High resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer

¹ CBS/INSERM/CNRS
² Institut Curie/CNRS

^* To whom correspondence should be addressed. E-mail: daniel.levy{at}curie.fr.

Submitted on May 9, 2006
Revised on June 29, 2006
Accepted on 11 July 2006

	Abstract

The heterologous expression and purification of membrane proteins represents a major limitation for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from one picomole of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl--maltoside or dodecyl--thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20° and at 4°C with three bacterial photosynthetic multi-subunits membrane proteins. Heights measurement by Atomic Force Microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrates that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and upon incubation diffuse and segregate into quasi-crystalline 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 2D crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nano-biomaterials involving membrane proteins.

Key Words: Atomic Force Microscopy, direct incorporation, membrane protein, photosynthetic complexes, sugar-based detergents, supported lipid bilayer