Synthesis of large lipid membranes with integrated membrane proteins from gas phase.

Abstract

In this experimental study I am presenting a new method to generate large, transportable membranes with integrated membrane proteins. These membranes can be used as an in-vitro system to study membrane protein function, investigate its interaction with lipids or study protein complex assembly in a system whose composition is controlled on a molecular basis. The method lends itself to produce anisotropic membranes in the lipid composition and protein orientation. Bio-membranes are generated by self-assembly. There are two established methods - self-assembly on a solid support and self-assembly within a lipo-protein ring: nanodiscs. Both methods solve the principal problem of bio-membrane assembly, their lack of stability in a hydrophilic environment, in a different way. However, solid support based membranes can not be moved from their support and nanodiscs can not be made larger than 17 nm in diameter. Here, large bio-membranes self assemble in a thin sheet of glycerol generated on a liquid surface. I use a nano-electrospray ion source to generate molecular beams of large molecules. The target area is the liquid surface of a container filled with buffer solution. The procedure consists of several steps: 1. formation of a lipid bi-layer; 2. formation of a thin glycerol layer; 3. formation of lipid mono-layer as template for the membrane assembly; 4. adding detergent solubilised membrane proteins and some more lipid; 5. sealing the layers off by some glycerol; 6. detergent extraction from the surface by SM-2 Biobeads over several days. This procedure leads to the formation of a large bio-membrane covering the entire liquid surface which can be transported for inspection or being used for the biochemical activity of the proteins.