Biophysical studies of membrane proteins/peptides. Interaction with ...

deactivating agents. Zwitterionic detergents combine the properties of ionic and nonionic
detergents (Seddon et al., 2004).
Several methods exist for insertion of integral membrane proteins in liposomes. The
most popular makes use of mediation by detergents. The choice of the best detergent
varies greatly between proteins, confirming the complexity of the interactions involved
in the reconstitution process. The simplest of the detergent-mediated reconstitution
method is a dilution approach. Liposomes are added to detergent micelles with protein
and the detergent is removed. Removal of detergent evolves until the concentration of
the detergent is below the critical micelle concentration (cmc) and micelles become
unstable inducing transfer of the protein to the liposomes. Removal of detergent can be
achieved by: i) dialysis, making use of a membrane with a pore size smaller than the
size of the proteoliposomes; ii) column chromatography; iii) incubation with detergent
adsorbing beads. However, pure liposomes are frequently impermeable to proteins and
often a destabilization of the bilayer is required for correct insertion of the protein
(Rigaud et al., 1995). Destabilization of the bilayer can be achieved by using mixtures
of lipids and detergents instead of pure liposomes. The resulting structures are more
receptive to protein insertion, and after this step the detergent can be removed by one of
the methods described above.
The required degree of detergent-destabilization of the liposomes depends on the
nature of the detergents. Several proteins were reconstituted through mediation of nonionic
detergents by destabilization of liposomes with the concentration of the detergent
set at saturation levels for the liposomes. This method frequently leads to asymmetrical
orientation of the proteins in the liposomes (a large fraction of the proteins face the
same direction after reconstitution) (Knol et al, 1998). On the other hand, reconstitution
of some proteins with sodium cholate required a concentration of detergent above the
saturation point for liposomes, i.e proteins in detergent micelles had to be mixed to
detergent micelles containing lipids (Seddon et al., 2004). This latter method usually
results in symmetrical orientations for the protein inside the proteoliposomes (Yeagle,
1993). In the case of bile acid salts, dialysis is a good detergent removal procedure.
These detergents present high cmc (high solubility as monomers) so dialysis can be
efficient at reasonable time scales.
Other reconstitution strategies include protein/peptide solubilization in organic
solvents. This procedure must be applied with caution as it often results in aggregation
and denaturation. Some protocols for proteins have been developed that make use of the
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