STRUCTURAL STUDIES OF FASCIN ACTIN BUNDLE

我们开发了一套组氨酸标记的分析系统 (组氨酸标记的)逆转录病毒核心蛋白(GAG)，体外组装 由卵磷脂酰胆碱(PC)和卵磷脂 新型脂类DHGN。DHGN对镍有原子螯合作用 吸收光谱，特别是含有DHGN的单分子膜 组氨酸标记蛋白质的结合金偶联物。使用PC+DHGN 单层，我们研究了N-末端的膜结合阵列 His标记的Moloney鼠白血病病毒(M-MuLV)衣壳(CA)蛋白， His-MoCA，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蛋白形成广泛的 二维(2D)蛋白质晶体，反射率达9.5埃 决议。His-MoCA阵列的图像分析二维投影 揭示了一个独特的笼状网络。经济增长的不对称性 网络的各个组成部分导致了两个 六角环的类型，周围不含蛋白质的笼孔。这些 结果预测Gag六聚体构成逆转录病毒核心。 子结构，且保持器孔大小定义了 逆转录病毒包膜蛋白或其他质膜的进入 蛋白质，变成病毒颗粒。我们相信2D 结晶法将使逆转录病毒的详细分析成为可能 GAG蛋白和其他His标记的蛋白。

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.