for the fast process, ~36.5 Æ 9.6 kJÁmol
À1 (~8.7 kcalÁmol
À1 ) for the slow one. When monitored by
fluorescence spectroscopy, these activation energies were ~8.8 Æ 2.3 kJÁmol
À1 for the fast process and
28.9 Æ 8.1 kJÁmol
À1 for the slow one. Within error margins, these values are comparable. They are
smaller than the activation energy of 46 kJÁmol
À1 reported for folding OmpA in small unilamellar
vesicles of dioleoylphosphatidylcholine (Kleinschmidt and Tamm 1996; Kleinschmidt 2015), which
may reflect the fact that reorganization of the protein in APols faces lower free energy barriers than in
lipids.
6.3.2
Why Are Amphipols a Good Medium for Membrane Protein Folding?
As described above, folding MPs in APols has been, to date, remarkably successful. This statement
must be qualified by noting that only a limited range of structural types has been explored so far: 7-αhelix bundles and single β-barrel outer membrane proteins (Table 6.1). In all cases that have been
tested to date, folding yields were at least as good as and usually better than those obtained, using
generally more complex procedures, in the presence of detergent or lipid/detergent mixed micelles.
Furthermore, no lengthy search for optimization was required, as is usually needed when endeavoring
to fold a new MP in a detergent environment. Indeed, two more or less universal protocols based on
urea dilution (β-barrel MPs) or PDS precipitation (α-helical ones) in the presence of APols give, as a
rule, satisfying results without departing very much from the conditions initially established for model
proteins, OmpA and FomA on the one hand, BR on the other (Pocanschi et al. 2006).
In a couple of cases where GPCRs did not fold efficiently in A8-35, the preparation in SDS
appeared heterogeneous upon SDS-PAGE (unpublished data). It is unsurprising that preparations in
which a large fraction of the material is either aggregated or misfolded do not fold in good yield.
Indeed, in early experiments in which PDS precipitation was used to refold and reassociate two BR
fragments in a lipid environment, satisfactory yields were not achieved before a way was devised to
transfer the fragments from organic solution to SDS without inducing any aggregation (Popot et al.
1987). Every effort should therefore be spent to obtain starting material that yields a single band upon
SDS-PAGE before investing too much time in refolding attempts. In difficult cases, one could consider
resorting to organic solvents to achieve complete unfolding, followed by transfer to SDS (Huang et al.
1981; Popot et al. 1987; Pocanschi et al. 2006; Dahmane et al. 2013). Replacing SDS with tetradecyl
sulfate, which is a stronger denaturant (Moosavi-Movahedi et al. 2003), is perhaps another option,
which has not been tested yet.
It is worth noting that, in all experiments published to date, no special precaution was taken to
control the reformation of disulfide bridges, if any. It is to be expected that control of the redox
potential, e.g. by folding first in a reducing medium, followed by oxidation, or by folding in the
presence of a mixture of reduced and oxidized glutathione, will turn out to be necessary in specific
cases.
In all cases where it has been tested, the presence of lipids (i) was not necessary to obtaining
good folding yields but (ii) improved the yield as compared to that observed in their absence. As lipids
were used in small amounts as compared to APols (typically in a 1:5 mass ratio), an effect on the
physical properties of the APol belt surrounding the refolding protein seems rather unlikely. More
probably, molecular interactions are at work. Crystallographic structures of MPs have revealed welldefined binding sites for lipids, often at the protein/lipid interface, sometimes within protein TM
domains (see Chap. 1, § 1.5.2). Lipids in biological membranes, should therefore not be considered as
a mere two-dimensional solvent: they also play the role of cofactors (for discussions, see e.g. Popot and
Engelman 2000; Lee 2003, 2011; Adamian et al. 2011; Aponte-Santamaríaa et al. 2012; Stansfeld et al.
2013). One way to understand their favorable effect on folding yields in APols is to assume that in the
350
6 Amphipol-Assisted Folding of Membrane Proteins
À1 (~8.7 kcalÁmol
À1 ) for the slow one. When monitored by
fluorescence spectroscopy, these activation energies were ~8.8 Æ 2.3 kJÁmol
À1 for the fast process and
28.9 Æ 8.1 kJÁmol
À1 for the slow one. Within error margins, these values are comparable. They are
smaller than the activation energy of 46 kJÁmol
À1 reported for folding OmpA in small unilamellar
vesicles of dioleoylphosphatidylcholine (Kleinschmidt and Tamm 1996; Kleinschmidt 2015), which
may reflect the fact that reorganization of the protein in APols faces lower free energy barriers than in
lipids.
6.3.2
Why Are Amphipols a Good Medium for Membrane Protein Folding?
As described above, folding MPs in APols has been, to date, remarkably successful. This statement
must be qualified by noting that only a limited range of structural types has been explored so far: 7-αhelix bundles and single β-barrel outer membrane proteins (Table 6.1). In all cases that have been
tested to date, folding yields were at least as good as and usually better than those obtained, using
generally more complex procedures, in the presence of detergent or lipid/detergent mixed micelles.
Furthermore, no lengthy search for optimization was required, as is usually needed when endeavoring
to fold a new MP in a detergent environment. Indeed, two more or less universal protocols based on
urea dilution (β-barrel MPs) or PDS precipitation (α-helical ones) in the presence of APols give, as a
rule, satisfying results without departing very much from the conditions initially established for model
proteins, OmpA and FomA on the one hand, BR on the other (Pocanschi et al. 2006).
In a couple of cases where GPCRs did not fold efficiently in A8-35, the preparation in SDS
appeared heterogeneous upon SDS-PAGE (unpublished data). It is unsurprising that preparations in
which a large fraction of the material is either aggregated or misfolded do not fold in good yield.
Indeed, in early experiments in which PDS precipitation was used to refold and reassociate two BR
fragments in a lipid environment, satisfactory yields were not achieved before a way was devised to
transfer the fragments from organic solution to SDS without inducing any aggregation (Popot et al.
1987). Every effort should therefore be spent to obtain starting material that yields a single band upon
SDS-PAGE before investing too much time in refolding attempts. In difficult cases, one could consider
resorting to organic solvents to achieve complete unfolding, followed by transfer to SDS (Huang et al.
1981; Popot et al. 1987; Pocanschi et al. 2006; Dahmane et al. 2013). Replacing SDS with tetradecyl
sulfate, which is a stronger denaturant (Moosavi-Movahedi et al. 2003), is perhaps another option,
which has not been tested yet.
It is worth noting that, in all experiments published to date, no special precaution was taken to
control the reformation of disulfide bridges, if any. It is to be expected that control of the redox
potential, e.g. by folding first in a reducing medium, followed by oxidation, or by folding in the
presence of a mixture of reduced and oxidized glutathione, will turn out to be necessary in specific
cases.
In all cases where it has been tested, the presence of lipids (i) was not necessary to obtaining
good folding yields but (ii) improved the yield as compared to that observed in their absence. As lipids
were used in small amounts as compared to APols (typically in a 1:5 mass ratio), an effect on the
physical properties of the APol belt surrounding the refolding protein seems rather unlikely. More
probably, molecular interactions are at work. Crystallographic structures of MPs have revealed welldefined binding sites for lipids, often at the protein/lipid interface, sometimes within protein TM
domains (see Chap. 1, § 1.5.2). Lipids in biological membranes, should therefore not be considered as
a mere two-dimensional solvent: they also play the role of cofactors (for discussions, see e.g. Popot and
Engelman 2000; Lee 2003, 2011; Adamian et al. 2011; Aponte-Santamaríaa et al. 2012; Stansfeld et al.
2013). One way to understand their favorable effect on folding yields in APols is to assume that in the
350
6 Amphipol-Assisted Folding of Membrane Proteins