Binding, reconstitution and 2D crystallization of membrane or soluble proteins onto functionalized lipid layer observed in situ by reflected light microscopy

Abstract

Monolayer of functionalized lipid spread at the air/water interface is used for the structural analysis of soluble and membrane proteins by electron crystallography and single particle analysis. This powerful approach lacks of a method for the screening of the binding of proteins to the surface of the lipid layer. Here, we described an optical method based on the use of reflected light microscopy to image, without the use of any labeling, the lipid layer at the surface of buffers in the Teflon wells used for 2D crystallization. Images revealed that the lipid layer was made of a monolayer coexisting with liposomes or aggregates of lipids floating at the surface. Protein binding led to an increase of the contrast and the decrease of the fluidity of the lipid surface, as demonstrated with the binding of soluble Shiga toxin B subunit, of purple membrane and of solubilized His-BmrA, a bacterial ABC transporter. Moreover the reconstitution of membrane proteins bound to the lipidic surface upon detergent removal can be followed through the appearance of large recognizable domains at the surface. Proteins binding and reconstitution were further confirmed by electron microcopy. Overall, this method provides a quick evaluation of the monolayer trials, a significant reduction in screening by transmission electron microscopy and new insights in the proteins binding and 2D crystallogenesis at the lipid surface.