the regeneration of the characteristic purple color of BR within minutes, indicating folding of BO and
rebinding of the retinal (Pocanschi et al. 2006). After dialyzing to remove residual DS, almost
quantitative refolding of BR (!90%) is observed for a mass ratio BR/A8-35 1:5 (Pocanschi et al.
2006; Dahmane et al. 2013) (Fig. 6.6). When refolded at such mass ratios, BR migrates as an A8-35trapped monomer, as determined by size exclusion chromatography (SEC). At a mass ratio of
BR/A8-35 ¼ 1:2, the yield is lower, and SEC reveals some aggregation of the renatured protein
(Fig. 6.5). Control experiments carried out using the same protocol but with detergents instead of
A8-35 show lower levels of BR refolding. Yields are in fact negligible with octylglucoside (OG),
octylthioglucoside (OTG), or C 8 E 4 , but reach ~66% in dodecylmaltoside (DDM) (Dahmane et al.
2013). BR refolded in A8-35 displays a fully functional photocycle. The light-adapted protein is
excited at 640 nm with a 5-ns laser flash. Transient absorbance changes, monitored from 10 ns to
100 ms in a spectral range from 370 to 500 nm, are found to be very similar to those observed with
lipid-associated native BR trapped in A8-35 (Pocanschi et al. 2006; Dahmane et al. 2013).
More stringent experiments were performed in which BO was totally unfolded in formic acid and
transferred to SDS after lipids and retinal had been removed by hydrophobic SEC, as described in path
II of Fig. 6.1. They established that completely delipidated BO (dBO) refolds in pure A8-35 to 60–80%
(Pocanschi et al. 2006; Dahmane et al. 2013).
Fig. 6.5 Schematic representation of typical protocols for amphipol-assisted folding of (A) α-helical and
(B) β-barrel membrane proteins. Green, SDS; light blue, APol; orange, concentrated urea; yellow, dilute
urea; PDS, potassium dodecyl sulfate. The cartoons on the right of each column show interpretations of
what is going on in the samples, some aspects of which are solidly supported by experimental data,
whereas others are more speculative.
6.3 Amphipol-Assisted Folding of Membrane Proteins
341
rebinding of the retinal (Pocanschi et al. 2006). After dialyzing to remove residual DS, almost
quantitative refolding of BR (!90%) is observed for a mass ratio BR/A8-35 1:5 (Pocanschi et al.
2006; Dahmane et al. 2013) (Fig. 6.6). When refolded at such mass ratios, BR migrates as an A8-35trapped monomer, as determined by size exclusion chromatography (SEC). At a mass ratio of
BR/A8-35 ¼ 1:2, the yield is lower, and SEC reveals some aggregation of the renatured protein
(Fig. 6.5). Control experiments carried out using the same protocol but with detergents instead of
A8-35 show lower levels of BR refolding. Yields are in fact negligible with octylglucoside (OG),
octylthioglucoside (OTG), or C 8 E 4 , but reach ~66% in dodecylmaltoside (DDM) (Dahmane et al.
2013). BR refolded in A8-35 displays a fully functional photocycle. The light-adapted protein is
excited at 640 nm with a 5-ns laser flash. Transient absorbance changes, monitored from 10 ns to
100 ms in a spectral range from 370 to 500 nm, are found to be very similar to those observed with
lipid-associated native BR trapped in A8-35 (Pocanschi et al. 2006; Dahmane et al. 2013).
More stringent experiments were performed in which BO was totally unfolded in formic acid and
transferred to SDS after lipids and retinal had been removed by hydrophobic SEC, as described in path
II of Fig. 6.1. They established that completely delipidated BO (dBO) refolds in pure A8-35 to 60–80%
(Pocanschi et al. 2006; Dahmane et al. 2013).
Fig. 6.5 Schematic representation of typical protocols for amphipol-assisted folding of (A) α-helical and
(B) β-barrel membrane proteins. Green, SDS; light blue, APol; orange, concentrated urea; yellow, dilute
urea; PDS, potassium dodecyl sulfate. The cartoons on the right of each column show interpretations of
what is going on in the samples, some aspects of which are solidly supported by experimental data,
whereas others are more speculative.
6.3 Amphipol-Assisted Folding of Membrane Proteins
341