precipitation (Dahmane et al. 2013; Elter et al. 2014) (Table 6.1, lines 3–6 and 9). Upon SDS removal
by dialysis, folding yields of BR increased with the A8-35/BO ratio, suggesting that aggregation is a
limiting factor (Dahmane et al. 2013).
BR was also successfully refolded by diluting a BO/SDS/A8-35 mixture with SDS-free buffer
(Table 6.1, lines 7–8), in which case larger mass ratios of A8-35/BO were required than observed using
either PDS precipitation or SDS dialysis: when folding was initiated by dilution, yields were highest at
a mass ratio of 25 (Dahmane et al. 2013). Two mechanisms may contribute to this effect. First, adding
more A8-35 reduces the proportion of SDS in the environment of the refolding proteins. Second, the
probability that the latter will establish intermolecular associations is reduced by diluting them with
more APol, making aggregation less likely (cf. Zoonens et al. 2007). Folding kinetics were faster at
higher concentrations of retinal, consistent with the view that refolded BO can pick up retinal very
rapidly if it is present in its associated APol belt, whereas the process is much slower if retinal uptake
depends on BO/APol complexes colliding with retinal-containing free particles of APol (cf. Zoonens
et al. 2007).
Folding of dBO in A8-35 by PDS precipitation was also successful in the absence of retinal
(Dahmane et al. 2013). However, retinal is known to stabilize BR (Kahn et al. 1992), and indeed
refolded BO is less stable than refolded BR: when retinal was added shortly after the transfer from SDS
to A8-35, the refolding yield was found to be as high as that observed when retinal is present during the
transfer (~80%, which is in the upper range of that observed with lipid-free BO preparations; Table 6.1,
line 2). If, on the other hand, retinal was first provided 3 days after the transfer, the percentage of
holoprotein in the renatured preparation dropped to ~30%. The most straightforward interpretation of
this observation is that, in the 3-day interval, ~2/3 of the BO that had initially refolded had denatured
again. This observation confirms (i) that BO folds efficiently in the absence of retinal (renatured BO
indeed features the same secondary structure as BR refolded from delipidated BO/SDS in the presence
of retinal; Dahmane et al. 2013) and (ii) that early enough rebinding of retinal is critical in stabilizing
the refolded state and, thereby, in determining the final yield of renaturation. This observation is
consistent with the general view that folding yields of fragile MPs can be improved in the presence of
their ligands.
Transfer from SDS to A8-35 can also be achieved by adsorbing SDS onto Bio-Beads, provided
aggregation is limited by immobilizing polyhistidine-tagged BR onto a nickel-bearing column (Elter
et al. 2014).
6.3.1.1.2 A8-35-Assisted Folding of GPCRs
The remarkable results achieved with BR prompted attempts at folding GPCRs, for which folding
using classic detergent/lipid systems, when successful, is typically limited to yields of 30% or less
(reviewed in Banères et al. 2011). The first GPCR whose folding in A8-35 was studied was BLT1, a
leukotriene receptor involved in the control of inflammatory processes. The protocol used was the most
successful of those tested on BR (Pocanschi et al. 2006; Dahmane et al. 2013) (Fig. 6.6A): BLT1, after
being solubilized in SDS from inclusion bodies, was supplemented with A8-35, most of DS
precipitated as PDS, and residual DS removed by dialysis. Ligand-binding assays indicated that the
receptor thus folded was functional, with a dissociation constant, K D % 9 nM, similar to that of native
BLT1 expressed in membrane fractions (Damian et al. 2006). Based on the number of binding sites per
mass of protein in the original SDS solution, the yield of folding was ~50% in pure A8-35 and 65–70%
in the presence of soybean lipids (asolectin; BLT1/A8-35/lipid mass ratio 1:5:1) (Table 6.1, lines
10–11). A8-35-folded BLT1 showed a similar pharmacological profile to the membrane-bound
receptor (Dahmane et al. 2009). Interestingly, in the absence of lipids, an optimal protein/APol
ratio of ~1:5 w/w was observed for BLT1 (Fig. 6.8, left), the possible reasons for which are discussed
in Box 6.2.
6.3 Amphipol-Assisted Folding of Membrane Proteins
343
by dialysis, folding yields of BR increased with the A8-35/BO ratio, suggesting that aggregation is a
limiting factor (Dahmane et al. 2013).
BR was also successfully refolded by diluting a BO/SDS/A8-35 mixture with SDS-free buffer
(Table 6.1, lines 7–8), in which case larger mass ratios of A8-35/BO were required than observed using
either PDS precipitation or SDS dialysis: when folding was initiated by dilution, yields were highest at
a mass ratio of 25 (Dahmane et al. 2013). Two mechanisms may contribute to this effect. First, adding
more A8-35 reduces the proportion of SDS in the environment of the refolding proteins. Second, the
probability that the latter will establish intermolecular associations is reduced by diluting them with
more APol, making aggregation less likely (cf. Zoonens et al. 2007). Folding kinetics were faster at
higher concentrations of retinal, consistent with the view that refolded BO can pick up retinal very
rapidly if it is present in its associated APol belt, whereas the process is much slower if retinal uptake
depends on BO/APol complexes colliding with retinal-containing free particles of APol (cf. Zoonens
et al. 2007).
Folding of dBO in A8-35 by PDS precipitation was also successful in the absence of retinal
(Dahmane et al. 2013). However, retinal is known to stabilize BR (Kahn et al. 1992), and indeed
refolded BO is less stable than refolded BR: when retinal was added shortly after the transfer from SDS
to A8-35, the refolding yield was found to be as high as that observed when retinal is present during the
transfer (~80%, which is in the upper range of that observed with lipid-free BO preparations; Table 6.1,
line 2). If, on the other hand, retinal was first provided 3 days after the transfer, the percentage of
holoprotein in the renatured preparation dropped to ~30%. The most straightforward interpretation of
this observation is that, in the 3-day interval, ~2/3 of the BO that had initially refolded had denatured
again. This observation confirms (i) that BO folds efficiently in the absence of retinal (renatured BO
indeed features the same secondary structure as BR refolded from delipidated BO/SDS in the presence
of retinal; Dahmane et al. 2013) and (ii) that early enough rebinding of retinal is critical in stabilizing
the refolded state and, thereby, in determining the final yield of renaturation. This observation is
consistent with the general view that folding yields of fragile MPs can be improved in the presence of
their ligands.
Transfer from SDS to A8-35 can also be achieved by adsorbing SDS onto Bio-Beads, provided
aggregation is limited by immobilizing polyhistidine-tagged BR onto a nickel-bearing column (Elter
et al. 2014).
6.3.1.1.2 A8-35-Assisted Folding of GPCRs
The remarkable results achieved with BR prompted attempts at folding GPCRs, for which folding
using classic detergent/lipid systems, when successful, is typically limited to yields of 30% or less
(reviewed in Banères et al. 2011). The first GPCR whose folding in A8-35 was studied was BLT1, a
leukotriene receptor involved in the control of inflammatory processes. The protocol used was the most
successful of those tested on BR (Pocanschi et al. 2006; Dahmane et al. 2013) (Fig. 6.6A): BLT1, after
being solubilized in SDS from inclusion bodies, was supplemented with A8-35, most of DS
precipitated as PDS, and residual DS removed by dialysis. Ligand-binding assays indicated that the
receptor thus folded was functional, with a dissociation constant, K D % 9 nM, similar to that of native
BLT1 expressed in membrane fractions (Damian et al. 2006). Based on the number of binding sites per
mass of protein in the original SDS solution, the yield of folding was ~50% in pure A8-35 and 65–70%
in the presence of soybean lipids (asolectin; BLT1/A8-35/lipid mass ratio 1:5:1) (Table 6.1, lines
10–11). A8-35-folded BLT1 showed a similar pharmacological profile to the membrane-bound
receptor (Dahmane et al. 2009). Interestingly, in the absence of lipids, an optimal protein/APol
ratio of ~1:5 w/w was observed for BLT1 (Fig. 6.8, left), the possible reasons for which are discussed
in Box 6.2.
6.3 Amphipol-Assisted Folding of Membrane Proteins
343