The Monoolein-Cholesterol Cubic Phase: Characterization by NMR Spectroscopy

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The lipid cubic phase (LCP) formed by particular mixtures of monoolein and water has received significant attention as a system for the crystallization of membrane proteins for structural characterization by x-ray crystallography (the so-called in meso method [Caffrey & Cherezov: Nature Protocols (2009) 4(5):706]). In several cases, the addition of specific lipids to the mesophase was necessary to promote the stabilization and crystallization of a protein. For example, the addition of cholesterol was instrumental for obtaining high-resolution structures of the beta2 adrenergic receptor [Cherezov et al: Science (2007) 318:1258], and of four other G protein-coupled receptors. Apart from the success of LCPs for supporting crystal growth, the bulk isotropic symmetry, rapid diffusion and localized bilayer-like environment make this system potentially useful for NMR studies. We demonstrate that several NMR techniques traditionally associated with solution phase samples (e.g., heteronuclear single quantum coherence, HSQC, and saturation transfer difference, STD) can be adapted for use in LCPs comprising monoolein with cholesterol and/or a transmembrane protein. Our data indicate that a range of advanced NMR techniques could be applied in LCPs, enabling studies of dynamics that will complement the x-ray structural data already obtainable using this phase and significantly expand the variety of tools available for the study of integral membrane proteins.