course of the conformational excursions experienced by a (re)folding protein, transient, partially folded
states appear, some of which may exhibit lipid-binding sites. Binding of lipids to such an intermediate
state will stabilize it, lengthening the period of time during which folding has a chance to proceed to
completion. In other words, binding of lipids to partially folded states may steer folding toward a
native-like conformation (Dahmane et al. 2013; Zoonens and Popot 2014). Once the native structure is
achieved, the binding of lipids will stabilize it, diminishing the frequency of conformational excursions
that could lead to denaturation, misfolding, and/or aggregation.
Why is it that folding of MPs in APols is, apparently, so efficient, even though this medium is so
unlike a lipid bilayer, both in its chemistry and in its organization? Among the various mechanisms of
stabilization that have been discussed in Chap. 5 (§ 5.6), the most relevant one may be their poorly
dissociating character. When folding is carried out in detergent, or in lipid/detergent mixtures, the
detergent – a surfactant that was initially selected for its dissociating properties – competes with
reforming protein/protein and protein/lipid interactions. In the scheme of Fig. 6.11A, this means that
folding to the native structure ① has to compete with partial folding ②, in which protein/surfactant
interactions replace some protein/protein ones, misfolding ③, in which non-native-like interactions
form intramolecularly, and aggregation ④, induced by intermolecular protein/protein interactions.
APols, because of their low detergency, can be expected to favor the productive path ①, which, being
more accessible, should more efficiently compete with the unproductive paths ②, ③, and ④
(Fig. 6.11B). Furthermore, most MPs are not particularly stable in detergent solutions, so that a protein
that has managed to reach a native-like state is at risk of denaturing again at a later stage. In SDS or
urea, it would go back to the “denatured” state, from which it can fold again. In a “non-denaturing”
detergent, however, which is not so dissociating, it may well reach a misfolded ③ and/or aggregated
④ state, from which it may not recover (Fig. 6.11A). In an APol environment, chances that a protein
that has reached a partially folded state ② will move to the native state ① can be expected to be higher,
because of the lesser competition of protein/surfactant interactions with native-like protein/protein and
protein/lipid ones. This, in turn, should diminish the risks of moving to the irreversible states ③ and ④
(Fig. 6.11B).
According to this view, APols could provide a good folding medium because, on the one hand,
they adsorb onto hydrophobic surfaces, keeping unfolded MPs from aggregating (or slowing down
their aggregation), while, on the other hand, they do not compete efficiently with the protein/protein
and protein/lipid interactions that define the native structure. Thus, they would substitute for SDS or
urea at the surface of the unfolded protein, keeping it soluble, but be progressively displaced from
those protein surfaces that can form stronger interactions either with other proteic elements or with
lipids. Although the term “molecular chaperones” has been overused and misused, it may be to some
extent appropriate here, in the sense that APols may slow down the formation of non-specific,
unproductive interactions between hydrophobic segments, which would lead to misfolding and/or
aggregation, while moving out of the way when specific interactions establish themselves.
Three types of APols have led to successful (re)folding of MPs to date: A8-35, SAPols, and
NAPols. Except for BR, for which comparable folding yields are achieved in A8-35 and in NAPols
(Pocanschi et al. 2006; Bazzacco et al. 2012; Dahmane et al. 2013), no comparative studies of the
folding yields achieved for one given MP using one or the other APol have been carried out yet. On the
basis of early experiments, it was proposed that A8-35 formed around refolding MPs a sort of
protective “bubble” that would allow folding to proceed while slowing down the formation of
nonproductive intermolecular interactions (Pocanschi et al. 2006) (Fig. 6.11B). One possible mechanism providing relative isolation of refolding proteins from one another could be the electrostatic
repulsion between complexes incorporating either A8-35 or SAPols: both of them are polyanions,
whose interactions indeed strongly depend on ionic strength (Zoonens et al. 2007). The fact that the
6.3 Amphipol-Assisted Folding of Membrane Proteins
351
states appear, some of which may exhibit lipid-binding sites. Binding of lipids to such an intermediate
state will stabilize it, lengthening the period of time during which folding has a chance to proceed to
completion. In other words, binding of lipids to partially folded states may steer folding toward a
native-like conformation (Dahmane et al. 2013; Zoonens and Popot 2014). Once the native structure is
achieved, the binding of lipids will stabilize it, diminishing the frequency of conformational excursions
that could lead to denaturation, misfolding, and/or aggregation.
Why is it that folding of MPs in APols is, apparently, so efficient, even though this medium is so
unlike a lipid bilayer, both in its chemistry and in its organization? Among the various mechanisms of
stabilization that have been discussed in Chap. 5 (§ 5.6), the most relevant one may be their poorly
dissociating character. When folding is carried out in detergent, or in lipid/detergent mixtures, the
detergent – a surfactant that was initially selected for its dissociating properties – competes with
reforming protein/protein and protein/lipid interactions. In the scheme of Fig. 6.11A, this means that
folding to the native structure ① has to compete with partial folding ②, in which protein/surfactant
interactions replace some protein/protein ones, misfolding ③, in which non-native-like interactions
form intramolecularly, and aggregation ④, induced by intermolecular protein/protein interactions.
APols, because of their low detergency, can be expected to favor the productive path ①, which, being
more accessible, should more efficiently compete with the unproductive paths ②, ③, and ④
(Fig. 6.11B). Furthermore, most MPs are not particularly stable in detergent solutions, so that a protein
that has managed to reach a native-like state is at risk of denaturing again at a later stage. In SDS or
urea, it would go back to the “denatured” state, from which it can fold again. In a “non-denaturing”
detergent, however, which is not so dissociating, it may well reach a misfolded ③ and/or aggregated
④ state, from which it may not recover (Fig. 6.11A). In an APol environment, chances that a protein
that has reached a partially folded state ② will move to the native state ① can be expected to be higher,
because of the lesser competition of protein/surfactant interactions with native-like protein/protein and
protein/lipid ones. This, in turn, should diminish the risks of moving to the irreversible states ③ and ④
(Fig. 6.11B).
According to this view, APols could provide a good folding medium because, on the one hand,
they adsorb onto hydrophobic surfaces, keeping unfolded MPs from aggregating (or slowing down
their aggregation), while, on the other hand, they do not compete efficiently with the protein/protein
and protein/lipid interactions that define the native structure. Thus, they would substitute for SDS or
urea at the surface of the unfolded protein, keeping it soluble, but be progressively displaced from
those protein surfaces that can form stronger interactions either with other proteic elements or with
lipids. Although the term “molecular chaperones” has been overused and misused, it may be to some
extent appropriate here, in the sense that APols may slow down the formation of non-specific,
unproductive interactions between hydrophobic segments, which would lead to misfolding and/or
aggregation, while moving out of the way when specific interactions establish themselves.
Three types of APols have led to successful (re)folding of MPs to date: A8-35, SAPols, and
NAPols. Except for BR, for which comparable folding yields are achieved in A8-35 and in NAPols
(Pocanschi et al. 2006; Bazzacco et al. 2012; Dahmane et al. 2013), no comparative studies of the
folding yields achieved for one given MP using one or the other APol have been carried out yet. On the
basis of early experiments, it was proposed that A8-35 formed around refolding MPs a sort of
protective “bubble” that would allow folding to proceed while slowing down the formation of
nonproductive intermolecular interactions (Pocanschi et al. 2006) (Fig. 6.11B). One possible mechanism providing relative isolation of refolding proteins from one another could be the electrostatic
repulsion between complexes incorporating either A8-35 or SAPols: both of them are polyanions,
whose interactions indeed strongly depend on ionic strength (Zoonens et al. 2007). The fact that the
6.3 Amphipol-Assisted Folding of Membrane Proteins
351