procedure. For the protein to adopt a TM position, some of its hydrophilic regions have to somehow
cross the bilayer, which can be a highly destabilizing process. It is reasonable to expect that the more
robust (or the simpler) the protein is, the greater are the chances that it can be transferred without
denaturation, or may be able to recover from it. Studying the transfer of a variety of MPs is clearly
needed before a general view of the usefulness of this procedure can be formed.
As will be discussed in Chap. 11, A8-35 – and, presumably, all APols carrying a net charge –
may not be suitable to forming well-diffracting MP crystals (Charvolin et al. 2014). However, A8-35
has been used by Valentin Gordeliy and colleagues to deliver BR to a lipidic mesophase, in which the
protein organized into 3D crystals diffracting to better than 2-Å resolution (Polovinkin et al. 2014b).
Crystallographic analysis showed that the APol had been totally displaced and was not present in the
crystals, whereas the purple membrane lipids introduced into the mesophase along with the protein had
remained associated to it. This experiment has been recently reproduced using SMA-trapped BR from
Haloquadratum walsbyi (Broecker et al. 2017). Delivering MPs and, in particular, GPCRs to lipidic
mesophases for crystallization is probably a very promising approach (see Chap. 11), particularly
given the high rate of success of APol-induced folding of GPCRs expressed in inclusion bodies
(Chap. 6).
Once an APol-trapped MP has been transferred to a detergent, any classical reconstitution
procedure can of course be resorted to. This has been exploited to transfer GPCRs that had been folded
in A8-35 to either nanodiscs (Damian et al. 2012; Casiraghi et al. 2016) or SMALPs (Logez et al.
2016). In the first case, the proteins were transferred from A8-35 to DDM, using IMAC to wash away
the APol, after which the detergent solution was supplemented with lipids and scaffold proteins, and
the nanodiscs formed by eliminating the detergent (Damian et al. 2012; Casiraghi et al. 2016; see § 5.9,
Protocol 5.3). In the second case, the APol-folded receptor was similarly transferred to DDM by IMAC
and then introduced into proteoliposomes, which were solubilized by SMA (Logez et al. 2016). These
procedures aim at providing the protein with a more bilayer-like environment, particularly with the
view of studying the regulatory effects of lipids.
Finally, a strange, totally foreign medium to which MPs have been transferred from an APoltrapped state is vacuum, that vacuum that reigns inside the cavity of mass spectrometers. Electron
spray ionization MS (ESI-MS) coupled with ion mobility spectrometry (IMS), in particular, has shown
that APols preserve better than detergents the folded state of MPs during the critical stage of transfer
from an aqueous solution to the gas phase (Calabrese et al. 2015; Watkinson et al. 2015). The
applications of APols to MS will be discussed in Chap. 14.
5.8
Conclusion
In this and the preceding chapter, I have tried to present an overview of the chemical structure and
solution properties of APols, of the modes of formation of MP/APol complexes, and of their organization, dynamics, and solution properties. In the following chapters, we will examine, first, how APols
may help to produce MPs amenable to in vitro studies, either by assisting their folding from a
denatured state such as can be obtained by dissolving inclusion bodies in a denaturing medium
(Chap. 6) or by acting as the folding medium for MPs expressed in vitro by cell-free expression
(Chap. 7). Chapters 8, 9, 10, 11, 12, 13, 14, and 15 will then be devoted to presenting and discussing
what has been and what can be done experimentally with MP/APol complexes, namely optical
spectroscopic studies (Chap. 8); solution studies by such approaches as radiation scattering, analytical
ultracentrifugation, etc. (Chap. 9); NMR spectroscopy (Chap. 10); radiocrystallography (Chap. 11);
electron microscopy (Chap. 12); studies exploiting tagged APols, such as solid-state ligand-binding
studies using APol-immobilized MPs (Chap. 13); proteomics, including MS (Chap. 14); and
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5 Formation and Properties of Membrane Protein/Amphipol Complexes
cross the bilayer, which can be a highly destabilizing process. It is reasonable to expect that the more
robust (or the simpler) the protein is, the greater are the chances that it can be transferred without
denaturation, or may be able to recover from it. Studying the transfer of a variety of MPs is clearly
needed before a general view of the usefulness of this procedure can be formed.
As will be discussed in Chap. 11, A8-35 – and, presumably, all APols carrying a net charge –
may not be suitable to forming well-diffracting MP crystals (Charvolin et al. 2014). However, A8-35
has been used by Valentin Gordeliy and colleagues to deliver BR to a lipidic mesophase, in which the
protein organized into 3D crystals diffracting to better than 2-Å resolution (Polovinkin et al. 2014b).
Crystallographic analysis showed that the APol had been totally displaced and was not present in the
crystals, whereas the purple membrane lipids introduced into the mesophase along with the protein had
remained associated to it. This experiment has been recently reproduced using SMA-trapped BR from
Haloquadratum walsbyi (Broecker et al. 2017). Delivering MPs and, in particular, GPCRs to lipidic
mesophases for crystallization is probably a very promising approach (see Chap. 11), particularly
given the high rate of success of APol-induced folding of GPCRs expressed in inclusion bodies
(Chap. 6).
Once an APol-trapped MP has been transferred to a detergent, any classical reconstitution
procedure can of course be resorted to. This has been exploited to transfer GPCRs that had been folded
in A8-35 to either nanodiscs (Damian et al. 2012; Casiraghi et al. 2016) or SMALPs (Logez et al.
2016). In the first case, the proteins were transferred from A8-35 to DDM, using IMAC to wash away
the APol, after which the detergent solution was supplemented with lipids and scaffold proteins, and
the nanodiscs formed by eliminating the detergent (Damian et al. 2012; Casiraghi et al. 2016; see § 5.9,
Protocol 5.3). In the second case, the APol-folded receptor was similarly transferred to DDM by IMAC
and then introduced into proteoliposomes, which were solubilized by SMA (Logez et al. 2016). These
procedures aim at providing the protein with a more bilayer-like environment, particularly with the
view of studying the regulatory effects of lipids.
Finally, a strange, totally foreign medium to which MPs have been transferred from an APoltrapped state is vacuum, that vacuum that reigns inside the cavity of mass spectrometers. Electron
spray ionization MS (ESI-MS) coupled with ion mobility spectrometry (IMS), in particular, has shown
that APols preserve better than detergents the folded state of MPs during the critical stage of transfer
from an aqueous solution to the gas phase (Calabrese et al. 2015; Watkinson et al. 2015). The
applications of APols to MS will be discussed in Chap. 14.
5.8
Conclusion
In this and the preceding chapter, I have tried to present an overview of the chemical structure and
solution properties of APols, of the modes of formation of MP/APol complexes, and of their organization, dynamics, and solution properties. In the following chapters, we will examine, first, how APols
may help to produce MPs amenable to in vitro studies, either by assisting their folding from a
denatured state such as can be obtained by dissolving inclusion bodies in a denaturing medium
(Chap. 6) or by acting as the folding medium for MPs expressed in vitro by cell-free expression
(Chap. 7). Chapters 8, 9, 10, 11, 12, 13, 14, and 15 will then be devoted to presenting and discussing
what has been and what can be done experimentally with MP/APol complexes, namely optical
spectroscopic studies (Chap. 8); solution studies by such approaches as radiation scattering, analytical
ultracentrifugation, etc. (Chap. 9); NMR spectroscopy (Chap. 10); radiocrystallography (Chap. 11);
electron microscopy (Chap. 12); studies exploiting tagged APols, such as solid-state ligand-binding
studies using APol-immobilized MPs (Chap. 13); proteomics, including MS (Chap. 14); and
310
5 Formation and Properties of Membrane Protein/Amphipol Complexes