and the so-called Rieske iron-sulfur protein, are directly involved in electron transfer. Cytochrome b 6
is a polytopic MP, with four TM α-helices, which carries two b-type and one c-type hemes;
cytochrome f and the Rieske protein are bitopic MPs, each of them featuring a single TM helix and
a functional domain, located in the lumen of the chloroplast thylakoids, which carries, respectively, a
c-type heme and an Fe 2 S 2 iron-sulfur center. The functional cycle of the b 6 f complex is somewhat
complicated and need not be described in detail here. For the sake of the present discussion, it is
sufficient to say that one of its branches involves the transfer of electrons in the following sequence:
PQH 2 ! b 6 ! Rieske ! f ! plastocyanin. Whereas all electron transfers involve tunneling, that from
b 6 to f via the Fe 2 S 2 center of the Rieske protein is peculiar, because it requires a physical movement
of the Rieske protein’s extramembrane domain from a site on cytochrome b 6 to one on cytochrome f.
Early attempts at purifying the complex from C. reinhardtii revealed a great instability in
detergent solutions (Pierre et al. 1995; Breyton et al. 1997). Indeed, exposure to detergent leads to
the detachment of the Rieske protein from the complex – and, therefore, to complete inactivation –
followed by the disaggregation of the superdimer into supermonomers that have lost both the Rieske
protein, one of the small subunits, PetL, and a cofactor, chlorophyll a. This is illustrated in Fig. 2.10A,
gradient to the right, and Fig. 2.10B, lower series of SDS-PAGE lanes. The various steps experimentally identified in the progressive disaggregation of the C. reinhardtii complex are schematized in
Fig. 2.10C.
Two ways around this problem were identified:
• A first approach is to expose the complex to a minimal concentration of detergent. This is
illustrated in Fig. 2.10A, gradient to the left, and Fig. 2.10B, upper row of lanes: decreasing
the DDM concentration from 3 or 5 to 0.2 mM, which is very close to the CMC of ~0.17 mM,
slowed down the disaggregation process sufficiently for the complex to stay mostly intact
during the 4 h (Fig. 2.10A) or 3 h (Fig. 2.10B) of ultracentrifugation.
• A second approach, illustrated in Fig. 2.11, is to supplement the detergent solution with lipids.
The detergent used in these experiments was Hecameg (HG), a short-chain analog of
octylglucoside (Fig. 2.1) with a high CMC of ~19.5 mM under the conditions used. Under
usual experimental conditions, the concentration of HG was kept at 20 mM, very close to the
CMC. In the experiments of Fig. 2.11, it was deliberately raised to very high concentrations,
either 50 or 100 mM, resulting in the rapid loss of electron transfer activity, which, after
30 min at 4
C, fell to ~25% or ~16% of its initial value, respectively. The inactivation
process could be considerably slowed down by supplementing the preparations with egg
phosphatidylcholine (egg PC) at a 1:10 molar ratio of lipid to micellar detergent and even
more so when raising this ratio to 1:5 (Fig. 2.11).
2.4 Why Are Membrane Proteins Unstable in Detergent Solutions?
73
is a polytopic MP, with four TM α-helices, which carries two b-type and one c-type hemes;
cytochrome f and the Rieske protein are bitopic MPs, each of them featuring a single TM helix and
a functional domain, located in the lumen of the chloroplast thylakoids, which carries, respectively, a
c-type heme and an Fe 2 S 2 iron-sulfur center. The functional cycle of the b 6 f complex is somewhat
complicated and need not be described in detail here. For the sake of the present discussion, it is
sufficient to say that one of its branches involves the transfer of electrons in the following sequence:
PQH 2 ! b 6 ! Rieske ! f ! plastocyanin. Whereas all electron transfers involve tunneling, that from
b 6 to f via the Fe 2 S 2 center of the Rieske protein is peculiar, because it requires a physical movement
of the Rieske protein’s extramembrane domain from a site on cytochrome b 6 to one on cytochrome f.
Early attempts at purifying the complex from C. reinhardtii revealed a great instability in
detergent solutions (Pierre et al. 1995; Breyton et al. 1997). Indeed, exposure to detergent leads to
the detachment of the Rieske protein from the complex – and, therefore, to complete inactivation –
followed by the disaggregation of the superdimer into supermonomers that have lost both the Rieske
protein, one of the small subunits, PetL, and a cofactor, chlorophyll a. This is illustrated in Fig. 2.10A,
gradient to the right, and Fig. 2.10B, lower series of SDS-PAGE lanes. The various steps experimentally identified in the progressive disaggregation of the C. reinhardtii complex are schematized in
Fig. 2.10C.
Two ways around this problem were identified:
• A first approach is to expose the complex to a minimal concentration of detergent. This is
illustrated in Fig. 2.10A, gradient to the left, and Fig. 2.10B, upper row of lanes: decreasing
the DDM concentration from 3 or 5 to 0.2 mM, which is very close to the CMC of ~0.17 mM,
slowed down the disaggregation process sufficiently for the complex to stay mostly intact
during the 4 h (Fig. 2.10A) or 3 h (Fig. 2.10B) of ultracentrifugation.
• A second approach, illustrated in Fig. 2.11, is to supplement the detergent solution with lipids.
The detergent used in these experiments was Hecameg (HG), a short-chain analog of
octylglucoside (Fig. 2.1) with a high CMC of ~19.5 mM under the conditions used. Under
usual experimental conditions, the concentration of HG was kept at 20 mM, very close to the
CMC. In the experiments of Fig. 2.11, it was deliberately raised to very high concentrations,
either 50 or 100 mM, resulting in the rapid loss of electron transfer activity, which, after
30 min at 4
C, fell to ~25% or ~16% of its initial value, respectively. The inactivation
process could be considerably slowed down by supplementing the preparations with egg
phosphatidylcholine (egg PC) at a 1:10 molar ratio of lipid to micellar detergent and even
more so when raising this ratio to 1:5 (Fig. 2.11).
2.4 Why Are Membrane Proteins Unstable in Detergent Solutions?
73