stable in the presence of APols than in that of detergents (see e.g. Fig. 5.26). In other terms,
for an equal volume of hydrophobic sink, APols are less destabilizing than detergents. This
may have several causes, which may contribute differently to the stabilization of different
proteins. For instance, the fact that lipids rebind (in the case of BR) or are likely to rebind
(in the case of the nAChR) upon transfer of a MP from detergent solution to APols suggests
that APols do not compete as efficiently as detergents do to displace lipids from TM protein
surfaces. Because lipids play an important role in stabilizing MP TM regions (see Chap. 2),
this, as observed, for example for BR (Fig. 5.27C) or for the leukotriene BLT1 receptor
(Fig. 5.27), will by itself have a stabilizing effect. By the same token, APols may be less able
to compete with the protein/protein interactions that keep together TM segments and TM
subunits. In keeping with this hypothesis, A8-35 has indeed been found to be less efficient
than detergents at dissociating, in the absence of lipids, the dimer formed by the TM αhelical anchor of glycophorin A (Stangl et al. 2014). Similarly, BR exposed to A8-35 in the
absence of lipids is much more stable than when exposed to OTG in their presence (compare
Fig. 5.27A, C; Dahmane et al. 2013).
(ii) Because APols do not spontaneously desorb from MPs unless they are displaced by another
surfactant (cf. Fig. 5.7), it is possible, circumstances permitting, to work in the presence of
very little or no free APols. Such is the case, for instance, when working with dilute protein
solutions. When using submicromolar concentrations of protein, for instance, as frequently
happens for functional or spectroscopic measurements, the total concentration of APol
(bound + free) can be kept in the range of tens of μg per mL without incurring protein
aggregation. This is lower than can be achieved with most detergents. When aggregation is
rendered impossible, as is the case for a MP immobilized on a chip or a column, or if a
limited degree of aggregation is tolerable, as can be the case for EM single-particle imaging
or for functional measurements, free APol can be dispensed with entirely. As seen in
Fig. 5.26B, lowering the APol concentration can, by diminishing the volume of the
hydrophobic sink, further slow down protein inactivation.
These two mechanisms are classical in the sense that they apply to any surfactant and are at the
basis of strategies for improving MP stability when working with detergents (Chap. 2). Several
observations suggest that a third mechanism may be involved in the case of APols, namely their
damping of the dynamics of MPs, which would affect both their stability and, in some cases, their
function. The evidence in favor of this (hypothetical) mechanism will be discussed in the next section.
5.6
Membrane Protein Dynamics and the Effects of Amphipols on Stability
and Function
The idea that damping by APols of MP dynamics may be involved in their effects on stability and
function (Popot et al. 2003) emerged as the outcome of studies on the sarcoplasmic calcium pump
(SERCA1a) (Champeil et al. 2000; Picard et al. 2006), compared to those on BR (Gohon et al. 2008)
and the nAChR (Martinez et al. 2002). It has received some support from MD studies of OmpX/A8-35
complexes (Perlmutter et al. 2014). We will start by discussing the experimental observations and then
confront to the MD data the hypothesis they led to.
296
5 Formation and Properties of Membrane Protein/Amphipol Complexes
for an equal volume of hydrophobic sink, APols are less destabilizing than detergents. This
may have several causes, which may contribute differently to the stabilization of different
proteins. For instance, the fact that lipids rebind (in the case of BR) or are likely to rebind
(in the case of the nAChR) upon transfer of a MP from detergent solution to APols suggests
that APols do not compete as efficiently as detergents do to displace lipids from TM protein
surfaces. Because lipids play an important role in stabilizing MP TM regions (see Chap. 2),
this, as observed, for example for BR (Fig. 5.27C) or for the leukotriene BLT1 receptor
(Fig. 5.27), will by itself have a stabilizing effect. By the same token, APols may be less able
to compete with the protein/protein interactions that keep together TM segments and TM
subunits. In keeping with this hypothesis, A8-35 has indeed been found to be less efficient
than detergents at dissociating, in the absence of lipids, the dimer formed by the TM αhelical anchor of glycophorin A (Stangl et al. 2014). Similarly, BR exposed to A8-35 in the
absence of lipids is much more stable than when exposed to OTG in their presence (compare
Fig. 5.27A, C; Dahmane et al. 2013).
(ii) Because APols do not spontaneously desorb from MPs unless they are displaced by another
surfactant (cf. Fig. 5.7), it is possible, circumstances permitting, to work in the presence of
very little or no free APols. Such is the case, for instance, when working with dilute protein
solutions. When using submicromolar concentrations of protein, for instance, as frequently
happens for functional or spectroscopic measurements, the total concentration of APol
(bound + free) can be kept in the range of tens of μg per mL without incurring protein
aggregation. This is lower than can be achieved with most detergents. When aggregation is
rendered impossible, as is the case for a MP immobilized on a chip or a column, or if a
limited degree of aggregation is tolerable, as can be the case for EM single-particle imaging
or for functional measurements, free APol can be dispensed with entirely. As seen in
Fig. 5.26B, lowering the APol concentration can, by diminishing the volume of the
hydrophobic sink, further slow down protein inactivation.
These two mechanisms are classical in the sense that they apply to any surfactant and are at the
basis of strategies for improving MP stability when working with detergents (Chap. 2). Several
observations suggest that a third mechanism may be involved in the case of APols, namely their
damping of the dynamics of MPs, which would affect both their stability and, in some cases, their
function. The evidence in favor of this (hypothetical) mechanism will be discussed in the next section.
5.6
Membrane Protein Dynamics and the Effects of Amphipols on Stability
and Function
The idea that damping by APols of MP dynamics may be involved in their effects on stability and
function (Popot et al. 2003) emerged as the outcome of studies on the sarcoplasmic calcium pump
(SERCA1a) (Champeil et al. 2000; Picard et al. 2006), compared to those on BR (Gohon et al. 2008)
and the nAChR (Martinez et al. 2002). It has received some support from MD studies of OmpX/A8-35
complexes (Perlmutter et al. 2014). We will start by discussing the experimental observations and then
confront to the MD data the hypothesis they led to.
296
5 Formation and Properties of Membrane Protein/Amphipol Complexes