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High-Resolution AFM of Membrane Proteins Directly Incorporated at High Density in Planar Lipid Bilayer

Milhiet, Pierre-Emmanuel; Gubellini, Francesca; Berquand, Alexandre; Dosset, Patrice; Rigaud, Jean-Louis; Le Grimellec, Christian; Lévy, Daniel
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica
EN
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The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-β-maltoside or dodecyl-β-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20°C and at 4°C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore...