Two-Dimensional Crystallization of Soluble Proteins on Planar Lipid Films

Abstract

Electron crystallography of protein two-dimensional (2D) crystals constitutes a fast-expanding method for determining the structure of macromolecules at near-atomic resolution (1, 2). The main limitation in the application and generalization of this approach remains in obtaining highly ordered 2D crystals, as is the case of 3D crystals in X-ray crystallography. Several methods of 2D crystallization are available which can be classified into two families, depending on the type of proteins under investigation, either membrane proteins (3, 4) or soluble proteins (5, 6). In both cases, 2D crystallization is a self-organization process which spontaneously occurs between macromolecules which are restricted to diffusing by translation and rotation in a 2D space, with a fixed orientation along the normal to this plane. The scope of this chapter is restricted to the 2D crystallization of soluble proteins on planar lipid films, by the so-called ‘lipid monlayer crystallization method’ (5). Our aim is to present a step-by-step description of the experimental procedures involved in the application of this method. The method of protein 2D crystallization on planar lipid films was introduced about 15 years ago (5) and has since been successfully applied to about 30 proteins. Its principle is based on the specific interaction between soluble proteins and lipid ligands inserted in a lipid monolayer, at an air-water interface. In practice, a lipid monolayer is formed by spreading lipids dissolved in an organic solvent on a water surface. Proteins present in the aqueous subphase bind to their ligand of lipidic nature and spontaneously form 2D domains and, in favourable cases, 2D crystals. The process of 2D crystal formation relies on three successive steps: (a) Molecular recognition between a protein and its ligand. (b) Diffusion and concentration of the protein-lipid complexes in the plane of the lipid film. (c) Self-organization of the proteins into 2D crystals. As indicated in Table 1, three different types of systems can be distinguished, depending on the nature of the lipid ligand: • natural lipids • synthetic lipids made of a protein ligand coupled to a lipid molecule • charged lipids.