High-resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer.
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-beta-maltoside or dodecyl-beta-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20 degrees C and at 4 degrees 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, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.
Milhiet
Pierre-Emmanuel
PE
Centre de Biochimie Structurale, Groupe Nanostructures et Complexes Membranaires, UMR 554 INSERM, UMR 5048 CNRS, Montpellier, France.
Gubellini
Francesca
F
Berquand
Alexandre
A
Dosset
Patrice
P
Rigaud
Jean-Louis
JL
Le Grimellec
Christian
C
Lévy
Daniel
D
D004548:Elasticity; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D013314:Stress, Mechanical
D008051:Lipid Bilayers; D008565:Membrane Proteins