STRUCTURAL BIOLOGY OF MACROMOLECULAR STRUCTURE

大分子结构的结构生物学

• 批准号: 3792014
• 负责人: A C STEVEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792014
• 关键词: capsid; conformation; covalent bond; crosslink; electron microscopy; epitope mapping; herpes simplex virus 1; image processing; immunoelectron microscopy; immunoglobulin structure; keratin; macromolecule; molecular weight; monoclonal antibody; protein folding; protein structure; protein structure function; scanning electron microscopy; virus DNA; virus protein

This laboratory aims to elucidate the regulatory mechanisms that govern the assembly of supramolecular complexes and the folding of macromolecules, as well as those that underlie the synthesis of organelles, cells, and tissues. In the past year, we have discovered that three-dimensional reconstruction from cryo-electron micrographs of antibody-labelled virus particles affords a method of mapping epitopes with remarkable, and unexpectedly high, precision. Previous forms of immuno-electron microscopy are indirect, detecting an electron-dense label (ferritin or colloidal gold) which may be 15-25nm from the epitope of interest: In contrast, our method directly visualizes the interaction between the Fab fragment and the underlying epitope, and may distinguish between epitopes as close as 1nm apart. It has been applied to three different monoclonal antibodies which bind to the outer surface of the capsid of herpes simplex virus. Two Mabs bind to distinct sites on the hexons, but not to pentons: the third binds to the protruding tips of pentons, but not to hexons. Taking into account our recent biochemical evidence that hexons and pentons are most likely composed of the same viral protein (VP5; 148 kda), these results indicate that there are major conformational differences between the same protein as deployed in pentons (at the capsid's vertices) and hexons (which form the rest of its shell). We have also devised techniques to quantitate the protein compositions of the cell envelopes of cornified epidermal keratinocytes, these structures are covalently cross-linked, rendering them inaccessible to conventional quantitation by gel electrophoresis. Thus we have found that in native epidermal envelopes, the primary constituent is loricrin (70-80%), whereas cultured cell envelopes contain little or no loricrin, but are mainly composed of involucrin, cystatin A and a cystein-rich protein. We infer that only the early stages of native cornification are induced under these in vitro conditions.

本实验室旨在阐明 超分子复合物的组装和折叠 大分子，以及那些基础的合成 细胞器细胞和组织在过去的一年里，我们发现 从冷冻电子显微镜照片的三维重建， 抗体标记的病毒颗粒提供了定位表位的方法 以惊人的，出乎意料的高精度以前的形式 免疫电子显微镜检查是间接，检测电子致密的 标记物（铁蛋白或胶体金），其可距离表位15- 25 nm 感兴趣的：相反，我们的方法直接可视化的互动 Fab片段和潜在表位之间的差异，并且可以区分 表位之间的距离仅为1 nm。已应用于三个 不同的单克隆抗体，其结合到细胞的外表面， 单纯疱疹病毒的衣壳。两种单克隆抗体结合到 六子，但不是五子：第三个结合到突出的尖端， 五子但不是六子考虑到我们最近的生物化学 证据表明，六子和五子最有可能是由相同的 病毒蛋白（VP 5; 148 kda），这些结果表明，有 部署在同一蛋白质之间的主要构象差异 五邻体（在衣壳的顶点）和六邻体（形成其余的 壳）。我们还发明了定量蛋白质的技术 角质层表皮角质形成细胞的细胞包膜的组成， 这些结构是共价交联的， 无法通过凝胶电泳进行常规定量。因此 我们已经发现，在天然表皮包膜中， 成分是兜甲蛋白（70-80%），而培养的细胞包膜 含有很少或不含兜甲蛋白，但主要由外皮蛋白组成， 半胱氨酸蛋白酶抑制剂A和富含半胱氨酸的蛋白。我们推断只有早期 在这些体外条件下诱导天然角化阶段 条件