9.5.2
Protocol 9.2: Size Exclusion Chromatography Analysis of Membrane
Protein/Amphipol Complexes
9.5.2.1 Introduction
Size exclusion chromatography (SEC) provides a very convenient tool to evaluate the outcome of
experiments in which membrane proteins (MPs) are trapped and/or folded in amphipols (APols).
Analytical SEC will give information on (i) the homogeneity and the size of the MP/APol complexes
(see e.g. Picard et al. 2006; Le Bon et al. 2014a), (ii) the fraction of free APol present in the preparation
(see e.g. Zoonens et al. 2007; Charvolin et al. 2014), (iii) the presence of protein aggregates (see
e.g. Pocanschi et al. 2006; Gohon et al. 2008), (iv) the mass ratio of MP and APol in the complexes
(Zoonens et al. 2007; Charvolin et al. 2014; cf. in Chap. 5, § 5.9.2, Protocol 5.2), and (v) the stability of
MP/APol complexes over time (Charvolin et al. 2014). In preparative SEC, peak fractionation provides
a purification step in view of structural studies, e.g. for cryo-electron microscopy (see e.g. Chap. 12,
§ 12.4, Protocol 12.1, based on Paulsen et al. 2015, or, for a more recent piece of work, Fitzpatrick
et al. 2017).
Bacteriorhodopsin (BR; see Chap. 1, § 1.6.1) was used as a model protein in several of the
experiments to be described below. BR is a particularly convenient model as it provides a visual
control of the protein’s state: when it is correctly folded and linked to its cofactor retinal via a Schiff
base, it presents a characteristic purple color; when it denatures and the Schiff base becomes
hydrolyzed, retinal is released, and the color of the sample turns to yellow. The ratio of the absorbance
at 554 and 280 nm is a convenient measure of the fraction of the protein that is in its native state. In the
following, details of the experimental steps that are specific to BR are marked with a ■ symbol, those
applying to other MPs with □. General comments and tips for optimization are in italics and marked
with a pointing hand sign ( ).
9.5.2.2 Biochemistry
■ Purple membrane purification: see Gohon et al. (2008).
■ □ Preparation of MP/APol complexes. MP/APol complexes can be obtained: (i) by transferring to APols a detergent-solubilized MP (see Chap. 5, § 5.9.1, Protocol 5.1) or (ii) by folding in APol a
denatured MP (see Chap. 6, § 6.4, Protocol 6.1). When first developing a trapping or folding protocol,
a set of samples will usually be prepared at different MP/APol mass ratios. SEC is a convenient tool to
determine the optimal ratio.
■ For BR/APol complexes, the usual mass ratios examined when first testing a new APol are
typically 1:2, 1:5, and 1:10. With A8-35, 1:5 w/w is a good ratio; BR binds ~2 g A8-35 per g protein,
the rest of the APol remaining free (Gohon et al. 2008).
□ For the transmembrane domain of outer membrane protein A from Escherichia coli (tOmpA),
which, like BR, has most of its mass in a TM position, 1:4 w/w is a good MP/APol ratio (Zoonens et al.
2005). For MPs whose extramembrane domains are comparatively more extended, less APol per mass
of protein is required, e.g. only 1:1.5 w/w MP/A8-35 in the case of cytochrome bc 1 (Charvolin et al.
2014). In principle, one would expect the mass of APol that binds to a given MP to be more or less
proportional to its membrane-exposed hydrophobic TM surface, but existing data are quite scattered
(see Chap. 5, Table 5.5 and Fig. 5.9).
444
9 Solution Studies of Membrane Protein/Amphipol Complexes
Protocol 9.2: Size Exclusion Chromatography Analysis of Membrane
Protein/Amphipol Complexes
9.5.2.1 Introduction
Size exclusion chromatography (SEC) provides a very convenient tool to evaluate the outcome of
experiments in which membrane proteins (MPs) are trapped and/or folded in amphipols (APols).
Analytical SEC will give information on (i) the homogeneity and the size of the MP/APol complexes
(see e.g. Picard et al. 2006; Le Bon et al. 2014a), (ii) the fraction of free APol present in the preparation
(see e.g. Zoonens et al. 2007; Charvolin et al. 2014), (iii) the presence of protein aggregates (see
e.g. Pocanschi et al. 2006; Gohon et al. 2008), (iv) the mass ratio of MP and APol in the complexes
(Zoonens et al. 2007; Charvolin et al. 2014; cf. in Chap. 5, § 5.9.2, Protocol 5.2), and (v) the stability of
MP/APol complexes over time (Charvolin et al. 2014). In preparative SEC, peak fractionation provides
a purification step in view of structural studies, e.g. for cryo-electron microscopy (see e.g. Chap. 12,
§ 12.4, Protocol 12.1, based on Paulsen et al. 2015, or, for a more recent piece of work, Fitzpatrick
et al. 2017).
Bacteriorhodopsin (BR; see Chap. 1, § 1.6.1) was used as a model protein in several of the
experiments to be described below. BR is a particularly convenient model as it provides a visual
control of the protein’s state: when it is correctly folded and linked to its cofactor retinal via a Schiff
base, it presents a characteristic purple color; when it denatures and the Schiff base becomes
hydrolyzed, retinal is released, and the color of the sample turns to yellow. The ratio of the absorbance
at 554 and 280 nm is a convenient measure of the fraction of the protein that is in its native state. In the
following, details of the experimental steps that are specific to BR are marked with a ■ symbol, those
applying to other MPs with □. General comments and tips for optimization are in italics and marked
with a pointing hand sign ( ).
9.5.2.2 Biochemistry
■ Purple membrane purification: see Gohon et al. (2008).
■ □ Preparation of MP/APol complexes. MP/APol complexes can be obtained: (i) by transferring to APols a detergent-solubilized MP (see Chap. 5, § 5.9.1, Protocol 5.1) or (ii) by folding in APol a
denatured MP (see Chap. 6, § 6.4, Protocol 6.1). When first developing a trapping or folding protocol,
a set of samples will usually be prepared at different MP/APol mass ratios. SEC is a convenient tool to
determine the optimal ratio.
■ For BR/APol complexes, the usual mass ratios examined when first testing a new APol are
typically 1:2, 1:5, and 1:10. With A8-35, 1:5 w/w is a good ratio; BR binds ~2 g A8-35 per g protein,
the rest of the APol remaining free (Gohon et al. 2008).
□ For the transmembrane domain of outer membrane protein A from Escherichia coli (tOmpA),
which, like BR, has most of its mass in a TM position, 1:4 w/w is a good MP/APol ratio (Zoonens et al.
2005). For MPs whose extramembrane domains are comparatively more extended, less APol per mass
of protein is required, e.g. only 1:1.5 w/w MP/A8-35 in the case of cytochrome bc 1 (Charvolin et al.
2014). In principle, one would expect the mass of APol that binds to a given MP to be more or less
proportional to its membrane-exposed hydrophobic TM surface, but existing data are quite scattered
(see Chap. 5, Table 5.5 and Fig. 5.9).
444
9 Solution Studies of Membrane Protein/Amphipol Complexes