exclusion chromatography (SEC) as sharper bands than A8-35 (Sharma et al. 2012) (see Chap. 4,
§ 4.3.2, Fig. 4.28), which is not the case for their heteropolymeric counterparts (Sharma et al. 2008;
Bazzacco et al. 2009). For further discussion of the source(s) of polydispersity of APol particles and
MP/APol complexes and possible ways to improvements, see Chap. 4, § 4.3.1.2.2.
It is interesting to note that trapping tOmpA with an A8-35/C 8 E 4 mixture results in the formation
of particles that migrate upon SEC with the same apparent R S as pure tOmpA/A8-35 complexes, but
are more homogeneous (Zoonens et al. 2007; see Chap. 9, § 9.3.2, Fig. 9.6). A tentative interpretation
of this observation is that detergent molecules provide the “small change” that permits optimization of
the volume of the surfactant belt, and relaxation toward a more uniform size. Because APols have a
stabilizing effect on MPs even when mixed with detergents (Champeil et al. 2000; Chap. 5, § 5.6.1,
Fig. 5.27), such mixtures could prove useful should particle homogeneity improve the quality of
sNMR signals.
Close attention must be paid to the fact that many factors can contribute to increasing the
polydispersity of MP/APol complexes, which can lead to signal degradation. Among those factors that
have been identified to date, on may cite:
(i) Deviations from the nominal composition of A8-35: a slightly higher hydrophobicity leads
to aggregation of APol particles and MP/APol complexes (see Gohon et al. 2004, 2006;
Chap. 4, § 4.3.1.2.2).
(ii) Incubation for a long time (days) at a pH too close to pH 7 (ibid.).
(iii) The presence of traces of calcium or other multivalent cations, which, presumably, bridge
MP/A8-35 complexes (Picard et al. 2006; Chap. 4, § 4.3.1.2.2, Fig. 4.16); indeed, adding
EDTA improves the quality of solution NMR spectra (Catoire et al. 2009, 2010b).
(iv) Removal of the excess of free APol that is required for efficient MP trapping. This is a
critical parameter. APols, as discussed in Chap. 5, are not very dissociating, and, in the
absence of a slight excess of them, MPs tend to form small oligomers (see Arunmanee et al.
2014; Gohon et al. 2008; Zoonens et al. 2007, Chap. 5, § 5.2.1, and Chap. 12, § 12.3.1 and
12.3.2). Solutions of A8-35-trapped tOmpA, for instance, appear monodisperse in SEC for
an overall MP/APol mass ratio of 1:4, whereas the protein is estimated to bind only about
half of that (Zoonens et al. 2007; see Chap. 5, § 5.2.1). As discussed in more detail in
Chap. 9, removing the excess APol by immobilized metal affinity chromatography leads to
the formation of small oligomers, which redissolve if the free polymer is added back
(Zoonens et al. 2007). The presence of oligomers does not preclude sNMR investigations,
but it can degrade the intensity and quality of the signals.
As described in Chaps. 5 and 9, checking for polydispersity can be done by SEC, by small-angle
X-ray or neutron scattering, or by AUC. AUC is a particularly sensitive and quantitative technique, as
illustrated by a detailed analysis of BR/A8-35 complexes (Gohon et al. 2008; see Chap. 9, § 9.3.7).
Another factor leading to improved resolution is to substitute unlabeled with deuterated APols.
Dipole-dipole coupling between amide protons (
1 H
N ) and nearby
1 H nuclei plays a major role in
1 H
N
spin relaxation phenomena. Among those, transverse relaxation can be substantially slowed down by
deuterium labeling (Anglister et al. 1993; Gardner and Kay 1998). Comparison of
1 H15
N TROSYHSQC spectra of tOmpA trapped in two forms of A8-35, HAPol, which is fully hydrogenated, and
DAPol, whose isopropylamine and octylamine chains are perdeuterated (Fig. 10.6), shows substantial
linewidth differences, whose analysis, as will be described in the next section, yielded the first
experimental insights into the relative arrangements of the protein and the polymer in MP/APol
complexes.
468
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins
§ 4.3.2, Fig. 4.28), which is not the case for their heteropolymeric counterparts (Sharma et al. 2008;
Bazzacco et al. 2009). For further discussion of the source(s) of polydispersity of APol particles and
MP/APol complexes and possible ways to improvements, see Chap. 4, § 4.3.1.2.2.
It is interesting to note that trapping tOmpA with an A8-35/C 8 E 4 mixture results in the formation
of particles that migrate upon SEC with the same apparent R S as pure tOmpA/A8-35 complexes, but
are more homogeneous (Zoonens et al. 2007; see Chap. 9, § 9.3.2, Fig. 9.6). A tentative interpretation
of this observation is that detergent molecules provide the “small change” that permits optimization of
the volume of the surfactant belt, and relaxation toward a more uniform size. Because APols have a
stabilizing effect on MPs even when mixed with detergents (Champeil et al. 2000; Chap. 5, § 5.6.1,
Fig. 5.27), such mixtures could prove useful should particle homogeneity improve the quality of
sNMR signals.
Close attention must be paid to the fact that many factors can contribute to increasing the
polydispersity of MP/APol complexes, which can lead to signal degradation. Among those factors that
have been identified to date, on may cite:
(i) Deviations from the nominal composition of A8-35: a slightly higher hydrophobicity leads
to aggregation of APol particles and MP/APol complexes (see Gohon et al. 2004, 2006;
Chap. 4, § 4.3.1.2.2).
(ii) Incubation for a long time (days) at a pH too close to pH 7 (ibid.).
(iii) The presence of traces of calcium or other multivalent cations, which, presumably, bridge
MP/A8-35 complexes (Picard et al. 2006; Chap. 4, § 4.3.1.2.2, Fig. 4.16); indeed, adding
EDTA improves the quality of solution NMR spectra (Catoire et al. 2009, 2010b).
(iv) Removal of the excess of free APol that is required for efficient MP trapping. This is a
critical parameter. APols, as discussed in Chap. 5, are not very dissociating, and, in the
absence of a slight excess of them, MPs tend to form small oligomers (see Arunmanee et al.
2014; Gohon et al. 2008; Zoonens et al. 2007, Chap. 5, § 5.2.1, and Chap. 12, § 12.3.1 and
12.3.2). Solutions of A8-35-trapped tOmpA, for instance, appear monodisperse in SEC for
an overall MP/APol mass ratio of 1:4, whereas the protein is estimated to bind only about
half of that (Zoonens et al. 2007; see Chap. 5, § 5.2.1). As discussed in more detail in
Chap. 9, removing the excess APol by immobilized metal affinity chromatography leads to
the formation of small oligomers, which redissolve if the free polymer is added back
(Zoonens et al. 2007). The presence of oligomers does not preclude sNMR investigations,
but it can degrade the intensity and quality of the signals.
As described in Chaps. 5 and 9, checking for polydispersity can be done by SEC, by small-angle
X-ray or neutron scattering, or by AUC. AUC is a particularly sensitive and quantitative technique, as
illustrated by a detailed analysis of BR/A8-35 complexes (Gohon et al. 2008; see Chap. 9, § 9.3.7).
Another factor leading to improved resolution is to substitute unlabeled with deuterated APols.
Dipole-dipole coupling between amide protons (
1 H
N ) and nearby
1 H nuclei plays a major role in
1 H
N
spin relaxation phenomena. Among those, transverse relaxation can be substantially slowed down by
deuterium labeling (Anglister et al. 1993; Gardner and Kay 1998). Comparison of
1 H15
N TROSYHSQC spectra of tOmpA trapped in two forms of A8-35, HAPol, which is fully hydrogenated, and
DAPol, whose isopropylamine and octylamine chains are perdeuterated (Fig. 10.6), shows substantial
linewidth differences, whose analysis, as will be described in the next section, yielded the first
experimental insights into the relative arrangements of the protein and the polymer in MP/APol
complexes.
468
10 Nuclear Magnetic Resonance Studies of Amphipol-Trapped Membrane Proteins