CHARACTERIZATION OF NOVEL LCP MATRICES FOR MEMBRANE PROTEIN CRYSTALLIZATION BY S

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 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 代表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，能够稳定蛋白质与大范围的大小和结构。在创新膜蛋白技术联合中心（JCIMPT），我们正在设计和合成这样的脂质。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。 我们建议克服与常规制备的脂质样品的X射线研究的障碍，通过制备样品在96孔夹心板和测量它们在原位的BioSAXS光束线。 这种方法将使我们能够在模拟结晶试验期间遇到的条件下，以高通量模式筛选洗涤剂、添加剂脂质、蛋白质以及各种各样的沉淀剂对结晶基质的影响。在此提议的持续时间内，我们预计将充分表征10-12个最有前途的新脂质，这些脂质是从更大的合成候选物库中预选出来的。所得结果对选择合适的脂类和指导介观结晶实验具有重要意义。

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. 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 large range of sizes and architectures. At the Joint Center for Innovative Membrane Protein Technologies (JCIMPT) we are working on design and synthesis of such lipids. The phase and structural behavior of novel lipid matrices should be thoroughly characterized prior to being used in specific applications. We propose to overcome the obstacles associated with conventional preparation of lipid samples for x-ray studies, by preparing samples in 96-well sandwich plates and measuring them in situ at the BioSAXS beamline. This approach will allow us to screen for effects of detergents, additive lipids, proteins as well as great variety of precipitants on the lipidic matrices in the high-throughput mode at conditions mimicking those encountered during crystallization trials. During the duration of this proposal we anticipate to fully characterize 10-12 most promising novel lipids pre-selected out of a larger pool of synthesized candidates. The obtained results will be indispensable for selection of proper lipids and guiding in meso crystallization experiments.