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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 膜蛋白的高分辨率结构研究受到衍射到高分辨率的可用性的限制。事实证明，脂质立方相（LCP）矩阵（或中）的结晶已被证明可以产生具有挑战性膜蛋白（例如人G蛋白偶联受体）的高质量晶体。在中等技术中的更广泛的应用需要鉴定具有具有特定相位特性的新脂质，该脂质能够稳定具有各种尺寸和体系结构的蛋白质。 TSRI设计和合成此类脂质的创新膜蛋白技术联合中心。在特定应用中使用之前，应彻底表征新型脂质矩阵的相位和结构行为。 JCIMPT开发了LCP夹心板，其中将少量的脂质 - 溶剂蛋白混合物固定在两个带有薄垫片的X射线透明的合成膜之间，以形成与标准96-Well MicroPlate的样品的8x12网格，用于膜结晶。 BTP的工作人员已为LCP三明治板建立​​了一个控制持有人，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了两个合成膜中的一部分，比另一个合成膜优于另一个合成膜，提供了低水平的合理平坦的散射背景。我们认为，我们的方法将使我们能够在高通量模式下研究脂质基质中的洗涤剂，添加剂脂质，蛋白质以及多种降水剂在模仿膜蛋白质结晶试验中遇到的脂质矩阵的影响。