complexes even more (see § 10.3.1). Smaller NDs have been engineered, but this may come
at the cost of moving MP/ND complexes away from a bilayer-like environment and imposing
new constraints on the protein (see Chap. 3, § 3.3.2). As regards the size of bicelles, it can be
modulated by playing on the proportion of plane and ring surfactant (Chap. 3, § 3.2).
• Sample preparation. MP/detergent complexes are the form under which most MPs are
initially obtained, and their transfer to APols is straightforward (Chap. 5). Alternatively, the
protein can be directly folded (Chap. 6) or expressed (Chap. 7) in the presence of APols.
Handling bicelles is more difficult, and the preparation of homogeneous preparations of
MP/ND complexes is work-intensive (Ritchie et al. 2009).
• Stability. Because of their dissociating properties, detergents provide the most unfriendly,
least membrane-like environment, and, as a result, the stability of most MPs is often poor,
limiting the duration of the experiments and the temperature at which they can be carried out.
The stability is almost always increased, most often dramatically so, upon transfer to APols.
This is in part due to the rebinding of lipids (see Chap. 5), which reconstitutes, to some extent,
interactions more akin to those the MP experiences in its native membrane environment than
it does in detergent solution. Bicelles offer a bilayer-like environment, but contaminated by
the ring surfactant, either a detergent or short-chain, detergent-like lipids, which partitions in
the bilayer. There are data suggesting that they provide a stabilizing environment (see
e.g. Poget et al. 2007), but those are scarce. NDs offer the environment closest to a lipid
bilayer, and the few comparative data available indicate that ND-trapped MPs can be even
more stable than APol-trapped ones (see e.g. Popot 2010; Etzkorn et al. 2013).
• Isotopic labeling. Finally, isotopic labeling is straightforward in detergent solution and
bicelles – that is, provided the protein is stable enough in the presence of those few surfactants
that exist in deuterated form (Hiruma-Shimizu et al. 2015). It is also relatively easy in the case
of APols, within the constraints discussed in § 10.3.2. There is nothing to prevent the
preparation of NDs formed of deuterated DMPC and deuterated scaffold proteins, but for
the cost and work involved.
One of the assets of APols is their great chemical versatility. Deuteration has already been
implemented as regards A8-35 and, as noted, could easily be extended to SAPols. SAPols themselves,
as discussed in § 10.3.2.1, could probably be improved by lowering the density of charges along the
chain, e.g. by replacing the carboxylate groups currently present along with the sulfonate ones
(Fig. 10.15) by isopropylamine. A SAPol whose charge density would be comparable to that of
A8-35 would likely be as mild as it toward fragile MPs, while remaining soluble at all pH, and it could
easily be obtained in perdeuterated form. It ought also to be possible, should it be desirable, to
synthesize
13 C-free APols.
Chemical modifications of APols cover a very wide range (Le Bon et al. 2014b; see Chap. 4,
§ 4.4, Table 4.5). Some of them could be exploited for NMR studies. Polyhistidine-tagged A8-35
(HistAPol), whose synthesis has been described and which has been validated for trapping and
immobilizing MPs (Giusti et al. 2015), could conceivably be used to bind traces of paramagnetic
ions in the vicinity of the TM region of HistAPol-trapped MPs. A spin-labeled APol, which has been
synthesized (F. Giusti and M. Zoonens, unpublished data), provides an alternative. Spin-labeled APols
could be of use to simplify NMR spectra and facilitate their interpretation by selectively suppressing, in
a controlled and tunable manner, the resonance peaks from TM residues. They could possibly also be
used to improve the sensitivity of NMR measurements by resorting to dynamic nuclear polarization
(magnetization transfer from the spin label to nuclei; see Griesinger et al. 2012). APols that carry tags
10.4 Challenges and Prospects
489
at the cost of moving MP/ND complexes away from a bilayer-like environment and imposing
new constraints on the protein (see Chap. 3, § 3.3.2). As regards the size of bicelles, it can be
modulated by playing on the proportion of plane and ring surfactant (Chap. 3, § 3.2).
• Sample preparation. MP/detergent complexes are the form under which most MPs are
initially obtained, and their transfer to APols is straightforward (Chap. 5). Alternatively, the
protein can be directly folded (Chap. 6) or expressed (Chap. 7) in the presence of APols.
Handling bicelles is more difficult, and the preparation of homogeneous preparations of
MP/ND complexes is work-intensive (Ritchie et al. 2009).
• Stability. Because of their dissociating properties, detergents provide the most unfriendly,
least membrane-like environment, and, as a result, the stability of most MPs is often poor,
limiting the duration of the experiments and the temperature at which they can be carried out.
The stability is almost always increased, most often dramatically so, upon transfer to APols.
This is in part due to the rebinding of lipids (see Chap. 5), which reconstitutes, to some extent,
interactions more akin to those the MP experiences in its native membrane environment than
it does in detergent solution. Bicelles offer a bilayer-like environment, but contaminated by
the ring surfactant, either a detergent or short-chain, detergent-like lipids, which partitions in
the bilayer. There are data suggesting that they provide a stabilizing environment (see
e.g. Poget et al. 2007), but those are scarce. NDs offer the environment closest to a lipid
bilayer, and the few comparative data available indicate that ND-trapped MPs can be even
more stable than APol-trapped ones (see e.g. Popot 2010; Etzkorn et al. 2013).
• Isotopic labeling. Finally, isotopic labeling is straightforward in detergent solution and
bicelles – that is, provided the protein is stable enough in the presence of those few surfactants
that exist in deuterated form (Hiruma-Shimizu et al. 2015). It is also relatively easy in the case
of APols, within the constraints discussed in § 10.3.2. There is nothing to prevent the
preparation of NDs formed of deuterated DMPC and deuterated scaffold proteins, but for
the cost and work involved.
One of the assets of APols is their great chemical versatility. Deuteration has already been
implemented as regards A8-35 and, as noted, could easily be extended to SAPols. SAPols themselves,
as discussed in § 10.3.2.1, could probably be improved by lowering the density of charges along the
chain, e.g. by replacing the carboxylate groups currently present along with the sulfonate ones
(Fig. 10.15) by isopropylamine. A SAPol whose charge density would be comparable to that of
A8-35 would likely be as mild as it toward fragile MPs, while remaining soluble at all pH, and it could
easily be obtained in perdeuterated form. It ought also to be possible, should it be desirable, to
synthesize
13 C-free APols.
Chemical modifications of APols cover a very wide range (Le Bon et al. 2014b; see Chap. 4,
§ 4.4, Table 4.5). Some of them could be exploited for NMR studies. Polyhistidine-tagged A8-35
(HistAPol), whose synthesis has been described and which has been validated for trapping and
immobilizing MPs (Giusti et al. 2015), could conceivably be used to bind traces of paramagnetic
ions in the vicinity of the TM region of HistAPol-trapped MPs. A spin-labeled APol, which has been
synthesized (F. Giusti and M. Zoonens, unpublished data), provides an alternative. Spin-labeled APols
could be of use to simplify NMR spectra and facilitate their interpretation by selectively suppressing, in
a controlled and tunable manner, the resonance peaks from TM residues. They could possibly also be
used to improve the sensitivity of NMR measurements by resorting to dynamic nuclear polarization
(magnetization transfer from the spin label to nuclei; see Griesinger et al. 2012). APols that carry tags
10.4 Challenges and Prospects
489