3.2
Bicelles
In the field of MP studies, bicelles have been used mostly for NMR investigations and for MP
crystallization, with a smattering of other applications. Because their use for NMR is intimately linked
to their mode of formation and phase diagram, we will deal with these two aspects along with NMR
applications.
3.2.1
The Formation and Phase Diagram of Bicelles and Their Use in Membrane
Protein NMR Spectroscopy
It has long been known that certain mixtures of lipids and detergent do not distribute more or less
randomly within mixed micelles but organize into patches of lipid bilayers surrounded and saturated
with the detergent. Such is the case of phosphatidylcholine/bile salt mixtures. In the case of bile
salts, this phenomenon is attributed to the laterally amphipathic nature of the sterol ring, one face of
which is rendered hydrophilic by the presence of hydroxyl moieties. This behavior explains the ability
of bile salts to dissolve high mass ratios of lipids (Small 1971; Carey and Small 1972). Before the
advent, in the 1980s, of sugar-based detergents such as octylglucoside (OG) and dodecylmaltoside
(DDM), bile salts were, along with Triton X-100 and Lubrol WX, among the “non-denaturing”
detergents most frequently used by biochemists. However, the peculiar structure of lipid/bile salt
mixed micelles was not exploited by membrane biophysicists until the 1990s, when it was realized,
most notably under the impulsion of James H. Prestegard and Charles R. Sanders, (i) that the size of
these assemblies could be modulated by playing on the lipid/detergent ratio; (ii) that they could be
obtained with other combinations of surfactants, e.g. dimyristoylphosphatidylcholine (DMPC,
diC 14:0 PC)/diC 6 PC mixtures (Sanders and Schwonek 1992); (iii) that the discs thus obtained, dubbed
“bicelles” – short for “bilayered micelles” (Sanders and Landis 1995) – could provide a bilayer-like
environment to MPs; and (iv) that, if large enough, they would, as shown previously for non-biological
systems (reviewed in Forrest and Reeves 1981), align in the magnetic field of NMR spectrometers
(Ram and Prestegard 1988; Sanders and Prestegard 1990; Sanders and Schwonek 1992; Sanders et al.
1994; Sanders and Landis 1995; Prosser et al. 1996, 1998; for reviews, see e.g. Sanders and Prosser
1998; Opella and Marassi 2004; Sanders and Sönnichsen 2006; Wang 2008; Kim et al. 2009; Qureshi
and Goto 2011; Warschawski et al. 2011; Dürr et al. 2013; Catoire et al. 2014; for a historical
perspective, see Sanders 2008).
Bicelles are rather easily obtained by mixing in aqueous solution one or more lipids and the
surfactant, generally CHAPSO or diC 6 PC, that will form the rim (Fig. 3.1). Solubilization may be slow
and may have to be accelerated by cycles of heating and cooling, freeze/thawing, and/or shearing (see
e.g. Sanders et al. 1994; Mäler and Gräslund 2009; Ujwal and Bowie 2011). This is somewhat timeconsuming, but, once formed, a preparation of bicelles can be stored frozen and used repeatedly. MPs
are usually incorporated by diluting concentrated bicelles with a detergent solution of the protein (see
e.g. Ujwal and Bowie 2011), but, depending on the protein, it is also possible to reconstitute it in lipid
vesicles formed by the long-chain lipid and dissolve them with the detergent, to add the long-chain
lipid to a solution of the protein in the detergent, to add buffer to a dried mixture of the protein and the
two surfactants, etc. (see e.g. Triba et al. 2006; De Angelis and Opella 2007; Poget and Girvin 2007;
Mäler and Gräslund 2009; Dürr et al. 2012; Morrison and Henzler-Wildman 2012, and references
therein). In Barrett et al. (2012), the polyhistidine-tagged target MP (the transmembrane C-terminal
domain of the amyloid precursor protein) was bound to an Ni:NTA column in detergent solution, the
solution exchanged for DMPC/diC 6 PC bicelles, and the bicelle-integrated protein eluted with a bicellecontaining solution.
3.2 Bicelles
99
Bicelles
In the field of MP studies, bicelles have been used mostly for NMR investigations and for MP
crystallization, with a smattering of other applications. Because their use for NMR is intimately linked
to their mode of formation and phase diagram, we will deal with these two aspects along with NMR
applications.
3.2.1
The Formation and Phase Diagram of Bicelles and Their Use in Membrane
Protein NMR Spectroscopy
It has long been known that certain mixtures of lipids and detergent do not distribute more or less
randomly within mixed micelles but organize into patches of lipid bilayers surrounded and saturated
with the detergent. Such is the case of phosphatidylcholine/bile salt mixtures. In the case of bile
salts, this phenomenon is attributed to the laterally amphipathic nature of the sterol ring, one face of
which is rendered hydrophilic by the presence of hydroxyl moieties. This behavior explains the ability
of bile salts to dissolve high mass ratios of lipids (Small 1971; Carey and Small 1972). Before the
advent, in the 1980s, of sugar-based detergents such as octylglucoside (OG) and dodecylmaltoside
(DDM), bile salts were, along with Triton X-100 and Lubrol WX, among the “non-denaturing”
detergents most frequently used by biochemists. However, the peculiar structure of lipid/bile salt
mixed micelles was not exploited by membrane biophysicists until the 1990s, when it was realized,
most notably under the impulsion of James H. Prestegard and Charles R. Sanders, (i) that the size of
these assemblies could be modulated by playing on the lipid/detergent ratio; (ii) that they could be
obtained with other combinations of surfactants, e.g. dimyristoylphosphatidylcholine (DMPC,
diC 14:0 PC)/diC 6 PC mixtures (Sanders and Schwonek 1992); (iii) that the discs thus obtained, dubbed
“bicelles” – short for “bilayered micelles” (Sanders and Landis 1995) – could provide a bilayer-like
environment to MPs; and (iv) that, if large enough, they would, as shown previously for non-biological
systems (reviewed in Forrest and Reeves 1981), align in the magnetic field of NMR spectrometers
(Ram and Prestegard 1988; Sanders and Prestegard 1990; Sanders and Schwonek 1992; Sanders et al.
1994; Sanders and Landis 1995; Prosser et al. 1996, 1998; for reviews, see e.g. Sanders and Prosser
1998; Opella and Marassi 2004; Sanders and Sönnichsen 2006; Wang 2008; Kim et al. 2009; Qureshi
and Goto 2011; Warschawski et al. 2011; Dürr et al. 2013; Catoire et al. 2014; for a historical
perspective, see Sanders 2008).
Bicelles are rather easily obtained by mixing in aqueous solution one or more lipids and the
surfactant, generally CHAPSO or diC 6 PC, that will form the rim (Fig. 3.1). Solubilization may be slow
and may have to be accelerated by cycles of heating and cooling, freeze/thawing, and/or shearing (see
e.g. Sanders et al. 1994; Mäler and Gräslund 2009; Ujwal and Bowie 2011). This is somewhat timeconsuming, but, once formed, a preparation of bicelles can be stored frozen and used repeatedly. MPs
are usually incorporated by diluting concentrated bicelles with a detergent solution of the protein (see
e.g. Ujwal and Bowie 2011), but, depending on the protein, it is also possible to reconstitute it in lipid
vesicles formed by the long-chain lipid and dissolve them with the detergent, to add the long-chain
lipid to a solution of the protein in the detergent, to add buffer to a dried mixture of the protein and the
two surfactants, etc. (see e.g. Triba et al. 2006; De Angelis and Opella 2007; Poget and Girvin 2007;
Mäler and Gräslund 2009; Dürr et al. 2012; Morrison and Henzler-Wildman 2012, and references
therein). In Barrett et al. (2012), the polyhistidine-tagged target MP (the transmembrane C-terminal
domain of the amyloid precursor protein) was bound to an Ni:NTA column in detergent solution, the
solution exchanged for DMPC/diC 6 PC bicelles, and the bicelle-integrated protein eluted with a bicellecontaining solution.
3.2 Bicelles
99