Membrane Protein Structure by Electron Crystallography

通过电子晶体学分析膜蛋白结构

• 批准号: 7658836
• 负责人: David L. Stokes
• 金额: $ 40.49万
• 依托单位: NEW YORK STRUCTURAL BIOLOGY CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-21 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7658836
• 关键词: Affinity Chromatography; Behavior; Cell Culture Techniques; Cells; Chemicals; Chimeric Proteins; Classification; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Databases; Detergents; Dialysis procedure; Dimensions; Drug Delivery Systems; Electron Microscope; Environment; Eukaryota; Eukaryotic Cell; Fostering; Gene Cluster; Genomics; Goals; Image; Integral Membrane Protein; Lead; Lipid Bilayers; Lipids; Liquid substance; Maps; Membrane; Membrane Lipids; Membrane Proteins; Metals; Methods; Microdialysis; Molecular Conformation; Morphology; NMR Spectroscopy; Negative Staining; New York; Orthologous Gene; Pharmaceutical Preparations; Process; Protein Structure Initiative; Proteins; Proteome; Research; Research Personnel; Resolution; Resources; Robot; Robotics; Roentgen Rays; Sampling; Screening procedure; Specialized Center; Staining method; Stains; Structure; Surveys; TEV protease; Techniques; Technology; Temperature; United States National Institutes of Health; X-Ray Crystallography; electron crystallography; expression vector; follow-up; high throughput screening; light microscopy; programs; protein complex; protein expression; protein structure; scale up; structural biology; success; therapeutic target; three dimensional structure; two-dimensional; vector

DESCRIPTION (provided by applicant): The overall goal of this proposal is to promote structure determination of integral membrane proteins by cryoelectron microscopy (cryo-EM) of 2D crystals. Membrane proteins are essential to all cells and favored drug targets, yet their 3D structures have proven difficult to ascertain. Cryo-EM has a demonstrated capability for structure determination at atomic resolution and the important advantage of maintaining integral membrane proteins within their native membrane environment. Nevertheless, the proliferation of high- throughput screening for 3D crystallization trials has given a distinct advantage to X-ray crystallography. We propose to implement analogous technologies for screening 2D crystallization trials of integral membrane proteins within lipid bilayers in order to reestablish cryo-EM as a viable alternative in the high-throughput, post-genomic era. We will form a partnership with the New York Consortium on Membrane Protein Structure (NYCOMPS), which is a Specialized Center of the NIH Protein Structure Initiative operating out of the New York Structural Biology Center. NYCOMPS will provide requested expression vectors from a large database of membrane proteins that they are screening for expression levels and homogeneity. After scaling up expression, we will use a robotic liquid handler to set up 2D crystallization trials by dialysis and prepare EM samples by negative stain in a 96-well format. Imaging currently represents a critical bottleneck for screening 2D crystal trials and we will implement technologies to automatically acquire images from these samples in the electron microscope. Thus, we expect to assess -50 different protein targets per year. The resulting information will be used to establish general principles governing the 2D crystallization process and, importantly, to produce 2D crystals that are suitable for structure determination at atomic resolution. Given a workable method to systematically search for and optimize 2D crystals, cryo-EM will become generally viable for high resolution structure determination and offer an important alternative to X-ray crystallography and NMR spectroscopy. In particular, the modest requirements for quantity and purity of proteins, as well as the natural environment provided by the lipid membrane, make cryo-EM an attractive alternative, especially as we move from bacterial proteomes towards large membrane protein complexes from eukaryotic cells that have proven more difficult to express and to maintain in a detergent solubilized state.

描述（由申请方提供）：本提案的总体目标是通过2D晶体的冷冻电子显微镜（cryo-EM）促进完整膜蛋白的结构测定。膜蛋白对所有细胞和药物靶点都是必不可少的，但它们的3D结构已被证明难以确定。Cryo-EM具有原子分辨率的结构测定能力，并且具有在其天然膜环境中维持完整膜蛋白的重要优势。然而，用于3D结晶试验的高通量筛选的激增已经为X射线晶体学提供了明显的优势。我们建议实施类似的技术来筛选脂质双层内的膜蛋白的2D结晶试验，以重建cryo-EM作为高通量，后基因组时代的一个可行的替代方案。我们将与纽约膜蛋白结构联盟（NYCOMPS）建立伙伴关系，NYCOMPS是美国国立卫生研究院蛋白质结构倡议的专业中心，在纽约结构生物学中心之外运作。NYCOMPS将从一个大型的膜蛋白数据库中提供所需的表达载体，他们正在筛选表达水平和同质性。在按比例放大表达后，我们将使用机器人液体处理器通过透析建立2D结晶试验，并通过阴性染色以96孔格式制备EM样品。成像目前是筛选2D晶体试验的关键瓶颈，我们将实施技术，在电子显微镜中自动获取这些样品的图像。因此，我们预计每年评估约50种不同的蛋白质目标。由此产生的信息将用于建立管理2D结晶过程的一般原则，重要的是，生产适合于原子分辨率结构测定的2D晶体。如果有一种可行的方法来系统地搜索和优化2D晶体，cryo-EM将成为高分辨率结构测定的普遍可行的方法，并为X射线晶体学和NMR光谱学提供一种重要的替代方法。特别是，对蛋白质的数量和纯度的适度要求，以及由脂质膜提供的自然环境，使cryo-EM成为一种有吸引力的替代方案，特别是当我们从细菌蛋白质组转向来自真核细胞的大膜蛋白复合物时，这些蛋白质复合物已被证明更难以表达和维持在去污剂溶解状态。