Amphipol-assisted cell-free expression of membrane proteins
(# 2018 by Francis Haraux)
In order to determine whether this inhibition was specific to MPs, expression of the soluble green
fluorescent protein (GFP) was tested under the same conditions (Fig. 7.5C). No detectable inhibition was
observed. This strongly suggests that the inhibition is not due to a general perturbation of the transcription/translation machineries but is specific to MPs. A possibility is that the inhibition is somehow related
to the binding of APols to hydrophobic segments of MPs emerging from the ribosome tunnel: negative
charges carried by A8-35 and SAPols bound to the emerging polypeptide may interact with basic side
chains at the surface of the ribosome or within the mouth of the ribosome tunnel, thus sterically slowing
down or blocking elongation, much as certain antibiotics seem to do by a different but equally steric
mechanism (see e.g. Arévalo et al. 1988; Bulkley et al. 2010; and references therein). The incomplete
inhibition observed with tLEP and MscL, at variance with the complete blockade of BR synthesis, may
perhaps be related to their different number of TM helices. In MLS B K antibiotic-resistant E. coli strains
that lack three critical basic residues in the tunnel-exposed β-hairpin loop of subunit L22, the mouth of
the tunnel is more widely open, which appears to account for the resistance to erythromycin, given that
the latter binds perfectly normally (Tu et al. 2005). The possibility that lysates derived from such mutants
would allow CFE of MPs in the presence of polyanionic APols is a far-fetched hypothesis but might
perhaps be worth testing (for further discussion, see § 7.4).
The hypothesis that the negative charges carried by A8-35 and SAPols are somehow responsible
for the inhibition is generally although not fully consistent with later work described in Studies 7.2–5
(Table 7.1). In Study 7.5, a large number of surfactants were examined for their ability to support cellfree synthesis and solubility of CrdS, the curdlan synthase from Agrobacterium sp., an ~72 kDa MP
predicted to comprise seven TM α-helices and a single, large cytoplasmic region inserted between
helices 3 and 4 (Hrmova et al. 2010). CFE in wheat germ lysates was found to be efficient in the
presence of Brij-58 or of liposomes but was inhibited by 5–40 gÁL
-1 concentrations of either A8-35 or
styrene-maleic acid (SMA) co-polymers, both of which are polyanionic polymers (Periasamy et al.
2013). It should be noted, however, that in Study 7.3, PMAL-B-100, an APol that is zwitterionic at and
368
7 Amphipol-Assisted Cell-Free Expression of Membrane Proteins
(# 2018 by Francis Haraux)
In order to determine whether this inhibition was specific to MPs, expression of the soluble green
fluorescent protein (GFP) was tested under the same conditions (Fig. 7.5C). No detectable inhibition was
observed. This strongly suggests that the inhibition is not due to a general perturbation of the transcription/translation machineries but is specific to MPs. A possibility is that the inhibition is somehow related
to the binding of APols to hydrophobic segments of MPs emerging from the ribosome tunnel: negative
charges carried by A8-35 and SAPols bound to the emerging polypeptide may interact with basic side
chains at the surface of the ribosome or within the mouth of the ribosome tunnel, thus sterically slowing
down or blocking elongation, much as certain antibiotics seem to do by a different but equally steric
mechanism (see e.g. Arévalo et al. 1988; Bulkley et al. 2010; and references therein). The incomplete
inhibition observed with tLEP and MscL, at variance with the complete blockade of BR synthesis, may
perhaps be related to their different number of TM helices. In MLS B K antibiotic-resistant E. coli strains
that lack three critical basic residues in the tunnel-exposed β-hairpin loop of subunit L22, the mouth of
the tunnel is more widely open, which appears to account for the resistance to erythromycin, given that
the latter binds perfectly normally (Tu et al. 2005). The possibility that lysates derived from such mutants
would allow CFE of MPs in the presence of polyanionic APols is a far-fetched hypothesis but might
perhaps be worth testing (for further discussion, see § 7.4).
The hypothesis that the negative charges carried by A8-35 and SAPols are somehow responsible
for the inhibition is generally although not fully consistent with later work described in Studies 7.2–5
(Table 7.1). In Study 7.5, a large number of surfactants were examined for their ability to support cellfree synthesis and solubility of CrdS, the curdlan synthase from Agrobacterium sp., an ~72 kDa MP
predicted to comprise seven TM α-helices and a single, large cytoplasmic region inserted between
helices 3 and 4 (Hrmova et al. 2010). CFE in wheat germ lysates was found to be efficient in the
presence of Brij-58 or of liposomes but was inhibited by 5–40 gÁL
-1 concentrations of either A8-35 or
styrene-maleic acid (SMA) co-polymers, both of which are polyanionic polymers (Periasamy et al.
2013). It should be noted, however, that in Study 7.3, PMAL-B-100, an APol that is zwitterionic at and
368
7 Amphipol-Assisted Cell-Free Expression of Membrane Proteins