2.4.3
The Dissociating Character of Detergents as a Major Cause of Membrane
Protein Inactivation
Such observations are by no means peculiar to the b 6 f complex. They are, actually, more the rule than
the exception. Thus, the functional reconstitution of the purified nicotinic acetylcholine receptor
(nAChR; introduced in Chap. 1, § 1.6.2) into lipid vesicles remained unsuccessful for many years
for want of supplementing with lipids the detergent solutions used for its purification (Huganir et al.
1979; Heidmann et al. 1980; Popot et al. 1981). Delipidation has been shown to be deleterious to the
sarcoplasmic reticulum calcium pump SERCA1a (described in § 1.6.3) (Lund et al. 1989), to the
serotonin 5-HT 1A G protein-coupled receptor (Banerjee et al. 1995), or to the red cell glucose
transporter Glut1 (Haneskog et al. 1996). Achieving a decent stability is of course critical for
crystallization attempts. The b 6 f complex from C. reinhardtii could be crystallized only by working
very close to the CMC of DDM and minimizing the number and duration of the purification steps, so
that crystallization drops were set up on the day of purification, leaving as little time as possible for
fragmentation to set in (Stroebel et al. 2003). Similar precautions against excessive delipidation were
essential to crystallizing rhodopsin (Palczewski et al. 2000). Crystallizing the b 6 f complex from the
thermophilic cyanobacterium Mastigocladus laminosus required the addition of lipids (Kurisu et al.
2003), as was also the case for SERCA1a (Toyoshima et al. 2000) or for the lactose permease from
E. coli (Guan et al. 2006). Inactivation of the shark rectal gland Na
+
,K
+
-ATPase by C 12 E 8 is critically
dependent on the concentration of detergent (Esmann 1986). Such examples could be multiplied (see
Hunte and Richers 2008 and references therein). As a general rule, the use of detergents with a low
CMC makes it easier to limit the volume of free micelles, which act as a hydrophobic sink, pushing
association/dissociation equilibria in the direction of dissociation.
These observations call for some comments:
• Supplementing a detergent solution with substantial amounts of lipids, as in the experiments
of Fig. 2.11, can be expected to change somewhat the physical properties of the belt of
surfactant the MP is associated with. The effect will be equivalent to increasing, on average,
the critical packing parameter v/a 0 ‧l c of the detergent, increasing the radius of curvature of the
surfactant/water interface, and pushing the equilibrium shape of the micelles away from the
spherical toward an elliptical shape, as would be the case if using a detergent with a smaller
polar head (see Chap. 1, Table 1.1, rows 1 and 2).
• When the concentration of detergent is changed in the presence of no or very low amounts of
lipids, as in the lipid-free experiments of Fig. 2.11, however, little effect can be expected, as a
rule, on the properties of micelles. Indeed, most of the detergents used in biology have been
selected so that their phase diagram comprises a broad L 1 phase (the free micelle phase), in
which micellar properties depend little on the concentration. Such is the case of DDM, the
detergent used in the experiments of Fig. 2.10, which remains in the L 1 phase in water up to
~45% w/w (Fig. 2.12) (Warr et al. 1986). As regards the aggregation number N of DDM
micelles, and therefore their size, they are nearly independent of concentration (from 2.5 to
10 gÁL
À1 ) and temperature (from 16 to 60
C), and the micelles interact very little one with
another (Aoudia and Zana 1998; see also Salvay et al. 2007). Under such conditions, it is hard
to argue that it is a physical effect that destabilizes the protein when the concentration of
DDM is raised.
• The same reasoning holds when the protein is exposed to a vast excess of mixed micelles with
a given composition. In the two series of experiments of Fig. 2.11, whatever residual native
lipids were present were diluted, if not bound to the protein, in a vast excess of PC/HG mixed
76
2 Extracting Membrane Proteins from Their Native Environment
The Dissociating Character of Detergents as a Major Cause of Membrane
Protein Inactivation
Such observations are by no means peculiar to the b 6 f complex. They are, actually, more the rule than
the exception. Thus, the functional reconstitution of the purified nicotinic acetylcholine receptor
(nAChR; introduced in Chap. 1, § 1.6.2) into lipid vesicles remained unsuccessful for many years
for want of supplementing with lipids the detergent solutions used for its purification (Huganir et al.
1979; Heidmann et al. 1980; Popot et al. 1981). Delipidation has been shown to be deleterious to the
sarcoplasmic reticulum calcium pump SERCA1a (described in § 1.6.3) (Lund et al. 1989), to the
serotonin 5-HT 1A G protein-coupled receptor (Banerjee et al. 1995), or to the red cell glucose
transporter Glut1 (Haneskog et al. 1996). Achieving a decent stability is of course critical for
crystallization attempts. The b 6 f complex from C. reinhardtii could be crystallized only by working
very close to the CMC of DDM and minimizing the number and duration of the purification steps, so
that crystallization drops were set up on the day of purification, leaving as little time as possible for
fragmentation to set in (Stroebel et al. 2003). Similar precautions against excessive delipidation were
essential to crystallizing rhodopsin (Palczewski et al. 2000). Crystallizing the b 6 f complex from the
thermophilic cyanobacterium Mastigocladus laminosus required the addition of lipids (Kurisu et al.
2003), as was also the case for SERCA1a (Toyoshima et al. 2000) or for the lactose permease from
E. coli (Guan et al. 2006). Inactivation of the shark rectal gland Na
+
,K
+
-ATPase by C 12 E 8 is critically
dependent on the concentration of detergent (Esmann 1986). Such examples could be multiplied (see
Hunte and Richers 2008 and references therein). As a general rule, the use of detergents with a low
CMC makes it easier to limit the volume of free micelles, which act as a hydrophobic sink, pushing
association/dissociation equilibria in the direction of dissociation.
These observations call for some comments:
• Supplementing a detergent solution with substantial amounts of lipids, as in the experiments
of Fig. 2.11, can be expected to change somewhat the physical properties of the belt of
surfactant the MP is associated with. The effect will be equivalent to increasing, on average,
the critical packing parameter v/a 0 ‧l c of the detergent, increasing the radius of curvature of the
surfactant/water interface, and pushing the equilibrium shape of the micelles away from the
spherical toward an elliptical shape, as would be the case if using a detergent with a smaller
polar head (see Chap. 1, Table 1.1, rows 1 and 2).
• When the concentration of detergent is changed in the presence of no or very low amounts of
lipids, as in the lipid-free experiments of Fig. 2.11, however, little effect can be expected, as a
rule, on the properties of micelles. Indeed, most of the detergents used in biology have been
selected so that their phase diagram comprises a broad L 1 phase (the free micelle phase), in
which micellar properties depend little on the concentration. Such is the case of DDM, the
detergent used in the experiments of Fig. 2.10, which remains in the L 1 phase in water up to
~45% w/w (Fig. 2.12) (Warr et al. 1986). As regards the aggregation number N of DDM
micelles, and therefore their size, they are nearly independent of concentration (from 2.5 to
10 gÁL
À1 ) and temperature (from 16 to 60
C), and the micelles interact very little one with
another (Aoudia and Zana 1998; see also Salvay et al. 2007). Under such conditions, it is hard
to argue that it is a physical effect that destabilizes the protein when the concentration of
DDM is raised.
• The same reasoning holds when the protein is exposed to a vast excess of mixed micelles with
a given composition. In the two series of experiments of Fig. 2.11, whatever residual native
lipids were present were diluted, if not bound to the protein, in a vast excess of PC/HG mixed
76
2 Extracting Membrane Proteins from Their Native Environment