6.4.3
Renaturation of b-Barrel MPs in APols
This step relies on diluting urea in the presence of APols (Fig. 6.6B).
1. Set, for example, to 1 mg the mass of protein to fold. Dilute the sample by a 10Â dilution
factor into urea-free buffer containing 5 mg of APols, so that the final MP/APol mass ratio is
1:5. Test also ratios 1:2 and 1:10. Note that the dilution factor and speed of dilution can be
optimized. If need be, incubate the samples at 40
C for 24 h.
2. Concentrate the samples using an ultrafiltration device and measure the optical density of
supernatants.
6.4.4
Completing the Renaturation
To increase the yield of folding, urea or DS traces can be further eliminated by a dialysis step.
1. Dialyze the sample for 24 h at room temperature using a standard dialysis membrane of
12–14 kDa MW cutoff. Note that APols are not needed in the external bath, but the presence
of 150 mM KCl is required to prevent redissolution of crystallites of PDS that may not have
been totally removed by centrifugation. The volume of the external bath is ~500Â larger than
the volume of the samples.
2. Recover the sample and centrifuge it for 5 min at the maximum speed of a benchtop
centrifuge.
3. Measure the optical density of the samples.
4. If the buffer needs to be exchanged, proceed to a second dialysis for 24 h at 4
C.
The solubility of MP is not a criterion of folding. The simplest and most direct proof that the
protein adopts its native conformation is to check its activity. If the activity assay is not easy to set up,
the yield of folding can be assessed by other approaches such as ligand-binding experiments using
equilibrium dialysis. In that case, ligand titration can be monitored by radioactivity measurements or
by following changes in the intensity of fluorescence emission or light absorbance. If the protein is
naturally colored in its native conformation due to the binding of a cofactor, such as retinal for BR, the
native state can be quantified by spectral absorbance changes. It is also possible to check the
homogeneity and size of the protein by SEC, its secondary structure by CD, the local environment
of tryptophan residues by CD and fluorescence measurements, the melting temperature by differential
scanning calorimetry, or by fluorescence thermal shift. In the case of porins, the folded state of the
protein can usually be assessed by SDS-PAGE, upon which, as a rule, folded and unfolded forms
exhibit different electrophoretic mobilities (cf. Figs. 6.9 and 6.10), by dot blots if an antibody
recognizing the native state of the protein is available, by protease digestion, etc.
Protocols prepared by Tassadite Dahmane and Manuela Zoonens, adapted from Zoonens
et al. (2014).
References
Adamian, L., Naveed, H., Liang, J. (2011) Lipid-binding surfaces of membrane proteins: Evidence from evolutionary
and structural analysis. Biochim. Biophys. Acta 1808:1092–1102.
356
6 Amphipol-Assisted Folding of Membrane Proteins
Renaturation of b-Barrel MPs in APols
This step relies on diluting urea in the presence of APols (Fig. 6.6B).
1. Set, for example, to 1 mg the mass of protein to fold. Dilute the sample by a 10Â dilution
factor into urea-free buffer containing 5 mg of APols, so that the final MP/APol mass ratio is
1:5. Test also ratios 1:2 and 1:10. Note that the dilution factor and speed of dilution can be
optimized. If need be, incubate the samples at 40
C for 24 h.
2. Concentrate the samples using an ultrafiltration device and measure the optical density of
supernatants.
6.4.4
Completing the Renaturation
To increase the yield of folding, urea or DS traces can be further eliminated by a dialysis step.
1. Dialyze the sample for 24 h at room temperature using a standard dialysis membrane of
12–14 kDa MW cutoff. Note that APols are not needed in the external bath, but the presence
of 150 mM KCl is required to prevent redissolution of crystallites of PDS that may not have
been totally removed by centrifugation. The volume of the external bath is ~500Â larger than
the volume of the samples.
2. Recover the sample and centrifuge it for 5 min at the maximum speed of a benchtop
centrifuge.
3. Measure the optical density of the samples.
4. If the buffer needs to be exchanged, proceed to a second dialysis for 24 h at 4
C.
The solubility of MP is not a criterion of folding. The simplest and most direct proof that the
protein adopts its native conformation is to check its activity. If the activity assay is not easy to set up,
the yield of folding can be assessed by other approaches such as ligand-binding experiments using
equilibrium dialysis. In that case, ligand titration can be monitored by radioactivity measurements or
by following changes in the intensity of fluorescence emission or light absorbance. If the protein is
naturally colored in its native conformation due to the binding of a cofactor, such as retinal for BR, the
native state can be quantified by spectral absorbance changes. It is also possible to check the
homogeneity and size of the protein by SEC, its secondary structure by CD, the local environment
of tryptophan residues by CD and fluorescence measurements, the melting temperature by differential
scanning calorimetry, or by fluorescence thermal shift. In the case of porins, the folded state of the
protein can usually be assessed by SDS-PAGE, upon which, as a rule, folded and unfolded forms
exhibit different electrophoretic mobilities (cf. Figs. 6.9 and 6.10), by dot blots if an antibody
recognizing the native state of the protein is available, by protease digestion, etc.
Protocols prepared by Tassadite Dahmane and Manuela Zoonens, adapted from Zoonens
et al. (2014).
References
Adamian, L., Naveed, H., Liang, J. (2011) Lipid-binding surfaces of membrane proteins: Evidence from evolutionary
and structural analysis. Biochim. Biophys. Acta 1808:1092–1102.
356
6 Amphipol-Assisted Folding of Membrane Proteins