title: Characterization of lipid matrices for membrane protein crystallization by high-throughput small angle X-ray scattering.
abstract: The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) - the most direct technique to identify lipid mesophases and measure their structural parameters - to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP-SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments.
authors: Department of Molecular Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd., La Jolla, CA 92037, USA.
authors: Jeremiah S
authors: JS
authors: Joseph
authors: Wei
authors: W
authors: Liu
authors: Joshua
authors: J
authors: Kunken
authors: Thomas M
authors: TM
authors: Weiss
authors: Hiro
authors: H
authors: Tsuruta
authors: Vadim
authors: V
authors: Cherezov
mesh_terms: D002784:Cholesterol / Q000737:chemistry; D019343:Citric Acid / Q000737:chemistry; D003460:Crystallization; D036681:Fluorescence Recovery After Photobleaching; D005989:Glycerides / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D050178:Monoglycerides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D015203:Reproducibility of Results; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D005989:Glycerides; D008565:Membrane Proteins; D050178:Monoglycerides; D019343:Citric Acid; D011092:Polyethylene Glycols; D002784:Cholesterol; C005953:monoolein