but not their ATPase activity, because SMALPs are destabilized by divalent metal ions (Gulati et al.
2014).
Redox reactions have been evidenced with several photosystems, respiratory complexes, and
other redox enzymes trapped in A8-35 (Kievit and Brudvig 2001; Nowaczyk et al. 2004; Althoff et al.
2011; Laursen et al. 2013; Charvolin et al. 2014; Shinzawa-Itoh et al. 2016) or SMALPs (Laursen et al.
2016). The oxidation of reduced cytochrome c by SMA-trapped cytochrome c oxidase can be observed
only after depleting the system of free SMA, which interferes with the binding of cytochrome c to the
oxidase (Smirnova et al. 2016).
A particularly interesting case is that of SERCA1a, the Ca
2+ -ATPase, which has been studied in
some details. SERCA1a, which is located in the sarcoplasmic reticulum of fast-twitch muscle, extracts
calcium ions from the cytosol. This relaxes actomyosin and, thereby, brings muscle contraction to an
end (see Chap. 1, § 1.6.3). Several APols (A8-35, PMAL-C12, PMALA-C12, and SAPols) have been
found to both protect SERCA1a against inactivation (see § 5.6) and to slow down its turnover
(Champeil et al. 2000; Picard et al. 2006). Intermediate effects are observed in mixtures of APols
and detergent. Trivial mechanisms, such as sequestration of Ca
2+ or interference with ATP binding, do
not account for the inhibition (Champeil et al. 2000; Picard et al. 2006). As will be discussed in § 5.6,
both the inhibition and stabilization of SERCA1a, and their intriguing correlation, can probably be
traced to a common cause, namely the influence of APols on protein dynamics.
Inhibition by A8-35 of the ATPase activity of the F 1 F O ATP synthase, reversible upon addition
of an excess of detergent, has also been reported, but not studied in any details (Wilkens et al. 2000).
5.5
Biochemical Stability of Amphipol-Trapped Membrane Proteins
The fact that transferring a MP from detergent solution to APols stabilizes it is a very general even
though not absolutely universal observation (see e.g. Tribet et al. 1996; Champeil et al. 2000; Picard
et al. 2006; Gohon et al. 2008; Dahmane et al. 2009; Tifrea et al. 2011; Bazzacco et al. 2012; Cocco
et al. 2013; Dahmane et al. 2013; Etzkorn et al. 2013; Pocanschi et al. 2013; Huynh et al. 2014;
reviewed in Kleinschmidt and Popot 2014; Zoonens and Popot 2014; Le Bon et al. 2018). It is
illustrated in Fig. 5.27 in the case of BR. Note that, as discussed in Chap. 2, § 2.4, one of the
mechanisms by which surfactants destabilize MPs is by diluting subunits or bound lipids in the
“phase” represented by the associated surfactant. In order to compare the intrinsic inactivating or
stabilizing properties of surfactants, one must therefore, as in the case of Fig. 5.26, pay attention to the
volume of non-monomeric, associated surfactant the protein is exposed to, so as to distinguish effects
that merely result from differences in the volume of the “hydrophobic sink” (see Chap. 2, § 2.4) from
those that reflect molecular interactions between the protein and the surfactant molecules that form the
layer surrounding its TM region.
The effects of trapping a fixed quantity of BR with increasing amounts of APols have been
examined in some details (Dahmane 2007). Native BR was extracted from PM with OTG along with
PM lipids (Gohon et al. 2008). Upon trapping it with A8-35 at BR/APol mass ratios ranging from 1:5
to 1:50, ternary BR/lipid/APol complexes formed. Control samples were stored in either 18 mM or
25 mM OTG. Under the experimental conditions used, the volume of the hydrophobic sink
(non-monomeric surfactant) was roughly comparable in 18 or 25 mM OTG and at BR/A8-35 ratios
of 1:10 or 1:20, respectively. At room temperature, BR is stable for at least a week whatever the
concentration of APol, whereas in OTG it denatures almost totally over the same period (Fig. 5.26,
left). Even at 40
C, BR trapped at low (1:5–1:10 w/w) APol ratios is highly stabilized by A8-35,
denaturing by <10% over a week (Fig. 5.26, right). At this temperature, the protein in OTG is totally
inactivated in less than a day. Whereas a large excess of APols (ratio 1:50) is well tolerated at 4
C and
290
5 Formation and Properties of Membrane Protein/Amphipol Complexes
2014).
Redox reactions have been evidenced with several photosystems, respiratory complexes, and
other redox enzymes trapped in A8-35 (Kievit and Brudvig 2001; Nowaczyk et al. 2004; Althoff et al.
2011; Laursen et al. 2013; Charvolin et al. 2014; Shinzawa-Itoh et al. 2016) or SMALPs (Laursen et al.
2016). The oxidation of reduced cytochrome c by SMA-trapped cytochrome c oxidase can be observed
only after depleting the system of free SMA, which interferes with the binding of cytochrome c to the
oxidase (Smirnova et al. 2016).
A particularly interesting case is that of SERCA1a, the Ca
2+ -ATPase, which has been studied in
some details. SERCA1a, which is located in the sarcoplasmic reticulum of fast-twitch muscle, extracts
calcium ions from the cytosol. This relaxes actomyosin and, thereby, brings muscle contraction to an
end (see Chap. 1, § 1.6.3). Several APols (A8-35, PMAL-C12, PMALA-C12, and SAPols) have been
found to both protect SERCA1a against inactivation (see § 5.6) and to slow down its turnover
(Champeil et al. 2000; Picard et al. 2006). Intermediate effects are observed in mixtures of APols
and detergent. Trivial mechanisms, such as sequestration of Ca
2+ or interference with ATP binding, do
not account for the inhibition (Champeil et al. 2000; Picard et al. 2006). As will be discussed in § 5.6,
both the inhibition and stabilization of SERCA1a, and their intriguing correlation, can probably be
traced to a common cause, namely the influence of APols on protein dynamics.
Inhibition by A8-35 of the ATPase activity of the F 1 F O ATP synthase, reversible upon addition
of an excess of detergent, has also been reported, but not studied in any details (Wilkens et al. 2000).
5.5
Biochemical Stability of Amphipol-Trapped Membrane Proteins
The fact that transferring a MP from detergent solution to APols stabilizes it is a very general even
though not absolutely universal observation (see e.g. Tribet et al. 1996; Champeil et al. 2000; Picard
et al. 2006; Gohon et al. 2008; Dahmane et al. 2009; Tifrea et al. 2011; Bazzacco et al. 2012; Cocco
et al. 2013; Dahmane et al. 2013; Etzkorn et al. 2013; Pocanschi et al. 2013; Huynh et al. 2014;
reviewed in Kleinschmidt and Popot 2014; Zoonens and Popot 2014; Le Bon et al. 2018). It is
illustrated in Fig. 5.27 in the case of BR. Note that, as discussed in Chap. 2, § 2.4, one of the
mechanisms by which surfactants destabilize MPs is by diluting subunits or bound lipids in the
“phase” represented by the associated surfactant. In order to compare the intrinsic inactivating or
stabilizing properties of surfactants, one must therefore, as in the case of Fig. 5.26, pay attention to the
volume of non-monomeric, associated surfactant the protein is exposed to, so as to distinguish effects
that merely result from differences in the volume of the “hydrophobic sink” (see Chap. 2, § 2.4) from
those that reflect molecular interactions between the protein and the surfactant molecules that form the
layer surrounding its TM region.
The effects of trapping a fixed quantity of BR with increasing amounts of APols have been
examined in some details (Dahmane 2007). Native BR was extracted from PM with OTG along with
PM lipids (Gohon et al. 2008). Upon trapping it with A8-35 at BR/APol mass ratios ranging from 1:5
to 1:50, ternary BR/lipid/APol complexes formed. Control samples were stored in either 18 mM or
25 mM OTG. Under the experimental conditions used, the volume of the hydrophobic sink
(non-monomeric surfactant) was roughly comparable in 18 or 25 mM OTG and at BR/A8-35 ratios
of 1:10 or 1:20, respectively. At room temperature, BR is stable for at least a week whatever the
concentration of APol, whereas in OTG it denatures almost totally over the same period (Fig. 5.26,
left). Even at 40
C, BR trapped at low (1:5–1:10 w/w) APol ratios is highly stabilized by A8-35,
denaturing by <10% over a week (Fig. 5.26, right). At this temperature, the protein in OTG is totally
inactivated in less than a day. Whereas a large excess of APols (ratio 1:50) is well tolerated at 4
C and
290
5 Formation and Properties of Membrane Protein/Amphipol Complexes