describe the state of the art of sNMR studies of MPs. In § 10.3, we will review existing NMR
experiments using APols and discuss the advantages and constraints associated to their use. In § 10.4,
we will compare them with those of other surfactants, and consider current challenges and prospects.
10.2 Context: Solution NMR Studies of Membrane Proteins
The technical difficulties associated with studying MPs by sNMR have been discussed in numerous
reviews, to which the reader is referred (see e.g. Sanders and Oxenoid 2000; Opella and Marassi 2004,
2017; Page et al. 2006; Sanders and Sönnichsen 2006; Poget and Girvin 2007; Wang 2008; Kim et al.
2009; Raschle et al. 2010; Nietlispach and Gautier 2011; Patching 2011; Qureshi and Goto 2011;
Tapaneeyakorn et al. 2011; Warschawski et al. 2011; Catoire et al. 2014; Kaplan et al. 2016; Mineev
and Nadezhdin 2017; Sim et al. 2017). We will concentrate on the two main ones, seen from the
biochemist’s perspective: (i) the production of sufficient amounts (0.1–1 mg) of isotopically labeled,
properly folded, functional MPs and (ii) their solubilization under a functional and stable form as
small, rapidly tumbling particles.
10.2.1 Producing the Protein
Most MPs are not abundant enough that it be practical to extract mg amounts of them from natural
sources. Overproducing them is, as discussed in Chaps. 1 and 6, a major stumbling block in
biochemical/biophysical studies. Furthermore, NMR studies require that appropriate isotopes be
introduced during the synthesis, those most frequently used being
2 H,
15 N, and
13 C. Various expression systems have been explored, the most prevalent ones being Escherichia coli, various yeasts, the
combination of insect cells and baculovirus, animal or human cells, and in vitro synthesis. When
dealing with eukaryotic MPs, there are rough, opposite gradients of convenience as regards, on the one
hand, expression yield, cost-efficiency, and ease of isotopic labeling (these considerations favoring
E. coli) and, on the other, folding efficiency and posttranslational modifications (favoring mammalian
cells), with the other methods lying somewhere in between (for discussions, see e.g. Kim et al. 2009;
Nietlispach and Gautier 2011; Tapaneeyakorn et al. 2011; for the special case of overproducing G
protein-coupled receptors for NMR studies, see the recent review of Milić and Veprintsev 2015).
The impact of APols, as regards the production of MP samples for NMR studies, may lie in two
principal directions, namely folding and stabilization (Fig. 10.1). As discussed in Chaps. 6 and 7,
APols make it easier to fold to their native state MPs that have been produced in vivo in E. coli – or,
conceivably, in yeasts – in an unfolded or misfolded form, such as in inclusion bodies. They can also
be used in the frame of in vitro, cell-free synthesis, whether they serve as the recipient medium in
which MPs fold to their native form, or they are used to fold a posteriori MPs that have been
synthesized in the absence of surfactant and solubilized in a denaturant. It appears that, in many
cases, posttranslational modifications, such as glycosylation, are not essential to the function of MPs
(they may be more important for governing their quality control and traffic in eukaryotic cells) and that
a functional MP can be obtained in E. coli, provided it can be convinced to adopt a native-like 3D
structure, which APols seem good at (see Chap. 6 and, for general discussions, Popot 2014; Popot and
Engelman 2016). Because good expression yields and the sophisticated isotopic labeling schemes that
are needed to study large proteins are more easily achieved and less costly in E. coli and during
cell-free expression, APols can probably be expected to be most helpful when using these two
modes of synthesis, but they could also be used to stabilize MPs produced under a native form in
eukaryotic cells.
10.2 Context: Solution NMR Studies of Membrane Proteins
455
experiments using APols and discuss the advantages and constraints associated to their use. In § 10.4,
we will compare them with those of other surfactants, and consider current challenges and prospects.
10.2 Context: Solution NMR Studies of Membrane Proteins
The technical difficulties associated with studying MPs by sNMR have been discussed in numerous
reviews, to which the reader is referred (see e.g. Sanders and Oxenoid 2000; Opella and Marassi 2004,
2017; Page et al. 2006; Sanders and Sönnichsen 2006; Poget and Girvin 2007; Wang 2008; Kim et al.
2009; Raschle et al. 2010; Nietlispach and Gautier 2011; Patching 2011; Qureshi and Goto 2011;
Tapaneeyakorn et al. 2011; Warschawski et al. 2011; Catoire et al. 2014; Kaplan et al. 2016; Mineev
and Nadezhdin 2017; Sim et al. 2017). We will concentrate on the two main ones, seen from the
biochemist’s perspective: (i) the production of sufficient amounts (0.1–1 mg) of isotopically labeled,
properly folded, functional MPs and (ii) their solubilization under a functional and stable form as
small, rapidly tumbling particles.
10.2.1 Producing the Protein
Most MPs are not abundant enough that it be practical to extract mg amounts of them from natural
sources. Overproducing them is, as discussed in Chaps. 1 and 6, a major stumbling block in
biochemical/biophysical studies. Furthermore, NMR studies require that appropriate isotopes be
introduced during the synthesis, those most frequently used being
2 H,
15 N, and
13 C. Various expression systems have been explored, the most prevalent ones being Escherichia coli, various yeasts, the
combination of insect cells and baculovirus, animal or human cells, and in vitro synthesis. When
dealing with eukaryotic MPs, there are rough, opposite gradients of convenience as regards, on the one
hand, expression yield, cost-efficiency, and ease of isotopic labeling (these considerations favoring
E. coli) and, on the other, folding efficiency and posttranslational modifications (favoring mammalian
cells), with the other methods lying somewhere in between (for discussions, see e.g. Kim et al. 2009;
Nietlispach and Gautier 2011; Tapaneeyakorn et al. 2011; for the special case of overproducing G
protein-coupled receptors for NMR studies, see the recent review of Milić and Veprintsev 2015).
The impact of APols, as regards the production of MP samples for NMR studies, may lie in two
principal directions, namely folding and stabilization (Fig. 10.1). As discussed in Chaps. 6 and 7,
APols make it easier to fold to their native state MPs that have been produced in vivo in E. coli – or,
conceivably, in yeasts – in an unfolded or misfolded form, such as in inclusion bodies. They can also
be used in the frame of in vitro, cell-free synthesis, whether they serve as the recipient medium in
which MPs fold to their native form, or they are used to fold a posteriori MPs that have been
synthesized in the absence of surfactant and solubilized in a denaturant. It appears that, in many
cases, posttranslational modifications, such as glycosylation, are not essential to the function of MPs
(they may be more important for governing their quality control and traffic in eukaryotic cells) and that
a functional MP can be obtained in E. coli, provided it can be convinced to adopt a native-like 3D
structure, which APols seem good at (see Chap. 6 and, for general discussions, Popot 2014; Popot and
Engelman 2016). Because good expression yields and the sophisticated isotopic labeling schemes that
are needed to study large proteins are more easily achieved and less costly in E. coli and during
cell-free expression, APols can probably be expected to be most helpful when using these two
modes of synthesis, but they could also be used to stabilize MPs produced under a native form in
eukaryotic cells.
10.2 Context: Solution NMR Studies of Membrane Proteins
455