Kraft et al. 2015). Stabilization upon transfer from detergent to APols has been observed with several
other MPs, including the sarcoplasmic Ca
2+ pump SERCA1a (Champeil et al. 2000; Picard et al. 2006;
see § 5.6), the respiratory complex cytochrome bc 1 (Charvolin et al. 2014), and the prokaryotic
voltage-gated sodium channel NavMs (Ireland et al. 2017). Less direct observations also suggest
stabilization of yet other α-helical MPs by APols as compared to detergents. A case in point is the ion
channel TRPV1, which has been studied by electron microscopy after trapping in A8-35. Galleries of
images of negatively stained particles indicate that their overall shape is much more reproducible in
A8-35 than it is in DDM, suggesting stabilization (Cao et al. 2013; Liao et al. 2013; see Chap. 12,
§ 12.3.4.2, Fig. 12.26).
APol-induced stabilization has also been noted for several β-barrel MPs, such as the major outer
membrane protein (MOMP) from Chlamydia trachomatis. At pH 7.4, the midpoint of the transition of
heat-induced unfolding of the MOMP trimer in Zwittergent 3–14 is ~52
C, whereas A8-35-trapped
MOMP does not unfold at all up to 78
C, indicating an exceptionally strong stabilization (Tifrea et al.
2011), which translates into a remarkable stability upon extended storage (Tifrea et al. 2011; Cocco
et al. 2013; see Chap. 15, Figs. 15.2 and 15.3).
Note that, whereas APols are generally used to stabilize MPs, extended exposure (from 40 min to
24 h and more at 4
C) to high concentrations (3.5%) of A8-35 has been exploited to gently and
progressively disassemble the dimeric F 1 F O ATP synthase from Polytomella sp. (Vázquez-Acevedo
et al. 2016).
A detailed thermodynamic study of the stabilization of E. coli OmpA by A8-35 carried out by
J.H. Kleinschmidt and colleagues has yielded interesting insights into stabilization mechanisms
(Pocanschi et al. 2013). Equilibrium folding/unfolding by urea was compared following trapping
with A8-35 vs. in LDAO solution, allowing the calculation and comparison of the free energies of
unfolding. In line with an earlier study of the stability of OmpA in sonicated lipid vesicles (Hong and
Tamm 2004), reversible folding/unfolding conditions were only achieved at pH 10. Unfolding and
folding titration curves superimposed only after very long incubation times: 30–40 days for OmpA/
A8-35 complexes and 18–25 days for OmpA/LDAO ones. It is the unfolding reaction that imposes
these long equilibration times. In urea at pH 10, OmpA is thermodynamically more stable in LDAO
than in A8-35. However, the activation free energy for unfolding OmpA is much higher in A8-35 than
in LDAO, as indicated by the slower kinetics. At pH 10, OmpA (pI ¼ 5.5) is strongly negatively
charged, because of the deprotonation of some of its 17 lysine (pK % 9.5–10.5) and 17 tyrosine (pK %
9.5–10) residues. The resulting increased negative charge leads to intermolecular repulsion and
stronger side-chain hydration, which prevents aggregation of the denatured proteins and ensures a
better solubility and reversibility. However, the increased net negative charge of OmpA may also lead
to less stable complexes with the negatively charged A8-35, because of charge-charge repulsion. This
might be the reason for the reduced thermodynamic stability of folded OmpA observed in A8-35 as
compared to LDAO. However, the activation energy of unfolding of OmpA being much higher in
A8-35 than in LDAO, it takes longer for OmpA to unfold, despite its lower thermodynamic stability
(Pocanschi et al. 2013). This is consistent with the view that part of the stabilizing properties of APols
results from their damping the conformational excursions of MPs, which slows down denaturation
(see § 5.6).
Trapping with SMA has been observed to strongly stabilize PagP (Knowles et al. 2009),
bacterial photosynthetic reaction centers (Swainsbury et al. 2014; Fig. 5.30A), or, more modestly,
the adenosine A 2A receptor (A 2A R) (Jamshad et al. 2015a, b; Fig. 5.30B). On the basis of NMR data,
the GPCR CRFR2β has been reported to be stable after transfer to NVoy (Klammt et al. 2011), but no
comparative studies of the stability of MPs complexed by this polymer vs. detergent-solubilized ones
have been reported yet.
Comparative studies about the relative ability of various APols to stabilize MPs remain scarce,
but they seem to point in at least one direction: the less charges APols bear, the more stabilizing they
294
5 Formation and Properties of Membrane Protein/Amphipol Complexes
other MPs, including the sarcoplasmic Ca
2+ pump SERCA1a (Champeil et al. 2000; Picard et al. 2006;
see § 5.6), the respiratory complex cytochrome bc 1 (Charvolin et al. 2014), and the prokaryotic
voltage-gated sodium channel NavMs (Ireland et al. 2017). Less direct observations also suggest
stabilization of yet other α-helical MPs by APols as compared to detergents. A case in point is the ion
channel TRPV1, which has been studied by electron microscopy after trapping in A8-35. Galleries of
images of negatively stained particles indicate that their overall shape is much more reproducible in
A8-35 than it is in DDM, suggesting stabilization (Cao et al. 2013; Liao et al. 2013; see Chap. 12,
§ 12.3.4.2, Fig. 12.26).
APol-induced stabilization has also been noted for several β-barrel MPs, such as the major outer
membrane protein (MOMP) from Chlamydia trachomatis. At pH 7.4, the midpoint of the transition of
heat-induced unfolding of the MOMP trimer in Zwittergent 3–14 is ~52
C, whereas A8-35-trapped
MOMP does not unfold at all up to 78
C, indicating an exceptionally strong stabilization (Tifrea et al.
2011), which translates into a remarkable stability upon extended storage (Tifrea et al. 2011; Cocco
et al. 2013; see Chap. 15, Figs. 15.2 and 15.3).
Note that, whereas APols are generally used to stabilize MPs, extended exposure (from 40 min to
24 h and more at 4
C) to high concentrations (3.5%) of A8-35 has been exploited to gently and
progressively disassemble the dimeric F 1 F O ATP synthase from Polytomella sp. (Vázquez-Acevedo
et al. 2016).
A detailed thermodynamic study of the stabilization of E. coli OmpA by A8-35 carried out by
J.H. Kleinschmidt and colleagues has yielded interesting insights into stabilization mechanisms
(Pocanschi et al. 2013). Equilibrium folding/unfolding by urea was compared following trapping
with A8-35 vs. in LDAO solution, allowing the calculation and comparison of the free energies of
unfolding. In line with an earlier study of the stability of OmpA in sonicated lipid vesicles (Hong and
Tamm 2004), reversible folding/unfolding conditions were only achieved at pH 10. Unfolding and
folding titration curves superimposed only after very long incubation times: 30–40 days for OmpA/
A8-35 complexes and 18–25 days for OmpA/LDAO ones. It is the unfolding reaction that imposes
these long equilibration times. In urea at pH 10, OmpA is thermodynamically more stable in LDAO
than in A8-35. However, the activation free energy for unfolding OmpA is much higher in A8-35 than
in LDAO, as indicated by the slower kinetics. At pH 10, OmpA (pI ¼ 5.5) is strongly negatively
charged, because of the deprotonation of some of its 17 lysine (pK % 9.5–10.5) and 17 tyrosine (pK %
9.5–10) residues. The resulting increased negative charge leads to intermolecular repulsion and
stronger side-chain hydration, which prevents aggregation of the denatured proteins and ensures a
better solubility and reversibility. However, the increased net negative charge of OmpA may also lead
to less stable complexes with the negatively charged A8-35, because of charge-charge repulsion. This
might be the reason for the reduced thermodynamic stability of folded OmpA observed in A8-35 as
compared to LDAO. However, the activation energy of unfolding of OmpA being much higher in
A8-35 than in LDAO, it takes longer for OmpA to unfold, despite its lower thermodynamic stability
(Pocanschi et al. 2013). This is consistent with the view that part of the stabilizing properties of APols
results from their damping the conformational excursions of MPs, which slows down denaturation
(see § 5.6).
Trapping with SMA has been observed to strongly stabilize PagP (Knowles et al. 2009),
bacterial photosynthetic reaction centers (Swainsbury et al. 2014; Fig. 5.30A), or, more modestly,
the adenosine A 2A receptor (A 2A R) (Jamshad et al. 2015a, b; Fig. 5.30B). On the basis of NMR data,
the GPCR CRFR2β has been reported to be stable after transfer to NVoy (Klammt et al. 2011), but no
comparative studies of the stability of MPs complexed by this polymer vs. detergent-solubilized ones
have been reported yet.
Comparative studies about the relative ability of various APols to stabilize MPs remain scarce,
but they seem to point in at least one direction: the less charges APols bear, the more stabilizing they
294
5 Formation and Properties of Membrane Protein/Amphipol Complexes