Biology Reference
In-Depth Information
3.
Bio-beads SM-2 (Bio-Rad) if using Bio-bead method
4.
Reagents for detergent concentration assay (optional)
5.
Reagents for phospholipid concentration determination (optional)
18.1.4.1.1
Liposome preparation
Saturating the liposomes with detergent prior to reconstitution improves membrane
protein incorporation into the vesicles from solution (
Knol, Sjollema, & Poolman,
1998; Paternostre, Roux, & Rigaud, 1988
). For each detergent and lipid mixture, there
is an effective detergent-lipid molar ratio (
R
eff
) at which the lipids are saturated but not
completely solubilized by the detergent. A table listing these parameters for several of
the most common detergents is available in
Rigaud and Levy (2003)
.ForDDM,
R
sat
is
1 (mol/mol), and
R
sol
1.6 (mol/mol). For a detailed description of the protocol, see
Lambert, Levy, Ranck, Leblanc, and Rigaud (1998)
. The appropriate amount of deter-
gent is added to the liposome solution and, in the case of DDM, allowed to incubate at
room temperature with stirring for 3 h. If using alternative detergents, such as sodium
cholate, incorporation into the liposomes may be more rapid.
18.1.4.1.2
Preparing Bio-beads
Bio-beads are thoroughly washed several times with methanol and then water and
can be stored at 4
C in water. Prior to use, they are washed in detergent-free buffer.
18.1.4.1.3
Incubation of lipid with receptor
After addition of the protein solution, the reaction mixture is incubated with gentle
mixing for
1 h before the addition of Bio-beads. This incubation period may be
shortened if the detergent used to solubilize the lipids is deleterious to the activity
of the protein.
18.1.4.1.4
Addition of Bio-beads
A wet Bio-bead-to-detergent ratio of 10 (w/w) is used, and the beads are added di-
rectly to the protein-lipid-detergent solution. The solution is incubated above the
phase transition temperature of the lipids for 1-2 h and then aspirated into fresh
Bio-beads and incubated with rotation overnight. Reconstitution can be monitored
using turbidity measurements, and a detergent assay can be performed to confirm
complete detergent removal. Proteoliposomes are isolated using sucrose density gra-
dient centrifugation (0-35% in liposome buffer) in 5% steps. The sample is centri-
fuged (100,000 g; 16 h) and fractionated. Proteoliposomes may be dialyzed or
pelleted to remove the sucrose.
18.1.4.1.5
Determining the lipid-to-protein ratio
The lipid-to-protein ratio used for FRET is very high (12,000:1) to avoid any
artifacts in the signal due to crowding effects. Both protein and lipid are lost
during the reconstitution process. Knowing the final lipid-protein ratio is useful
for estimating receptor density in the proteoliposomes and thus likely resonance
transfer. We use a number of ways to estimate the lipid-protein ratio in reconstituted
samples. The first is a simple comparison of the
A
280
(corrected for liposome
