and stabilizing MPs (Bazzacco et al. 2012; Sharma et al. 2012; see Chap. 4, § 4.2.3.1, Chap. 5, § 5.5,
and Chaps. 6 and 7).
In Study 10.7 (Table 10.1; Bazzacco et al. 2012), OmpX was used as a model MP to examine the
usefulness of NAPols for sNMR studies. 2D
1 H15 N HSQC spectra of OmpX/NAPol complexes
feature a high resolution and a wide spectral dispersion in both dimensions, equivalent to those
observed with OmpX/A8-35 (Catoire et al. 2009, 2010b; Fig. 10.17A) and tOmpA/SAPol complexes
(Dahmane et al. 2011; Figs. 10.16 and 10.17). The small size and monodispersity of MP/NAPol
complexes, observed in SEC, are comparable to MP/A8-35 ones (Sharma et al. 2012), which gives rise
to a good sensitivity and resolution of the 2D HSQC spectrum at a
1 H Larmor frequency of 700 MHz,
without the need for transverse relaxation-optimized spectroscopy. Both amide
1 H and
15 N chemical
shifts are very similar in the spectra of the NAPol- and A8-35-trapped samples recorded at pH 6.8 and
8.0, respectively (Fig. 10.18A), consistent with the view that, in both cases, it is mainly the alkyl chains
that interact with the protein (see Chap. 5, § 5.3.3). On the basis of the assumption that the closest
peaks observed in NAPols and A8-35 vs. those in diC 6 PC correspond to the same residue, and if one
excepts residue Ala-10 (located toward the end of the first TM β-strand), the
1 H and
15 N weightaverage chemical shift differences between NAPol- and A8-35-complexed OmpX lie between 0.001
and 0.20 ppm, with an average of 0.04 ppm (Bazzacco et al. 2012). Keeping in mind that (i) the
chemical shifts of OmpX/A8-35 samples are themselves very close to those observed in diC 6 PC
(Catoire et al. 2010b), in which the NMR structure was obtained (Fernández et al. 2001a, b), and
(ii) the NMR structure is similar to the X-ray one, obtained in C 8 E 4 (Vogt and Schulz 1999), this
indicates that NAPol-trapped OmpX also is properly folded.
For residues that are not exposed to the solvent, the linewidth of the peaks in the 2D spectrum of
OmpX/NAPol complexes at pH 6.8 is very similar to that for OmpX/A8-35 complexes at pH 8.0 (see
e.g. residue G81 in Fig. 10.18B, left). On the contrary, peak intensities are substantially higher at pH
6.8 for solvent-exposed residues, such as those located in the loops or turns, whose
1 H chemical shifts
lie roughly between 7 and 8.5 ppm (see e.g. residue G22 in Fig. 10.18B, right). As a result, the
spectrum of the OmpX/NAPol sample at pH 6.8 shows additional cross-peaks in this region
(Fig. 10.18A, B, right). NAPols have also been used for a sNMR study on a non-transmembrane,
intrinsically disordered viral protein (Sólyom et al. 2015).
The high quality of NMR signals observed with OmpX/NAPol complexes opens perspectives of
complete structural determination of MP/NAPol complexes, provided the size of the MP of interest is
compatible with sNMR studies. APols thus join bicelles (Vold and Prosser 1996; Sanders and Prosser
1998; see Chap. 3, § 3.2) and NDs (Bayburt et al. 2002; Denisov et al. 2004; Etzkorn et al. 2013; Hagn
et al. 2013; Chap. 3, § 3.3) as useful alternatives to detergents for sNMR studies of MPs.
Media milder than detergents, whether they provide a bilayer-like environment, like bicelles and
NDs, or favor the retention of MP-bound lipids, as APols seem to do (Martinez et al. 2002; Gohon et al.
2008; Dahmane et al. 2013; see Chap. 5, § 5.3.1.2), are likely to better preserve native-like structural
features. This is suggested by differences in protein signal chemical shifts between MP/detergent and
either MP/bicelle (Chou et al. 2002; Lee et al. 2008) or MP/ND (Glück et al. 2009; Raschle et al. 2009,
2010; Shenkarev et al. 2010; Etzkorn et al. 2013) complexes. It will be interesting to examine how well
APols fare in this respect. A notable advantage of APols is the simplicity of preparation and handling
of MP/APol complexes. MP/bicelle complexes indeed require a strict control of the ratio of aggregated
long-chain vs. short-chain lipids or detergents all along the preparation and data collection to ensure
the presence of small and monodisperse bicellar particles (Triba et al. 2005; Sanders and Sönnichsen
2006; cf. Chap. 3, § 3.2). This control is far from trivial when buffer exchange or protein concentration
steps are required. MP/ND complexes are simpler to handle once they have been formed; however,
their preparation involves the production and purification of substantial amounts of lipoproteins,
10.3 Solution NMR Studies of Amphipol-Trapped Membrane Proteins
477
and Chaps. 6 and 7).
In Study 10.7 (Table 10.1; Bazzacco et al. 2012), OmpX was used as a model MP to examine the
usefulness of NAPols for sNMR studies. 2D
1 H15 N HSQC spectra of OmpX/NAPol complexes
feature a high resolution and a wide spectral dispersion in both dimensions, equivalent to those
observed with OmpX/A8-35 (Catoire et al. 2009, 2010b; Fig. 10.17A) and tOmpA/SAPol complexes
(Dahmane et al. 2011; Figs. 10.16 and 10.17). The small size and monodispersity of MP/NAPol
complexes, observed in SEC, are comparable to MP/A8-35 ones (Sharma et al. 2012), which gives rise
to a good sensitivity and resolution of the 2D HSQC spectrum at a
1 H Larmor frequency of 700 MHz,
without the need for transverse relaxation-optimized spectroscopy. Both amide
1 H and
15 N chemical
shifts are very similar in the spectra of the NAPol- and A8-35-trapped samples recorded at pH 6.8 and
8.0, respectively (Fig. 10.18A), consistent with the view that, in both cases, it is mainly the alkyl chains
that interact with the protein (see Chap. 5, § 5.3.3). On the basis of the assumption that the closest
peaks observed in NAPols and A8-35 vs. those in diC 6 PC correspond to the same residue, and if one
excepts residue Ala-10 (located toward the end of the first TM β-strand), the
1 H and
15 N weightaverage chemical shift differences between NAPol- and A8-35-complexed OmpX lie between 0.001
and 0.20 ppm, with an average of 0.04 ppm (Bazzacco et al. 2012). Keeping in mind that (i) the
chemical shifts of OmpX/A8-35 samples are themselves very close to those observed in diC 6 PC
(Catoire et al. 2010b), in which the NMR structure was obtained (Fernández et al. 2001a, b), and
(ii) the NMR structure is similar to the X-ray one, obtained in C 8 E 4 (Vogt and Schulz 1999), this
indicates that NAPol-trapped OmpX also is properly folded.
For residues that are not exposed to the solvent, the linewidth of the peaks in the 2D spectrum of
OmpX/NAPol complexes at pH 6.8 is very similar to that for OmpX/A8-35 complexes at pH 8.0 (see
e.g. residue G81 in Fig. 10.18B, left). On the contrary, peak intensities are substantially higher at pH
6.8 for solvent-exposed residues, such as those located in the loops or turns, whose
1 H chemical shifts
lie roughly between 7 and 8.5 ppm (see e.g. residue G22 in Fig. 10.18B, right). As a result, the
spectrum of the OmpX/NAPol sample at pH 6.8 shows additional cross-peaks in this region
(Fig. 10.18A, B, right). NAPols have also been used for a sNMR study on a non-transmembrane,
intrinsically disordered viral protein (Sólyom et al. 2015).
The high quality of NMR signals observed with OmpX/NAPol complexes opens perspectives of
complete structural determination of MP/NAPol complexes, provided the size of the MP of interest is
compatible with sNMR studies. APols thus join bicelles (Vold and Prosser 1996; Sanders and Prosser
1998; see Chap. 3, § 3.2) and NDs (Bayburt et al. 2002; Denisov et al. 2004; Etzkorn et al. 2013; Hagn
et al. 2013; Chap. 3, § 3.3) as useful alternatives to detergents for sNMR studies of MPs.
Media milder than detergents, whether they provide a bilayer-like environment, like bicelles and
NDs, or favor the retention of MP-bound lipids, as APols seem to do (Martinez et al. 2002; Gohon et al.
2008; Dahmane et al. 2013; see Chap. 5, § 5.3.1.2), are likely to better preserve native-like structural
features. This is suggested by differences in protein signal chemical shifts between MP/detergent and
either MP/bicelle (Chou et al. 2002; Lee et al. 2008) or MP/ND (Glück et al. 2009; Raschle et al. 2009,
2010; Shenkarev et al. 2010; Etzkorn et al. 2013) complexes. It will be interesting to examine how well
APols fare in this respect. A notable advantage of APols is the simplicity of preparation and handling
of MP/APol complexes. MP/bicelle complexes indeed require a strict control of the ratio of aggregated
long-chain vs. short-chain lipids or detergents all along the preparation and data collection to ensure
the presence of small and monodisperse bicellar particles (Triba et al. 2005; Sanders and Sönnichsen
2006; cf. Chap. 3, § 3.2). This control is far from trivial when buffer exchange or protein concentration
steps are required. MP/ND complexes are simpler to handle once they have been formed; however,
their preparation involves the production and purification of substantial amounts of lipoproteins,
10.3 Solution NMR Studies of Amphipol-Trapped Membrane Proteins
477