In summary, the comparative study of BR/A8-35 complexes by a broad array of methods has
identified some caveats in using biophysical approaches to determine the mass, size, composition, and
shape of MP/APol complexes, of which the two most important appear to be the following:
• SEC is useful to examine the homogeneity of the preparations, but the R S values it provides in
the conditions used in Studies 9.5 and 9.9 are overestimated. If the SEC-derived value of R S is
used to derive the molecular mass of monomeric BR/A8-35 complexes from AUC sedimentation velocity data, it leads to strongly overestimating it (~130 kDa, whereas the actual mass
is ~90 kDa).
• When using SANS or SAXS to study MP/APol complexes, great attention should be paid to
the monodispersity of the samples. In Gohon et al. (2006, 2008) and Zoonens et al. (2007),
two causes of polydispersity were identified:
(i) Unplanned-for excessive hydrophobicity of A8-35, due to the artifactual grafting
of a hydrophobic secondary product during the synthesis (see Chap. 4, § 4.2.1); this
problem has been brought under control by careful optimization of the synthesis
protocol (see Gohon et al. 2006, and Protocol 4.1 in Chap. 4, § 4.5).
(ii) Oligomerization of the complexes upon complete removal of free APol (Zoonens
et al. 2007; Gohon et al. 2008; Arunmanee et al. 2014; Charvolin et al. 2014; see
Chap. 5, § 5.2.1). This problem can be avoided by working in the presence of a
sufficient concentration of free APol (see e.g. Zoonens et al. 2007; Charvolin et al.
2014). For particularly fragile MP complexes, it may however be difficult to find
conditions that will prevent oligomerization while avoiding any degree of disaggregation (see e.g. Study 9.41).
Whatever the cause of aggregation, if any, it must be carefully documented, so as to prevent any
overestimation of the mass and dimensions of the monomeric complexes. AUC used in the sedimentation velocity mode is, by far, the most performing way to quantify the relative proportion of the
various forms (cf. Fig. 9.12).
Other SANS Studies of MP/APol Complexes
Also relevant are the following SANS studies of MP/APol complexes:
• In Study 9.7, the solution behavior of cytochrome bc 1 /A8-35 complexes has been studied
by both SAXS and SANS (see § 9.3.8.3). SANS studies were conducted in buffers containing
17% D 2 O, so as to eliminate most of the contribution to scattering of polyethylene glycol,
detergent, and/or A8-35 (Charvolin et al. 2014).
• In Study 9.16, the mass, size, and shape of complexes between BR and a non-ionic APol have
been studied by SANS. Scattering data at wide angles were exploited (cf. Box 9.2) to yield an
envelope of the particle (Fig. 9.21).
• In Study 9.41, the composition and organization of ExbB/ExbD complexes have been studied
by a broad array of techniques, including SANS and SAXS, and envelopes of the particles
calculated from wide-angle data and compared to reconstructed EM envelopes (see § 9.3.8.3,
Fig. 9.22).
436
9 Solution Studies of Membrane Protein/Amphipol Complexes
identified some caveats in using biophysical approaches to determine the mass, size, composition, and
shape of MP/APol complexes, of which the two most important appear to be the following:
• SEC is useful to examine the homogeneity of the preparations, but the R S values it provides in
the conditions used in Studies 9.5 and 9.9 are overestimated. If the SEC-derived value of R S is
used to derive the molecular mass of monomeric BR/A8-35 complexes from AUC sedimentation velocity data, it leads to strongly overestimating it (~130 kDa, whereas the actual mass
is ~90 kDa).
• When using SANS or SAXS to study MP/APol complexes, great attention should be paid to
the monodispersity of the samples. In Gohon et al. (2006, 2008) and Zoonens et al. (2007),
two causes of polydispersity were identified:
(i) Unplanned-for excessive hydrophobicity of A8-35, due to the artifactual grafting
of a hydrophobic secondary product during the synthesis (see Chap. 4, § 4.2.1); this
problem has been brought under control by careful optimization of the synthesis
protocol (see Gohon et al. 2006, and Protocol 4.1 in Chap. 4, § 4.5).
(ii) Oligomerization of the complexes upon complete removal of free APol (Zoonens
et al. 2007; Gohon et al. 2008; Arunmanee et al. 2014; Charvolin et al. 2014; see
Chap. 5, § 5.2.1). This problem can be avoided by working in the presence of a
sufficient concentration of free APol (see e.g. Zoonens et al. 2007; Charvolin et al.
2014). For particularly fragile MP complexes, it may however be difficult to find
conditions that will prevent oligomerization while avoiding any degree of disaggregation (see e.g. Study 9.41).
Whatever the cause of aggregation, if any, it must be carefully documented, so as to prevent any
overestimation of the mass and dimensions of the monomeric complexes. AUC used in the sedimentation velocity mode is, by far, the most performing way to quantify the relative proportion of the
various forms (cf. Fig. 9.12).
Other SANS Studies of MP/APol Complexes
Also relevant are the following SANS studies of MP/APol complexes:
• In Study 9.7, the solution behavior of cytochrome bc 1 /A8-35 complexes has been studied
by both SAXS and SANS (see § 9.3.8.3). SANS studies were conducted in buffers containing
17% D 2 O, so as to eliminate most of the contribution to scattering of polyethylene glycol,
detergent, and/or A8-35 (Charvolin et al. 2014).
• In Study 9.16, the mass, size, and shape of complexes between BR and a non-ionic APol have
been studied by SANS. Scattering data at wide angles were exploited (cf. Box 9.2) to yield an
envelope of the particle (Fig. 9.21).
• In Study 9.41, the composition and organization of ExbB/ExbD complexes have been studied
by a broad array of techniques, including SANS and SAXS, and envelopes of the particles
calculated from wide-angle data and compared to reconstructed EM envelopes (see § 9.3.8.3,
Fig. 9.22).
436
9 Solution Studies of Membrane Protein/Amphipol Complexes