5.9.3
Protocol 5.3. Transferring a MP from A8-35 to Nanodiscs
5.9.3.1 Exchange of A8-35 for DDM
The interest of conducting MP studies in nanodiscs (NDs) rather than in APols lies in the ability of
NDs to provide a bilayer-like environment of defined size and lipid composition (see Chap. 3, § 3.3).
Even if the target MP is active and stable when trapped in APols, as is the case of the BLT2 GPCR used
here (Dahmane et al. 2009; Catoire et al. 2010a), reintroducing it into a membrane environment is
preferable for certain studies, especially those aimed at investigating the impact of the composition and
biophysical properties of the lipid environment. When starting from a detergent-solubilized MP, the
incorporation into NDs is straightforward, as NDs form spontaneously upon detergent removal from a
mixture comprising the scaffold protein (MSP), lipids, and the target MP in a detergent solution. In the
case of a MP trapped in APols, an additional step is required in order to remove the APol before
proceeding to ND reconstitution. This is achieved by displacing the APol with an excess of detergent
(Tribet et al. 1997; Zoonens et al. 2007). In our example, A8-35 is exchanged for DDM. Sodium cholate
has been tested as well, as this detergent is frequently used to solubilize the ND reconstitution mixture.
However, in our experience, sodium cholate is inefficient at displacing A8-35 from the transmembrane
surface of BLT2. DDM, on the contrary, completely removes the APol (Zoonens et al. 2007), which has
been checked by fluorescence spectroscopy, mass spectrometry, and NMR spectroscopy measurements
(Casiraghi et al. 2016). The exchange is performed on a Ni:NTA-charged resin, to which BLT2 is bound
via its polyhistidine tag. The addition of Ca
2+ facilitates APol removal, because it decreases A8-35
solubility (Picard et al. 2006; Diab et al. 2007) (Chap. 4, § 4.3.1.2.2). The exchange is conducted in the
presence of 0.2 gÁL
À1 cholesteryl hemisuccinate (CHS), a soluble analog of cholesterol that has a
stabilizing effect on GPCRs (Rosenbaum et al. 2007; Kuszak et al. 2009).
Briefly, the procedure is carried out as follows. Following A8-35-assisted folding of BLT2 (see
Chap. 6), both the receptor/APol complex and the Ni:NTA resin are incubated in a buffer containing a
high concentration of DDM (3 gÁL
À1 ). Following binding of BLT2 to the resin, two washing steps are
applied, first at 2 gÁL
À1 DDM in the presence of CaCl 2 , then with the same buffer but without Ca
2+ .
Once the exchange of surfactants is complete, the resin, carrying the bound BLT2/DDM complexes, is
collected to proceed to ND reconstitution. Experiments conducted in the presence of FAPol NBD , a
fluorescent version of A8-35 (Zoonens et al. 2007) (see Chap. 4, § 4.4), whose absorbance was followed
at 476 nm during FPLC and gel filtration analyses, attested to the complete removal of the APol.
• Protocol
1. Collect the APol-folded BLT2 (~1 mg for routine assays, ~6 mg for NMR samples)
from the dialysis bag in which it was freed of the last traces of dodecyl sulfate (see
Chap. 6, Protocol 6.1), and incubate at 4
C for 2 h under gentle stirring with 3 g‧L
À1
DDM, 0.2 g‧L
À1 CHS in 50 mM Tris/HCl, 150 mM NaCl buffer, pH 8.
2. It is advisable to use a FPLC chromatography system, if possible, so as to be able to
monitor the OD 280 during the exchange of surfactants. Set up the chromatography instrument at 4
C, pour in a XK 16/20 Column (GE Healthcare Life Sciences) the Ni:NTA
resin (Ni-NTA Superflow, Qiagen, 2–3 mL of resin per mg of BLT2), wash with water
purified on a Millipore Milli-Q Advantage A10 system (Milli-Q water), and equilibrate
with 3 g‧L
À1 DDM, 0.2 g‧L
À1 CHS in 50 mM Tris/HCl, 150 mM NaCl buffer, pH 8.
3. Pour the BLT2/A8-35/DDM solution incubated at stage 1 onto the chromatography
column washed and equilibrated at stage 2. Wash the resin with 20 column volumes of
5.9 Protocols
319
Protocol 5.3. Transferring a MP from A8-35 to Nanodiscs
5.9.3.1 Exchange of A8-35 for DDM
The interest of conducting MP studies in nanodiscs (NDs) rather than in APols lies in the ability of
NDs to provide a bilayer-like environment of defined size and lipid composition (see Chap. 3, § 3.3).
Even if the target MP is active and stable when trapped in APols, as is the case of the BLT2 GPCR used
here (Dahmane et al. 2009; Catoire et al. 2010a), reintroducing it into a membrane environment is
preferable for certain studies, especially those aimed at investigating the impact of the composition and
biophysical properties of the lipid environment. When starting from a detergent-solubilized MP, the
incorporation into NDs is straightforward, as NDs form spontaneously upon detergent removal from a
mixture comprising the scaffold protein (MSP), lipids, and the target MP in a detergent solution. In the
case of a MP trapped in APols, an additional step is required in order to remove the APol before
proceeding to ND reconstitution. This is achieved by displacing the APol with an excess of detergent
(Tribet et al. 1997; Zoonens et al. 2007). In our example, A8-35 is exchanged for DDM. Sodium cholate
has been tested as well, as this detergent is frequently used to solubilize the ND reconstitution mixture.
However, in our experience, sodium cholate is inefficient at displacing A8-35 from the transmembrane
surface of BLT2. DDM, on the contrary, completely removes the APol (Zoonens et al. 2007), which has
been checked by fluorescence spectroscopy, mass spectrometry, and NMR spectroscopy measurements
(Casiraghi et al. 2016). The exchange is performed on a Ni:NTA-charged resin, to which BLT2 is bound
via its polyhistidine tag. The addition of Ca
2+ facilitates APol removal, because it decreases A8-35
solubility (Picard et al. 2006; Diab et al. 2007) (Chap. 4, § 4.3.1.2.2). The exchange is conducted in the
presence of 0.2 gÁL
À1 cholesteryl hemisuccinate (CHS), a soluble analog of cholesterol that has a
stabilizing effect on GPCRs (Rosenbaum et al. 2007; Kuszak et al. 2009).
Briefly, the procedure is carried out as follows. Following A8-35-assisted folding of BLT2 (see
Chap. 6), both the receptor/APol complex and the Ni:NTA resin are incubated in a buffer containing a
high concentration of DDM (3 gÁL
À1 ). Following binding of BLT2 to the resin, two washing steps are
applied, first at 2 gÁL
À1 DDM in the presence of CaCl 2 , then with the same buffer but without Ca
2+ .
Once the exchange of surfactants is complete, the resin, carrying the bound BLT2/DDM complexes, is
collected to proceed to ND reconstitution. Experiments conducted in the presence of FAPol NBD , a
fluorescent version of A8-35 (Zoonens et al. 2007) (see Chap. 4, § 4.4), whose absorbance was followed
at 476 nm during FPLC and gel filtration analyses, attested to the complete removal of the APol.
• Protocol
1. Collect the APol-folded BLT2 (~1 mg for routine assays, ~6 mg for NMR samples)
from the dialysis bag in which it was freed of the last traces of dodecyl sulfate (see
Chap. 6, Protocol 6.1), and incubate at 4
C for 2 h under gentle stirring with 3 g‧L
À1
DDM, 0.2 g‧L
À1 CHS in 50 mM Tris/HCl, 150 mM NaCl buffer, pH 8.
2. It is advisable to use a FPLC chromatography system, if possible, so as to be able to
monitor the OD 280 during the exchange of surfactants. Set up the chromatography instrument at 4
C, pour in a XK 16/20 Column (GE Healthcare Life Sciences) the Ni:NTA
resin (Ni-NTA Superflow, Qiagen, 2–3 mL of resin per mg of BLT2), wash with water
purified on a Millipore Milli-Q Advantage A10 system (Milli-Q water), and equilibrate
with 3 g‧L
À1 DDM, 0.2 g‧L
À1 CHS in 50 mM Tris/HCl, 150 mM NaCl buffer, pH 8.
3. Pour the BLT2/A8-35/DDM solution incubated at stage 1 onto the chromatography
column washed and equilibrated at stage 2. Wash the resin with 20 column volumes of
5.9 Protocols
319