CHARACTERIZATION OF NOVEL LIPID CUBIC PHASE MATRICES FOR MEMBRANE PROTEIN CRYSTA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. High-resolution structural studies of membrane proteins are limited by the availability of crystals diffracting to high resolution. Crystallization in lipidic cubic phase (LCP) matrices (or in meso) has proven to yield high quality crystals of challenging membrane proteins, such as human G protein-coupled receptors. Broader applications of in meso techniques require identification of new lipids with specific phase properties capable of stabilizing proteins with a wide range of sizes and architectures. The Joint Center for Innovative Membrane Protein Technologies at TSRI designs and synthesizes such lipids. The phase and structural behavior of novel lipid matrices should be thoroughly characterized prior to being used in specific applications. JCIMPT has developed the LCP sandwich plates in which small drops of lipid-solvent-protein mixture are held between two x-ray transparent synthetic films with a thin spacer to form 8x12 grids of samples similar to the standard 96-well microplate format for membrane crystallization. The BTP staff has built a temperature controlled holder for the LCP sandwich plate and conducted first trial measurements on BL4-2. One of the two synthetic films was found superior to the other for x-ray scattering studies, providing a low-level of reasonably flat scattering background. We believe our approach will allow us to study effects of detergents, additive lipids, proteins as well as great variety of precipitants on the lipidic matrices in a high-throughput mode under the conditions mimicking those encountered during membrane protein crystallization trials.