Chemically engineered bilayers for cryoEM imaging of membrane proteins in continuous membranes

用于连续膜中膜蛋白冷冻电镜成像的化学工程双层

• 批准号: 10091731
• 负责人: Aviv Paz
• 金额: $ 29.1万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091731
• 关键词: 3-Dimensional; Address; Affinity; Biochemical; Biological; Biological Models; Caliber; Carbon; Cell membrane; Cell physiology; Cells; Chemical Engineering; Chemicals; Cryoelectron Microscopy; Crystallization; Data; Data Analyses; Destinations; Detergents; Development; Electrons; Engineering; Environment; Event; Face; Film; Financial compensation; Generations; Heterogeneity; Ice; Image; Inositol; Ion Channel; Label; Laboratories; Ligands; Lipid Bilayers; Lipids; Membrane; Membrane Lipids; Membrane Proteins; Methods; Microscopic; Miniaturization; Modeling; Molecular Conformation; Phase; Physiological; Plant Roots; Play; Post-Translational Protein Processing; Procedures; Process; Proteins; Publishing; Recombinants; Resolution; Role; Signal Transduction; Structure; Surface; System; Technology; Testing; Thick; Tissues; Vesicle; Visualization; Work; base; chemical group; density; electron crystallography; improved; membrane reconstitution; milligram; mimetics; nanodisk; nanometer; nanoscale; new technology; particle; protein complex; receptor; reconstitution; reconstruction; success; technology development; unilamellar vesicle

Cells interact with their environments through membrane proteins. Structural and functional studies of membrane proteins are thus very important. Structure determination of eukaryotic membrane proteins in membrane however remains difficult despite substantial progresses. Part of the challenge comes from the fact that many eukaryotic membrane proteins undergo a complicated intracellular maturation process and carry different post-translational modifications before reaching their final destinations. Current high throughput crystallization and cryoEM single particle reconstruction are largely carried out with proteins in detergents, or in membrane-mimetic systems such as bicelles, nanodiscs, lipid-cubic phases or amphipols, where there are still significant differences in comparison with a native membrane. New technologies are needed to overcome these problems. We propose here to develop two new technologies for cryoEM study of membranes proteins in continuous membrane using type 1 IP3 receptor (IP3R) as a working model. The premise of these two methods is partly based on our recent work of a chemical engineering procedure that is suitable for functionalizing nanometer-thick carbon films and of a bead-supported spherical unilamellar membrane (bSUM) system that allows the generation of stable giant unilamellar vesicles. With milligram amounts of IP3R proteins, we will produce a nanometer-bSUM (nm-bSUM) and a carbon-supported planar unilamellar membrane (cPUM). These two systems will be prepared for the cryoEM visualization of the IP3Rs in continuous membrane where the proteins are fully immersed in a lipid bilayer, and will allow us to resolve the receptor structure from images of membrane-integrated molecules. Images of the receptors in nm-bSUMs will be used for random spherically constrained (RSC) reconstruction. Receptors in cPUMs will be imaged at high tilt angles for 3D reconstruction with corrections for changes in defocus levels across the imaging field. Both methods will rely on chemical engineering and membrane reconstitution at the nanometer scale and will result in efficient unidirectional insertion of membrane proteins at sub-nM concentrations, which will be particularly beneficial for selecting specifically labeled mature functional membrane proteins or enriching low-abundance membrane protein complexes at sub-nM concentrations. Results of the proposed studies will create new windows of opportunities for cryo-EM study of various membrane protein complexes in membrane and for using nanoscale membrane systems in other bioanalytical or biomedical applications. ! 1!

细胞通过膜蛋白与环境相互作用。结构和功能研究 因此，膜蛋白非常重要。真核膜蛋白的结构测定 然而，尽管取得了长足的进展，膜仍然很困难。挑战的一部分来自事实 许多真核膜蛋白都经历了复杂的细胞内成熟过程，并携带 在到达最终目的地之前，不同的翻译后修改。当前高通量 结晶和冷冻单粒子重建在很大程度上是用洗涤剂中的蛋白质进行的，或者在 膜模拟系统，例如双壳，纳米盘，脂质 - 烟酸相或埋有埋葬的系统，仍然存在 与天然膜比较的显着差异。需要新技术来克服 这些问题。我们在这里建议开发两种新技术，用于膜蛋白的冷冻研究 在连续膜上使用类型1 IP3受体（IP3R）作为工作模型。这两个的前提 方法部分基于我们最近的化学工程程序的工作 功能化纳米厚的碳膜和珠子支撑的球形膜（BSUM） 允许生成稳定的巨型单层囊泡的系统。毫克量的IP3R蛋白质， 我们将产生纳米泡沫（NM-bsum）和碳支持的平面Unilamellar膜（CPUM）。 这两个系统将为连续膜中IP3R的冷冻可视化准备准备 蛋白质完全浸入脂质双层中，将使我们能够从图像中解析受体结构 膜积分分子的。 NM-bsums中受体的图像将用于随机球形 受约束（RSC）重建。 CPUM中的受体将以高倾斜角度成像，以进行3D重建 通过校正整个成像场的散焦水平的变化。两种方法都将依靠化学 工程和膜的重构以纳米量表为单位，将导致有效的单向 在亚NM浓度下插入膜蛋白，这对选择特别有益 特定标记的成熟功能膜蛋白或丰富低丰度膜蛋白 亚NM浓度的复合物。拟议研究的结果将创造新的机会窗口 用于膜中各种膜蛋白复合物和使用纳米级膜的冷冻EM研究 其他生物分析或生物医学应用中的系统。 呢1！