signals between 0.8 and 2.6 ppm (Fig. 10.19B), creating important distortions of the baseline of the
NOESY spectrum. Baseline corrections carried out after the Fourier transform can affect the volumes
of weak ligand cross-peaks that are located close to DAPol ones, which affects the accuracy of peak
volume measurements. With perDAPol, no such baseline treatment will be needed.
As regards MP structural studies, APol signals can interfere with those from α-carbon-linked
protons and CH n moieties of amino acid side chains, as illustrated in Fig. 10.20. The use of perDAPol
should be very advantageous, especially when protein
13 C signals need to be observed above ~25 ppm
(Fig. 10.20). Among various approaches that emerged during the past 10 years to study large proteins
or protein complexes, one of the best strategies is the use of
13 CH 3 methyl groups immersed in a
perdeuterated environment (Sprangers et al. 2007; Plevin and Boisbouvier 2012). Unfortunately, most
of the
13 CH 3 methyl groups in amino acids resonate between 0 and 2.5 ppm in the
1 H dimension and
between 10 and 30 ppm in the
13 C one (Fig. 10.20), i.e. in a region where many signals from the CH n
groups of the alkyl chains of surfactants are localized. In the case of methyl-TROSY-based
experiments, the use of perDAPol rather than DAPol would not make much of a difference, because
in this region, i.e. between 0.6 and 1.4 ppm in the
1 H dimension, the remaining
1 H signals are more or
less the same for the two polymers.
Previous work has shown that the resolution of 2D
1 H15 N TROSY spectra collected on APoltrapped MPs is sufficient to obtain insights into membrane protein structure and dynamics at least up to
the size of BR (27 kDa) (see § 10.3.1). A8-35 permits the identification of most amide protons, but not,
however, of those whose exchange with the solvent is too rapid at the slightly alkaline pH (typically,
pH 8) that is optimal for use of this APol. Hence, sequence-specific assignment of backbone
1 H
N ,
15
N,
and
13 C
α chemical shifts using TROSY-based HNCA (Salzmann et al. 1999) and HNCOCA (Kay et al.
1990) experiments should be realized with MPs trapped in a pH-insensitive APol, such as SAPol or
NAPol, at least for amide protons located in solvent-exposed loops. Concerning the identification of
intramolecular
1 H dipolar interactions through 3D
15 N-edited and
13 C-edited NOESY experiments, the
choice of the APol to be used would again depend on how exchangeable the protons investigated are.
DAPol and perDAPol would be highly recommended to observe methyl-methyl interactions, for
instance, whereas NAPol or SAPol would be needed to observe NOEs involving loop
1 H
N backbone
protons, provided that the NOE cross-peaks are not hidden by NAPol or SAPol signals. This begs the
question of SAPol and NAPol perdeuteration. As noted in § 10.3.2.1, because their solubility is
ensured by sugar residues, NAPols would be extremely costly to perdeuterate. SAPols, on the other
hand, could be perdeuterated following the route used for obtaining perDAPol. A remaining concern is
that SAPols, probably because of their high charge density, appear less stabilizing than A8-35 or
NAPol (see Chap. 5, § 5.5). They will probably be adequate for many MPs, but, as mentioned above, a
higher stabilizing power could probably be achieved by lowering their charge density to the level of
that of A8-35. This, however, would require the development of a different synthesis protocol.
10.3.3 Biologically Oriented Studies
Most of the sNMR studies involving APols that have been published to date – 8 out of the 12 listed in
Table 10.1 – have been methodological in their intent, aiming to understand the structure and dynamics
of MP/APol complexes, define optimal protocols for their preparation and study, develop APols
optimized for NMR applications, clarify the advantages, pitfalls, and limitations of the approach,
etc. Studies in which a biological problem is the main issue are however starting to trickle in. Four of
them, of unequal breadth, are listed in Table 10.1. The first of them (Study 10.3) investigates the
dynamics of OmpX, whose role as an outer membrane ion channel is disputed. The second and most
extensive study (Study 10.4) exploits APols to fold a GPCR and to establish, using transferred NOE
482
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins
NOESY spectrum. Baseline corrections carried out after the Fourier transform can affect the volumes
of weak ligand cross-peaks that are located close to DAPol ones, which affects the accuracy of peak
volume measurements. With perDAPol, no such baseline treatment will be needed.
As regards MP structural studies, APol signals can interfere with those from α-carbon-linked
protons and CH n moieties of amino acid side chains, as illustrated in Fig. 10.20. The use of perDAPol
should be very advantageous, especially when protein
13 C signals need to be observed above ~25 ppm
(Fig. 10.20). Among various approaches that emerged during the past 10 years to study large proteins
or protein complexes, one of the best strategies is the use of
13 CH 3 methyl groups immersed in a
perdeuterated environment (Sprangers et al. 2007; Plevin and Boisbouvier 2012). Unfortunately, most
of the
13 CH 3 methyl groups in amino acids resonate between 0 and 2.5 ppm in the
1 H dimension and
between 10 and 30 ppm in the
13 C one (Fig. 10.20), i.e. in a region where many signals from the CH n
groups of the alkyl chains of surfactants are localized. In the case of methyl-TROSY-based
experiments, the use of perDAPol rather than DAPol would not make much of a difference, because
in this region, i.e. between 0.6 and 1.4 ppm in the
1 H dimension, the remaining
1 H signals are more or
less the same for the two polymers.
Previous work has shown that the resolution of 2D
1 H15 N TROSY spectra collected on APoltrapped MPs is sufficient to obtain insights into membrane protein structure and dynamics at least up to
the size of BR (27 kDa) (see § 10.3.1). A8-35 permits the identification of most amide protons, but not,
however, of those whose exchange with the solvent is too rapid at the slightly alkaline pH (typically,
pH 8) that is optimal for use of this APol. Hence, sequence-specific assignment of backbone
1 H
N ,
15
N,
and
13 C
α chemical shifts using TROSY-based HNCA (Salzmann et al. 1999) and HNCOCA (Kay et al.
1990) experiments should be realized with MPs trapped in a pH-insensitive APol, such as SAPol or
NAPol, at least for amide protons located in solvent-exposed loops. Concerning the identification of
intramolecular
1 H dipolar interactions through 3D
15 N-edited and
13 C-edited NOESY experiments, the
choice of the APol to be used would again depend on how exchangeable the protons investigated are.
DAPol and perDAPol would be highly recommended to observe methyl-methyl interactions, for
instance, whereas NAPol or SAPol would be needed to observe NOEs involving loop
1 H
N backbone
protons, provided that the NOE cross-peaks are not hidden by NAPol or SAPol signals. This begs the
question of SAPol and NAPol perdeuteration. As noted in § 10.3.2.1, because their solubility is
ensured by sugar residues, NAPols would be extremely costly to perdeuterate. SAPols, on the other
hand, could be perdeuterated following the route used for obtaining perDAPol. A remaining concern is
that SAPols, probably because of their high charge density, appear less stabilizing than A8-35 or
NAPol (see Chap. 5, § 5.5). They will probably be adequate for many MPs, but, as mentioned above, a
higher stabilizing power could probably be achieved by lowering their charge density to the level of
that of A8-35. This, however, would require the development of a different synthesis protocol.
10.3.3 Biologically Oriented Studies
Most of the sNMR studies involving APols that have been published to date – 8 out of the 12 listed in
Table 10.1 – have been methodological in their intent, aiming to understand the structure and dynamics
of MP/APol complexes, define optimal protocols for their preparation and study, develop APols
optimized for NMR applications, clarify the advantages, pitfalls, and limitations of the approach,
etc. Studies in which a biological problem is the main issue are however starting to trickle in. Four of
them, of unequal breadth, are listed in Table 10.1. The first of them (Study 10.3) investigates the
dynamics of OmpX, whose role as an outer membrane ion channel is disputed. The second and most
extensive study (Study 10.4) exploits APols to fold a GPCR and to establish, using transferred NOE
482
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins