suggests, in keeping with SEC data (§ 9.3.2; Fig. 9.8), that traces of Ca
2+ degrade the resolution of
solution NMR spectra by bridging some of the particles via their A8-35 belt.
9.3.7
Analytical Ultracentrifugation
Analytical ultracentrifugation (AUC) is a particularly powerful method for determining the size and
mass of solutes. It can be used at equilibrium, by subjecting the sample to a relatively low gravity field
and examining its distribution throughout the buffer column once it has reached final equilibration, or
as a sedimentation velocity technique, in which case the redistribution of the various solutes is
followed throughout the experiment (see Box 9.1; for reviews, see e.g. Laue and Stafford 1999;
Carruthers et al. 2000; Schuck 2000; Padrick et al. 2010; Ebel 2011; and references therein). The latter
approach is particularly powerful when dealing with mixtures, as is usually the case of MP/surfactant
preparations, which generally contain both MP/surfactant complexes and free surfactant particles.
Detection can be achieved either by absorbance spectroscopy at multiple wavelengths or by measuring
the refractive index of the solution, measurements being carried out at each point along the buffer
column as a function of time. The data are then decomposed to yield the sedimentation coefficients and
relative concentrations of the various species present in the solution, as well as, under favorable
circumstances, the ratio of various components in a particle. AUC has been applied to eight
APol-trapped MPs, ranging from small (BR; ~27 kDa) to moderately large (AcrB trimer;
~360 kDa), trapped in A8-35, in one of its fluorescent or deuterated derivatives, in NAPols, or in
SMA (see Table 9.1).
As a first example of the kind of data obtained, Fig. 9.12 presents the results of AUC analyses of
two BR/A8-35 samples (Study 9.5; Gohon et al. 2008). In one case, BR was trapped using a batch of
unlabeled A8-35 (HAPol), which, upon SEC, was found to form monodisperse particles
(cf. Fig. 4.14A). In keeping with SEC data (Fig. 9.7A), AUC showed that the preparation of HAPoltrapped BR comprised only two types of particles, pure HAPol particles, migrating with a sedimentation coefficient s 20,w ¼ 1.6 S, as observed with pure A8-35 preparations (Gohon et al. 2006), and
monomeric BR/APol particles, with s 20,w ¼ 3.2 S, representing ~98% of the BR present in the sample
(Fig. 9.12C; cf. the SEC analysis presented in Fig. 9.7A). The ratio A 280 /A 550 ¼ 1.6 in the BR peak
indicates that BR is in its native state. From a comparison of interference measurements (J), which
detect all species with a refractive index different from that of the solution, with absorbance
measurements at 280 nm (protein) and 550 nm (BR in its native state), one can deduce that
BR/A8-35 complexes comprise ~2 g A8-35 per g of BR (taking into account the contribution of
lipids, whose mass ratio in the complexes was determined by biochemical analysis; Gohon et al. 2008)
(Table 9.2).
A second sample was prepared using a batch of deuterated A8-35 (DAPol), which, for reasons
unrelated to the deuteration, turned out to be more hydrophobic than standard A8-35 and, upon SEC,
formed slightly polydisperse particles. AUC of the preparation of BR trapped using this batch showed
the presence of free DAPol particles, with s 20,w % 2 S, a majority of monomeric DAPol-trapped BR
(s 20,w % 4), a small fraction (~11%) of dimers, and traces (~2%) of trimers (Fig. 9.12C
0 ; see also SEC
analysis in Fig. 9.7A). The ratio A 280 /A 550 ¼ 1.6 in the BR peaks again indicates that the protein is in its
native state. Some of the data deduced from these two series of experiments are compiled in Table 9.2.
Sedimentation velocity experiments were completed with equilibrium sedimentation ones
(Fig. 9.13). Analyses carried out in buffers prepared in either H 2 O, D 2 O, or D 2
18 O yielded information
about the composition of BR/A8-35 particles through the determination of their buoyant molar mass
(M b ), which, in a given solvent, depends on the particle mass and density (see Box 9.1). Data were
9.3 A Walk Through Selected Solution Studies
423
2+ degrade the resolution of
solution NMR spectra by bridging some of the particles via their A8-35 belt.
9.3.7
Analytical Ultracentrifugation
Analytical ultracentrifugation (AUC) is a particularly powerful method for determining the size and
mass of solutes. It can be used at equilibrium, by subjecting the sample to a relatively low gravity field
and examining its distribution throughout the buffer column once it has reached final equilibration, or
as a sedimentation velocity technique, in which case the redistribution of the various solutes is
followed throughout the experiment (see Box 9.1; for reviews, see e.g. Laue and Stafford 1999;
Carruthers et al. 2000; Schuck 2000; Padrick et al. 2010; Ebel 2011; and references therein). The latter
approach is particularly powerful when dealing with mixtures, as is usually the case of MP/surfactant
preparations, which generally contain both MP/surfactant complexes and free surfactant particles.
Detection can be achieved either by absorbance spectroscopy at multiple wavelengths or by measuring
the refractive index of the solution, measurements being carried out at each point along the buffer
column as a function of time. The data are then decomposed to yield the sedimentation coefficients and
relative concentrations of the various species present in the solution, as well as, under favorable
circumstances, the ratio of various components in a particle. AUC has been applied to eight
APol-trapped MPs, ranging from small (BR; ~27 kDa) to moderately large (AcrB trimer;
~360 kDa), trapped in A8-35, in one of its fluorescent or deuterated derivatives, in NAPols, or in
SMA (see Table 9.1).
As a first example of the kind of data obtained, Fig. 9.12 presents the results of AUC analyses of
two BR/A8-35 samples (Study 9.5; Gohon et al. 2008). In one case, BR was trapped using a batch of
unlabeled A8-35 (HAPol), which, upon SEC, was found to form monodisperse particles
(cf. Fig. 4.14A). In keeping with SEC data (Fig. 9.7A), AUC showed that the preparation of HAPoltrapped BR comprised only two types of particles, pure HAPol particles, migrating with a sedimentation coefficient s 20,w ¼ 1.6 S, as observed with pure A8-35 preparations (Gohon et al. 2006), and
monomeric BR/APol particles, with s 20,w ¼ 3.2 S, representing ~98% of the BR present in the sample
(Fig. 9.12C; cf. the SEC analysis presented in Fig. 9.7A). The ratio A 280 /A 550 ¼ 1.6 in the BR peak
indicates that BR is in its native state. From a comparison of interference measurements (J), which
detect all species with a refractive index different from that of the solution, with absorbance
measurements at 280 nm (protein) and 550 nm (BR in its native state), one can deduce that
BR/A8-35 complexes comprise ~2 g A8-35 per g of BR (taking into account the contribution of
lipids, whose mass ratio in the complexes was determined by biochemical analysis; Gohon et al. 2008)
(Table 9.2).
A second sample was prepared using a batch of deuterated A8-35 (DAPol), which, for reasons
unrelated to the deuteration, turned out to be more hydrophobic than standard A8-35 and, upon SEC,
formed slightly polydisperse particles. AUC of the preparation of BR trapped using this batch showed
the presence of free DAPol particles, with s 20,w % 2 S, a majority of monomeric DAPol-trapped BR
(s 20,w % 4), a small fraction (~11%) of dimers, and traces (~2%) of trimers (Fig. 9.12C
0 ; see also SEC
analysis in Fig. 9.7A). The ratio A 280 /A 550 ¼ 1.6 in the BR peaks again indicates that the protein is in its
native state. Some of the data deduced from these two series of experiments are compiled in Table 9.2.
Sedimentation velocity experiments were completed with equilibrium sedimentation ones
(Fig. 9.13). Analyses carried out in buffers prepared in either H 2 O, D 2 O, or D 2
18 O yielded information
about the composition of BR/A8-35 particles through the determination of their buoyant molar mass
(M b ), which, in a given solvent, depends on the particle mass and density (see Box 9.1). Data were
9.3 A Walk Through Selected Solution Studies
423