CHARACTERIZATION OF NOVEL LIPID CUBIC PHASE MATRICES FOR MEMBRANE PROTEIN CRYSTA

膜蛋白晶体新型脂质立方相基质的表征

• 批准号: 8362060
• 负责人: HIROTSUGU TSURUTA
• 金额: $ 0.19万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362060
• 关键词: Architecture; Behavior; Crystallization; Detergents; Drops; Film; Funding; G-Protein-Coupled Receptors; Grant; Human; Joints; Lipids; Measurement; Membrane; Membrane Proteins; National Center for Research Resources; Phase; Principal Investigator; Property; Proteins; Radiation; Research; Research Infrastructure; Resolution; Resources; Sampling; Solvents; Source; Techniques; Technology; Temperature; United States National Institutes of Health; cost; design; innovation; novel; structural biology

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 膜蛋白的高分辨率结构研究受到高分辨率晶体衍射的可用性的限制。事实证明，在脂质立方相 (LCP) 基质（或内消旋）中结晶可以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。内消旋技术的更广泛应用需要鉴定具有特定相特性的新脂质，这些脂质能够稳定各种尺寸和结构的蛋白质。 TSRI 创新膜蛋白技术联合中心设计并合成了此类脂质。在用于特定应用之前，应彻底表征新型脂质基质的相和结构行为。 JCIMPT 开发了 LCP 夹心板，其中小滴脂溶剂蛋白质混合物被固定在两个带有薄垫片的 X 射线透明合成膜之间，形成 8x12 样品网格，类似于用于膜结晶的标准 96 孔微孔板格式。 BTP 工作人员为 LCP 夹层板建造了一个温控支架，并在 BL4-2 上进行了首次试验测量。在 X 射线散射研究中，发现两种合成薄膜中的一种优于另一种，提供了低水平的相当平坦的散射背景。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过程中遇到的条件下，以高通量模式研究去垢剂、添加剂脂质、蛋白质以及各种沉淀剂对脂质基质的影响。