STRUCTURE DETERMINATION OF VPU FROM HIV 1

节目的这一方面的目的是描述频道的特征 重组人免疫缺陷病毒1型VPU的特性研究 蛋白质，以建立寻找所需的功能数据库 结构-功能关系。实现这一目标的全部力量 这种方法需要高分辨率的结构。因此，功能 信息将与通过以下方式获得的结构数据相结合 核磁共振谱、X射线结晶学、中子衍射和 构象能计算。该计划的优势在于 基于专注于这一单一的多学科方法 似乎对病毒释放很重要的分子实体 感染艾滋病毒的细胞。 VPU通道特性的生物物理表征包括 重组蛋白在脂质双层中的重组。这些 特性包括单通道电导、离子选择性、 饱和，以及开启和关闭通道的寿命。航道封锁 将被用来筛选潜在的拦截者。 特定残基在确定通道活性中的意义 VPU及其对阻滞剂的敏感性将通过设计 以及合成特定部位的替代物。《纽约时报》的贡献 N-末端为谷氨酸2，C-末端为丝氨酸23。 VPU跨膜结构域的末端为阳离子 VPU跨膜多肽的选择性将测定为 分别用谷氨酰胺或丙氨酸的代用品进行检查。 通道的形成被认为是由低聚的 VPU。暴露在低聚物的通道管腔中的残留物将 测定膜的渗透和阻隔性能- 嵌入式VPU通道。VPU的结构和模型 计算将提出进一步诱变的候选残基 然后进行突变体重组后的功能分析 脂双层中的蛋白质。这一周期的改进将提供一个 对造孔结构的蓝图绘制，这应该是 VPU专用通道阻滞剂的设计。离子通道的活性 因此，VPU为药物干预提供了潜在的靶点。 基于VPU特异性发展的艾滋病管理 通道阻滞剂。

The purpose of this facet of the program is to characterize the channel properties of recombinant human immunodeficiency virus type 1 Vpu protein in order to build up the functional data base required to seek structure-function relationships. Realization of the full power of this approach requires a high resolution structure. Thus, the functional information will be combined with the structural data obtained by NMR spectroscopy, X-ray crystallography, neutron diffraction and conformational energy calculations. The strength of the program is based on the multidisciplinary approach focused on this single molecular entity that appears to be important for virus release from HIV infected cells. Biophysical characterization of the channel properties of Vpu involves reconstitution of the recombinant protein in lipid bilayers. These properties include single channel conductance, ionic selectivity, saturation, and open and closed channel lifetimes. Channel blockade will be used to screen for potential blockers. The significance of specific residues in determining the channel activity of Vpu and its sensitivity to blockers will be evaluated by designing and synthesizing site-specific replacements. The contribution of the glutamic acid 2 at the N-terminal end, and of serine 23 at the C- terminal end of the Vpu transmembrane domain to the cationic selectivity determined for the Vpu transmembrane peptide will be examined by substitution for glutamine or alanine, respectively. Channel formation is envisioned to arise from the oligomerization of Vpu. The residues exposed to the channel lumen of the oligomer will determine the permeation and blockade properties of the membrane- embedded Vpu channel. The structure of Vpu and the model calculations will suggest candidate residues for further mutagenesis followed by functional analysis after reconstitution of the mutant protein in lipid bilayers. This cycle of refinements will provide a bluepring for the pore-forming structure that should be pivotal for the design of Vpu-specific channel blockers. The ion channel activity of Vpu, therefore, provides a potential target for drug intervention in the management of AIDS based on the development of Vpu-specific channel blockers.