More of micelles and porins….and symmetry.

A while ago I blogged about the incorporation of porins, channel-forming proteins, into synthetic polymer membranes and in particular into micelles and the binding of phages to the incorporated porins.

I did not mention that this is usually done through simple diffusion processes: one can either preform the membrane over a hole on a substrate and add a drop of a solution of porins to this or one mixes the block-copolymer solution and the porin prior to forming micelles. While this is a well-established way of doing things and has been demonstrated to be effective in a number of experiments, it is not very good when trying to imitate biological systems.

Why? Well, proteins have both an amino-terminus and a carboxy terminus. In nature, porins are “vectorial” molecules, they have very distinct intracellular and extracellular parts. In “real” biological systems, the amino-terminus of a porin is located in the cytoplasm, i.e. inside the cell. Phages, however, typically bind to the carboxy-terminus, i.e. the part of the protein outside the cell. Incorporation of porins in the synthetic membranes by diffusion is essentially a statistical process and hence one would expect only half of the porins to be incorporated into a membrane in the physiologically correct orientation. So what to do? Well, the structure of natural membranes may hold a clue.

Meier and colleagues noted, that a lot of physicochemical and immunological properties of cell membranes are dependent on the lipid composition of both membrane leaflets and that this composition is usually asymmetric.(DOI).[1] They furthermore noted, that all of the polymers used in membrane/porin experiments are symmetric with respect to their mid-planes if one disregards curvature effects for a moment. For this reason, they prepard both a symmetric poly(2-methyl-2-oxazoline)-block-poly(dimethyl siloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-PDMS-PMOXA) copolymer, as well as asymmetric poly(ethylene oxide)-block-poly(dimethyl siloxane)-block-poly(2-methyl-2-oxazoline) (PEO-PDMS-PMOXA) polymers. The latter was present in two forms containing both a large (PEO25-PDMS40-PMOXA110) and a small (PEO67-PDMS40-PMOXA45) poly(2-methyl-2-oxazoline) block.

All of the polymers form vesicles when dissolved in water, with the hydrophobic block being covered by the hydrophilic blocks on both the outside and the inside of the vesicle wall with the more voluminous hydrobilic block on the outside of the micelle. For this reason, the PEO25-PDMS40-PMOXA110 polymer is expected to give rise to an ABC motive, and PEO67-PDMS40-PMOXA45 to a CBA orientation.

aquaporin0.gif
Figure 1: The asymmetric unit of aquaporin0.

Aquaporin0, labelled with histidine tags on the amino-terminus was subsequently incorporated into the vesicles formed by all three polymers. An antibody assay, using an antibody specific to histidine, showed that in the case of the symmetric PMOXA-PDMS-PMOXA polymer, the aquaporin did indeed incorporate statistically into the vesicle membrane, with an equal amount of histidine tags located on the outside and inside walls of the vesicle wall. The ABC motif, by contrast, led to a physiological incorporation of the porins, with approximately 80 % of the histidine tags inside the vesicle (corresponding to the cytoplasm in a cell). For the CBA motif, the situation was reversed: 70 % of the histidine residues are now located on the outside of vesicle walls.

In this way the authors have clearly demonstrated, that breaking the symmetry of a synthetic membrane system results in the directed insertion of membrane proteins.

[1] Stoenescu, R.; Graff, A.; Meier, W. Macromol. Biosci. 2004, 4, 930-935

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