STRUCTURAL STUDIES OF FASCIN ACTIN BUNDLE

肌成束蛋白肌动蛋白束的结构研究

• 批准号: 6120881
• 负责人: Joseph Bryan
• 金额: $ 1.53万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120881
• 关键词: AIDS; biomedical resource; immunology; infection; inflammation; lymphatic system; neoplasm /cancer; proteins; virus

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinctive cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.

我们已经开发了一个用于组氨酸标签的系统 （His标记的）逆转录病毒核（GAG）蛋白，在体外组装 脂质单层由鸡蛋磷脂酰胆碱（PC）和 新颖的脂质DHGN。 DHGN被证明是通过原子螯合镍的 专门的吸收光谱法和含DHGN的单层 绑定了他标记的蛋白质的黄金结合物。 使用PC + DHGN 单层，我们检查了N端的膜结合阵列 用他的标签莫洛尼鼠白血病病毒（M-Mulv）capsid（CA）蛋白， His-moca和体内研究表明，体外衍生的His-Moca 阵列反映了发生在 组装病毒颗粒。 His-moca蛋白形成了广泛的 二维（2D）蛋白晶体，反射到9.5 a 解决。 图像分析的His-Moca阵列的2D投影 揭示了一个独特的笼子状网络。 不对称的 网络的个别构建块导致了两个 六聚体环，周围无蛋白质的笼孔。 这些 结果预测，hexamer构成逆转录病毒核心 子结构，该笼孔的大小定义了一个排除限制 逆转录病毒包膜蛋白或其他质膜的进入 蛋白质，进入病毒颗粒。 我们相信2D 结晶方法将允许对逆转录病毒进行详细分析 插科打蛋白和其他用his标记的蛋白质。