Structure and Functional Characterization of Membrane Integral Proteins in the Lipid Cubic Phase.

The lipid cubic phase (LCP) has been used extensively as a medium for crystallizing membrane proteins. It is an attractive environment in which to perform such studies because it incorporates a lipid bilayer. It is therefore considered a useful and a faithful biomembrane mimetic. Here, we bring together evidence that supports this view. Biophysical characterizations are described demonstrating that the cubic phase is a porous medium into and out of which water-soluble molecules can diffuse for binding to and reaction with reconstituted proteins. The proteins themselves are shown to be functionally reconstituted into and to have full mobility in the bilayered membrane, a prerequisite for LCP crystallogenesis. Spectroscopic methods have been used to characterize the conformation and disposition of proteins in the mesophase. Procedures for performing activity assays on enzymes directly in the cubic phase have been reported. Specific examples described here include a kinase and two transferases, where quantitative kinetics and mechanism-defining measurements were performed directly or via a coupled assay system. Finally, ligand-binding assays are described, where binding to proteins in the mesophase membrane was monitored directly by eye and indirectly by fluorescence quenching, enabling binding constant determinations for targets with affinity values in the micromolar and nanomolar range. These results make a convincing case that the lipid bilayer of the cubic mesophase is an excellent membrane mimetic and a suitable medium in which to perform not only crystallogenesis but also biochemical and biophysical characterizations of membrane proteins.

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