CHARACTERIZATION OF NOVEL LIPID CUBIC PHASE MATRICES FOR MEMBRANE PROTEIN CRYSTA

该子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 膜蛋白的高分辨率结构研究受到衍射到高分辨率的晶体的可用性的限制。在介晶立方相（LCP）基质（或内消旋）中结晶已被证明产生具有挑战性的膜蛋白（例如人G蛋白偶联受体）的高质量晶体。更广泛的应用，在介观技术需要识别新的脂质与特定的相特性，能够稳定蛋白质的范围广泛的大小和结构。TSRI的创新膜蛋白技术联合中心设计和合成这种脂质。新型脂质基质的相和结构行为在用于特定应用之前应彻底表征。JCIMPT开发了LCP夹心板，其中小滴的脂质-溶剂-蛋白质混合物被保持在两个X射线透明的合成膜之间，具有薄的间隔物，以形成类似于用于膜结晶的标准96孔微孔板格式的8x 12样品网格。 BTP工作人员为LCP夹层板构建了一个温控保持器，并对BL 4 -2进行了首次试验测量。发现两种合成膜中的一种在X射线散射研究中比另一种优越上级，提供了低水平的合理平坦的散射背景。我们相信，我们的方法将使我们能够研究洗涤剂，添加剂脂质，蛋白质以及各种各样的沉淀剂在高通量模式下，在模拟膜蛋白质结晶试验过程中遇到的条件下，对蛋白质基质的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.