ELECTRON MICROSCOPIC ANALYSIS OF GP120/GP160

GP120/GP160的电子显微镜分析

• 批准号: 2751240
• 负责人: KENNETH H ROUX
• 金额: $ 19.05万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2751240
• 关键词: HIV envelope protein gp120; HIV envelope protein gp160; cold temperature; cryoelectron microscopy; electron microscopy; epitope mapping; human immunodeficiency virus 1; immune complex; ligands; molecular site; monoclonal antibody; protein structure; protein structure function; recombinant proteins

DESCRIPTION: (Adapted from applicant's abstract): A detailed understanding of the structural and functional aspects of the molecules involved in the interaction, binding, and fusion of the HIV-1 virus with its target cells is fundamental to an understanding of the disease process in AIDS and to the rational design of vaccines and other therapeutic and diagnostic agents. The one molecule that is arguably the most important in the entire infectious process, namely the envelope glycoprotein, gpl20, remains the least well characterized. This proposal describes investigations aimed at determining the 3-D morphology of the HIV-1 envelope glycoproteins, gpl20, and its precursor, gpl60. This will be accomplished by determining its overall shape and generating a 3-D epitope and reactive site map of the surface of the molecule. Two forms of high resolution electron microscopy will be used to analyze the envelope glycoproteins alone and in combination with various ligands and monoclonal antibodies to give a characterization of domain organization and a 3-D surface map of epitopes and reactive sites. Negative stain electron microscopy will yield images of molecules and immune complexes as they adhere to a carbon substrate. This is a well established technique for analyzing the structure of macromolecules though it is rarely used for detailed analysis of molecules as small as gpl20. The second approach will be the use of cryo-electron microscopy where unstained molecules are viewed suspended in ice. Analysis of such small molecules may be at the cutting edge of this technology. Extensive use will be made of previously defined and well characterized reagents. The information generated may: 1) reveal the overall domain configuration, 2) give an indication of the intramolecular (segmental) flexibility, and 3) reveal the topological locations of immunological, functional, and sequence-defined reactive sites and epitopes. With this information, it may be possible to propose more detailed mechanistic explanations for envelope glycoprotein function and for the ability of some antibodies but not others to neutralize HIV-1.

描述：（改编自申请人的摘要）：详细 了解分子的结构和功能方面 参与HIV-1病毒与 它的靶细胞是对疾病的理解至关重要的 艾滋病的过程以及疫苗和其他疫苗的合理设计 治疗和诊断剂。一个可以说是 在整个感染过程中最重要，即信封 糖蛋白GPL20仍然是最不明显的特征。 这 提案描述了旨在确定3-D的调查 HIV-1信封糖蛋白GPL20及其的形态 前体，GPL60。 这将通过确定其整体来实现 塑造并产生3-D表位和反应性位点图 分子。 两种形式的高分辨率电子显微镜将 用于单独分析包膜糖蛋白和组合 带有各种配体和单克隆抗体，可提供 域组织的表征和3D表面图 表位和反应性位点。负污渍电子显微镜将 在粘附到A时产生分子和免疫复合物的图像 碳底物。 这是一项良好的分析技术 大分子的结构虽然很少用于详细 分子分析与GPL20一样小。 第二种方法将是 观察未染色分子的冷冻电子显微镜的使用 悬浮在冰上。 对这种小分子的分析可能是在 这项技术的最前沿。 大量使用将由 先前定义且特征良好的试剂。 生成的信息可能：1）揭示整个域 配置，2）指示分子内（分段） 灵活性和3）揭示了免疫学的拓扑位置， 功能和序列定义的反应性位点和表位。 与此 信息，可能会提出更详细的机理 信封糖蛋白功能的解释和能力 一些抗体，但没有其他抗体来中和HIV-1。