis
of MPs, possibly by interacting with cationic sites at the surface of the translation apparatus (Park et al.
2011; Periasamy et al. 2013). Only glucose-based NAPols (Bazzacco et al. 2012) and NVoy (Guild
et al. 2011; Klammt et al. 2011), both of which are non-ionic, have been validated to date for use in MP
cell-free expression. Using APols for this application will be discussed in Chap. 7.
5.2 Forming Membrane Protein/Amphipol Complexes
265