detergent concentration. It will, by the same token, be strongly dependent on the concentration of membranes in the sample, which must be kept constant from one experiment to the
next if the solubilization conditions are to be reproducible.
• High-CMC detergents, e.g. OG or CHAPS, which have less hydrophobic tails, will tend, on
the contrary, to partition in the aqueous phase more than in the membrane one. Their effect
will therefore depend on the absolute concentration of detergent more than on that of
membrane fragments, which bind little detergent.
Whatever the detergent, not much may be observed at that stage, but some extrinsic MPs may
detach from the membrane, and the detergent that partitions into it may affect the function of some
intrinsic proteins (for a careful analysis of why different detergents seem to have very different
functional effects at that stage, whereas they actually act at similar molar ratios in the membrane,
see the examination of the dependence of the inhibition of the Na
+ ,K
+
-ATPase on detergent concentration in Brotherus et al. 1979).
At step 2, the concentration of detergent is high enough for mixed micelles to form. Because
these micelles are not pure detergent but incorporate lipids extracted from the membrane, they are in
equilibrium with a concentration of free detergent molecules slightly lower than the CMC. Most
often, pores form in the membrane, which has two important consequences. First, the membrane
becomes leaky, which may affect functional measurements (see e.g. in Chap. 5, § 5.6.1, the case of
the sarcoplasmic Ca
2+ -ATPase). Second, the detergent can use the rim of the pores to flip from the
external leaflet of the membrane fragments – which usually form closed vesicles – to the inner leaflet.
This is an important step, as membrane solubilization by detergents that do not undergo this process
can be extremely slow (see e.g. Kragh-Hansen et al. 1998).
The ability of detergents to destabilize the target membrane and extract different types of lipids
can vary widely from one detergent to another (see e.g. Banerjee et al. 1995). In some cases, the
process may stop at stage 1 or 2. For instance, purple membrane fragments exposed to deoxycholate
yield a contracted form of the native 2D BR crystals (see § 1.6.1), from which the lipid layer that
ensures the contacts between BR trimers in the native membrane has been extracted (Henderson et al.
1982; Glaeser et al. 1985).
Mixed micelles are generally disordered. However, in the case of bile salts, the peculiar
molecular structure of the detergent, whose hydrophobic moiety is itself laterally amphipathic (see §
2.2.1), causes the formation of disc-like micelles in which the detergent covers the rim of a small patch
of bilayer, into which it partitions (see Carey and Small 1972; Marrink and Mark 2002) – a structure
similar to that proposed for the bicelles formed by mixtures of lipids and DHPC, CHAPS, or CHAPSO
(see e.g. Sanders and Landis 1995; Triba et al. 2005; Poget and Girvin 2007; Kim et al. 2009, and
Chap. 3, § 3.2).
Step 3, sometimes very useful from a practical point of view, may see a selective extraction of
some MPs. It may provide an opportunity to enrich one’s sample with the target protein, whether in the
supernatant or in the pellet. MPs solubilized at that stage find themselves associated with a lipid-rich
mixture of detergent and lipids, which, as discussed below, is a stabilizing factor.
At step 4, a major fraction of the membrane is solubilized, at step 5, all or nearly all of it. Some
detergents stop at step 4, whereas the “strongest” (most disruptive) ones reach step 5, the effect of
course depending on the detergent’s nature, its concentration and its mass ratio to the membrane
fragments, the nature of the latter, but also on such factors as the temperature and buffer conditions:
ionic strength, presence or not of calcium chelators, pH, redox potential, etc. If one’s target protein is
extracted quantitatively enough at step 4, there is no need to push the solubilization further, at the risk
of inactivating it. Indeed, as more and more detergent is supplied, the mixture of detergent and lipids
associated with the solubilized MPs becomes increasingly poorer in lipids, which is generally to be
avoided (§ 2.4).
2.3 Solubilizing Membrane Proteins with Detergents
67
next if the solubilization conditions are to be reproducible.
• High-CMC detergents, e.g. OG or CHAPS, which have less hydrophobic tails, will tend, on
the contrary, to partition in the aqueous phase more than in the membrane one. Their effect
will therefore depend on the absolute concentration of detergent more than on that of
membrane fragments, which bind little detergent.
Whatever the detergent, not much may be observed at that stage, but some extrinsic MPs may
detach from the membrane, and the detergent that partitions into it may affect the function of some
intrinsic proteins (for a careful analysis of why different detergents seem to have very different
functional effects at that stage, whereas they actually act at similar molar ratios in the membrane,
see the examination of the dependence of the inhibition of the Na
+ ,K
+
-ATPase on detergent concentration in Brotherus et al. 1979).
At step 2, the concentration of detergent is high enough for mixed micelles to form. Because
these micelles are not pure detergent but incorporate lipids extracted from the membrane, they are in
equilibrium with a concentration of free detergent molecules slightly lower than the CMC. Most
often, pores form in the membrane, which has two important consequences. First, the membrane
becomes leaky, which may affect functional measurements (see e.g. in Chap. 5, § 5.6.1, the case of
the sarcoplasmic Ca
2+ -ATPase). Second, the detergent can use the rim of the pores to flip from the
external leaflet of the membrane fragments – which usually form closed vesicles – to the inner leaflet.
This is an important step, as membrane solubilization by detergents that do not undergo this process
can be extremely slow (see e.g. Kragh-Hansen et al. 1998).
The ability of detergents to destabilize the target membrane and extract different types of lipids
can vary widely from one detergent to another (see e.g. Banerjee et al. 1995). In some cases, the
process may stop at stage 1 or 2. For instance, purple membrane fragments exposed to deoxycholate
yield a contracted form of the native 2D BR crystals (see § 1.6.1), from which the lipid layer that
ensures the contacts between BR trimers in the native membrane has been extracted (Henderson et al.
1982; Glaeser et al. 1985).
Mixed micelles are generally disordered. However, in the case of bile salts, the peculiar
molecular structure of the detergent, whose hydrophobic moiety is itself laterally amphipathic (see §
2.2.1), causes the formation of disc-like micelles in which the detergent covers the rim of a small patch
of bilayer, into which it partitions (see Carey and Small 1972; Marrink and Mark 2002) – a structure
similar to that proposed for the bicelles formed by mixtures of lipids and DHPC, CHAPS, or CHAPSO
(see e.g. Sanders and Landis 1995; Triba et al. 2005; Poget and Girvin 2007; Kim et al. 2009, and
Chap. 3, § 3.2).
Step 3, sometimes very useful from a practical point of view, may see a selective extraction of
some MPs. It may provide an opportunity to enrich one’s sample with the target protein, whether in the
supernatant or in the pellet. MPs solubilized at that stage find themselves associated with a lipid-rich
mixture of detergent and lipids, which, as discussed below, is a stabilizing factor.
At step 4, a major fraction of the membrane is solubilized, at step 5, all or nearly all of it. Some
detergents stop at step 4, whereas the “strongest” (most disruptive) ones reach step 5, the effect of
course depending on the detergent’s nature, its concentration and its mass ratio to the membrane
fragments, the nature of the latter, but also on such factors as the temperature and buffer conditions:
ionic strength, presence or not of calcium chelators, pH, redox potential, etc. If one’s target protein is
extracted quantitatively enough at step 4, there is no need to push the solubilization further, at the risk
of inactivating it. Indeed, as more and more detergent is supplied, the mixture of detergent and lipids
associated with the solubilized MPs becomes increasingly poorer in lipids, which is generally to be
avoided (§ 2.4).
2.3 Solubilizing Membrane Proteins with Detergents
67