In the following two sections, some of the results obtained by SANS and SAXS analyses of
MP/APol complexes are presented, with particular attention being paid to the problems and opportunities specific to the use of APols.
9.3.8.2 Small-Angle Neutron Scattering
SANS Studies of BR/A8-35 Complexes
The first SANS study of an APol-trapped MP was that of BR complexed by either unlabeled
A8-35 (“HAPol”) or a partially deuterated version of it (“DAPol”) obtained by derivation of unlabeled
polyacrylic acid (PAA) with deuterated octyl- and isopropylamine (Gohon et al. 2004, 2006). This
study (Study 9.5) is particularly instructive because, being the first of its kind, it had the privilege of
discovering – by the virtue of falling into them – quite a few of the traps that should be paid attention to
when carrying out such investigations. It is therefore worth presenting in some details, without
smoothing away its wrinkles.
Figure 9.19A presents the variation of the neutron scattering intensity, I(Q), by solutions of
BR/DAPol complexes as a function of Q
2 . In the particular case of Fig. 9.19A, I(Q), which is directly
proportional to the concentration of particles, has been divided by c BR , the concentration of BR, so that
the shape of the scattering curves collected at different BR concentrations can be compared. Three
points are worth noting:
(i) The scattering curves appear linear over a large part of the Guinier region (marked by
dashed vertical lines in Panel B), which is consistent with the preparations being monodisperse, but does not prove that they are strictly so. As a matter of fact, they are not,
sedimentation velocity experiments similar to those of Fig. 9.12 showing that the preparation actually comprises ~75% monomers, ~20% dimers, and ~5% trimers. This polydispersity must be taken into account in the analysis, or conclusions about the mass and R g of the
particles will be off the mark.
(ii) The slope of the linear region does not change with the concentration, which indicates that
no association/dissociation process takes place as the concentration varies.
(iii) In the very small-angle region, a downward deflection is seen at high concentration
(10 gÁL
À1 ), which progressively disappears as the preparation is diluted. This feature
indicates that at high concentration, particles repulse each other. Electrostatic repulsion
between the negatively charged MP/A8-35 complexes and A8-35 particles has indeed
been observed to control the rate of exchange between free and protein-bound APols
(Zoonens et al. 2007) and is also observed, using SAXS, between cytochrome bc 1 /A8-35
complexes (Charvolin et al. 2014; see Chap. 11, Fig. 11.13). The data in Panel A indicates
that 1.9 gÁL
À1 , the lowest concentration tested, provides data of sufficient quality without
any such perturbation in the Guinier region.
In the experiments presented in Fig. 9.19B, the concentration of BR has been kept at 1.9 gÁL
À1 ,
and the percentage of D 2 O in the buffer has been varied between 0% and 100%, with intermediate
concentrations of 40% (diamonds), at which BR is contrast-matched; 65% (squares), at which the
whole complex is matched; and 84.5% (inverted triangles), at which DAPol is matched. The shape of
the scattering curves is seen to vary with the D 2 O content, because the relative contributions of the
complexes and of the free DAPol particles that are also present in the preparation depend on their
respective contrast with the solvent. When BR (diamonds) or the BR/DAPol complexes (squares) are
contrast-matched, increased scattering at very small angles (Q
2 < 0.001 Å
À2 ) reveals the presence of
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9 Solution Studies of Membrane Protein/Amphipol Complexes
MP/APol complexes are presented, with particular attention being paid to the problems and opportunities specific to the use of APols.
9.3.8.2 Small-Angle Neutron Scattering
SANS Studies of BR/A8-35 Complexes
The first SANS study of an APol-trapped MP was that of BR complexed by either unlabeled
A8-35 (“HAPol”) or a partially deuterated version of it (“DAPol”) obtained by derivation of unlabeled
polyacrylic acid (PAA) with deuterated octyl- and isopropylamine (Gohon et al. 2004, 2006). This
study (Study 9.5) is particularly instructive because, being the first of its kind, it had the privilege of
discovering – by the virtue of falling into them – quite a few of the traps that should be paid attention to
when carrying out such investigations. It is therefore worth presenting in some details, without
smoothing away its wrinkles.
Figure 9.19A presents the variation of the neutron scattering intensity, I(Q), by solutions of
BR/DAPol complexes as a function of Q
2 . In the particular case of Fig. 9.19A, I(Q), which is directly
proportional to the concentration of particles, has been divided by c BR , the concentration of BR, so that
the shape of the scattering curves collected at different BR concentrations can be compared. Three
points are worth noting:
(i) The scattering curves appear linear over a large part of the Guinier region (marked by
dashed vertical lines in Panel B), which is consistent with the preparations being monodisperse, but does not prove that they are strictly so. As a matter of fact, they are not,
sedimentation velocity experiments similar to those of Fig. 9.12 showing that the preparation actually comprises ~75% monomers, ~20% dimers, and ~5% trimers. This polydispersity must be taken into account in the analysis, or conclusions about the mass and R g of the
particles will be off the mark.
(ii) The slope of the linear region does not change with the concentration, which indicates that
no association/dissociation process takes place as the concentration varies.
(iii) In the very small-angle region, a downward deflection is seen at high concentration
(10 gÁL
À1 ), which progressively disappears as the preparation is diluted. This feature
indicates that at high concentration, particles repulse each other. Electrostatic repulsion
between the negatively charged MP/A8-35 complexes and A8-35 particles has indeed
been observed to control the rate of exchange between free and protein-bound APols
(Zoonens et al. 2007) and is also observed, using SAXS, between cytochrome bc 1 /A8-35
complexes (Charvolin et al. 2014; see Chap. 11, Fig. 11.13). The data in Panel A indicates
that 1.9 gÁL
À1 , the lowest concentration tested, provides data of sufficient quality without
any such perturbation in the Guinier region.
In the experiments presented in Fig. 9.19B, the concentration of BR has been kept at 1.9 gÁL
À1 ,
and the percentage of D 2 O in the buffer has been varied between 0% and 100%, with intermediate
concentrations of 40% (diamonds), at which BR is contrast-matched; 65% (squares), at which the
whole complex is matched; and 84.5% (inverted triangles), at which DAPol is matched. The shape of
the scattering curves is seen to vary with the D 2 O content, because the relative contributions of the
complexes and of the free DAPol particles that are also present in the preparation depend on their
respective contrast with the solvent. When BR (diamonds) or the BR/DAPol complexes (squares) are
contrast-matched, increased scattering at very small angles (Q
2 < 0.001 Å
À2 ) reveals the presence of
432
9 Solution Studies of Membrane Protein/Amphipol Complexes