CHARACTERIZATION OF NOVEL LIPID CUBIC PHASE MATRICES FOR MEMBRANE PROTEIN CRYSTA

这个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。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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型高分辨率晶体可用性的限制。在脂质立方相(LCP)基质中(或在介观中)结晶已被证明可以产生高质量的具有挑战性的膜蛋白晶体，如人G蛋白偶联受体。介观技术的广泛应用要求鉴定具有特定相性质的新脂类，这些新脂类能够稳定各种大小和结构的蛋白质。TSRI创新膜蛋白技术联合中心设计和合成这种脂类。在用于特定应用之前，应对新型脂质基质的相和结构行为进行彻底的表征。JCIMPT开发了LCP夹心板，在夹心板中，将小滴的脂类-溶剂-蛋白质混合物保存在两个具有薄间隔物的X射线透明合成薄膜之间，形成类似于标准96孔膜结晶微板格式的8x12格子样品。BTP工作人员已经为LCP夹层板制造了一个温控夹持器，并对BL4-2进行了初步测试。两种合成薄膜中的一种被发现在X射线散射研究中优于另一种，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在模拟膜蛋白结晶试验中遇到的条件下，以高通量模式研究洗涤剂、添加剂脂类、蛋白质以及各种沉淀剂对脂质基质的影响。

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.