STRUCTURE DETERMINATION OF VPU FROM HIV 1

该计划的这一方面的目的是描述渠道的特点 重组人免疫缺陷病毒1型Vpu的特性 蛋白质，以建立所需的功能数据库寻求 结构-功能关系。 实现这一切的全部力量 方法需要高分辨率结构。 因此，函数 信息将与结构数据相结合， 核磁共振光谱学、X射线晶体学、中子衍射和 构象能计算 该计划的优势在于 基于多学科的方法，专注于这一单一的 分子实体，似乎是重要的病毒释放， HIV感染细胞 Vpu通道特性的生物物理表征涉及 重组蛋白在脂质双层中的重构。 这些 特性包括单通道电导，离子选择性， 饱和度以及开放和闭合沟道寿命。 通道阻滞 将用于筛选潜在的阻滞剂。 特定残基在决定通道活性中的意义 的Vpu及其对阻滞剂的敏感性将通过设计 和合成位点特异性替代物。 的贡献 谷氨酸2在N-末端，丝氨酸23在C-末端， Vpu跨膜结构域的末端与阳离子 对Vpu跨膜肽测定的选择性将是 通过分别替换谷氨酰胺或丙氨酸进行检查。 设想通道形成由以下的寡聚化引起： Vpu。 暴露于低聚物通道内腔的残留物将 确定膜的渗透和阻断性质- 嵌入式Vpu通道。 Vpu的结构和模型 计算将建议用于进一步诱变的候选残基 然后在突变体重建后进行功能分析 脂质双层中的蛋白质。 这一周期的改进将提供一个 造孔结构的蓝图，这应该是关键的 Vpu专用通道阻断器的设计。 离子通道活性 因此，Vpu为药物干预提供了一个潜在的靶点， 艾滋病管理的基础上开发专用的vpu 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.