APols (Chap. 5, § 5.2.1, and Fig. 9.7D). Such measurements should therefore be carried out
either in the presence of enough free APol to prevent these phenomena, or by focusing on the
composition of the monomeric fraction of the preparations, as can be done, for instance, by
multiwavelength analysis in AUC.
• Performed under carefully controlled conditions, SANS and SAXS have the potential to
provide detailed data about the dimensions and shape of APol-trapped MPs. SANS is
particularly powerful, because contrast-matching makes it possible to eliminate the
contributions to scattering of both free and protein-bound APols. Comparing models built
using data gathered with and without contrast-matching of the APol may help in their
interpretation and that of electron microscopy images.
A detailed knowledge and control of the composition of MP/APol preparations is a strong asset
when designing experiments aiming at collecting more detailed data by NMR, crystallography, or
electron microscopy. In NMR, for instance (Chap. 10), the resolution of spectra can be improved by
adding EDTA, a treatment that prevents traces of divalent ions present in the buffers from cross-linking
MP/A8-35 complexes (Catoire et al. 2010). Homogeneity is a critical parameter for crystallization
from solutions (Chap. 11, § 11.3.1). It may not be so important for crystallization in lipidic mesophases
(ibid., § 11.3.2). However, the dispersity of the complexes before they are transferred to the mesophase
will likely be a parameter worth controlling. In single-particle cryo-EM (Chap. 12), it is always
possible to sort out the images of particles that are sufficiently different from one another, such as
monomers and dimers, but this can become more difficult, for instance, with oligomers featuring
different subunit stoichiometries. Previous knowledge of the homogeneity or heterogeneity of the
preparations is definitely desirable.
The next three chapters will be devoted to a review and discussion of the studies of APol-trapped
MPs by NMR, X-ray crystallography, and electron microscopy and the prospects they present.
9.5
Protocols
9.5.1
Protocol 9.1: Analytical Ultracentrifugation of Membrane Protein/
Amphipol Complexes
9.5.1.1 Introduction
Analytical ultracentrifugation (AUC) examines the behavior of macromolecules in solution when
subjected to a centrifugal field. Absorbance and interference fringe shifts measured as a function of the
distance to the rotation axis reflect the evolution of the distribution of the particles. Sedimentation
velocity (SV) experiments are performed at high speed, sedimentation profiles being measured as a
function of time. The different types of macromolecules (in the case of MPs, MP/surfactant complexes,
and free surfactant particles) sediment at different velocities depending on their sedimentation coefficient s. Analysis of AUC data yields the distribution c(s) of the particles present in a sample as a
function of s. SV-AUC is a very powerful tool to assess sample homogeneity, the amount of free and
bound surfactant, and the occurrence of protein-protein association equilibria. If, as is the case for
bacteriorhodopsin (BR), the UV-visible spectra of the native and denatured protein are different, the
two states can be distinguished and the extent to which the protein has retained its native state can be
precisely assessed.
AUC is routinely used for the characterization of MP/detergent complexes (for reviews, see
e.g. Ebel 2011; Le Roy et al. 2014, 2015). It is a priori applicable to any MP, because surfactant particles
440
9 Solution Studies of Membrane Protein/Amphipol Complexes
either in the presence of enough free APol to prevent these phenomena, or by focusing on the
composition of the monomeric fraction of the preparations, as can be done, for instance, by
multiwavelength analysis in AUC.
• Performed under carefully controlled conditions, SANS and SAXS have the potential to
provide detailed data about the dimensions and shape of APol-trapped MPs. SANS is
particularly powerful, because contrast-matching makes it possible to eliminate the
contributions to scattering of both free and protein-bound APols. Comparing models built
using data gathered with and without contrast-matching of the APol may help in their
interpretation and that of electron microscopy images.
A detailed knowledge and control of the composition of MP/APol preparations is a strong asset
when designing experiments aiming at collecting more detailed data by NMR, crystallography, or
electron microscopy. In NMR, for instance (Chap. 10), the resolution of spectra can be improved by
adding EDTA, a treatment that prevents traces of divalent ions present in the buffers from cross-linking
MP/A8-35 complexes (Catoire et al. 2010). Homogeneity is a critical parameter for crystallization
from solutions (Chap. 11, § 11.3.1). It may not be so important for crystallization in lipidic mesophases
(ibid., § 11.3.2). However, the dispersity of the complexes before they are transferred to the mesophase
will likely be a parameter worth controlling. In single-particle cryo-EM (Chap. 12), it is always
possible to sort out the images of particles that are sufficiently different from one another, such as
monomers and dimers, but this can become more difficult, for instance, with oligomers featuring
different subunit stoichiometries. Previous knowledge of the homogeneity or heterogeneity of the
preparations is definitely desirable.
The next three chapters will be devoted to a review and discussion of the studies of APol-trapped
MPs by NMR, X-ray crystallography, and electron microscopy and the prospects they present.
9.5
Protocols
9.5.1
Protocol 9.1: Analytical Ultracentrifugation of Membrane Protein/
Amphipol Complexes
9.5.1.1 Introduction
Analytical ultracentrifugation (AUC) examines the behavior of macromolecules in solution when
subjected to a centrifugal field. Absorbance and interference fringe shifts measured as a function of the
distance to the rotation axis reflect the evolution of the distribution of the particles. Sedimentation
velocity (SV) experiments are performed at high speed, sedimentation profiles being measured as a
function of time. The different types of macromolecules (in the case of MPs, MP/surfactant complexes,
and free surfactant particles) sediment at different velocities depending on their sedimentation coefficient s. Analysis of AUC data yields the distribution c(s) of the particles present in a sample as a
function of s. SV-AUC is a very powerful tool to assess sample homogeneity, the amount of free and
bound surfactant, and the occurrence of protein-protein association equilibria. If, as is the case for
bacteriorhodopsin (BR), the UV-visible spectra of the native and denatured protein are different, the
two states can be distinguished and the extent to which the protein has retained its native state can be
precisely assessed.
AUC is routinely used for the characterization of MP/detergent complexes (for reviews, see
e.g. Ebel 2011; Le Roy et al. 2014, 2015). It is a priori applicable to any MP, because surfactant particles
440
9 Solution Studies of Membrane Protein/Amphipol Complexes