STRUCTURE DETERMINATION OF VPU FROM HIV 1

HIV 1 中 VPU 的结构测定

• 批准号: 6430500
• 负责人: MAURICIO S MONTAL
• 金额: $ 16.56万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6430500
• 关键词: electrical measurement; gene mutation; human immunodeficiency virus 1; ion channel blocker; membrane channels; membrane potentials; membrane proteins; protein structure function; recombinant proteins; site directed mutagenesis; structural biology; virus protein

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.

该程序的这个方面的目的是表征频道 重组人免疫缺陷病毒1型VPU的特性 蛋白质以建立寻求的功能数据库 结构功能关系。 实现全部力量 方法需要高分辨率结构。 因此，功能 信息将与通过 NMR光谱，X射线晶体学，中子衍射和 构象能计算。 该程序的优势是 基于多学科方法的重点 分子实体似乎对于从病毒释放很重要 HIV感染的细胞。 VPU的通道特性的生物物理表征涉及 脂质双层中重组蛋白的重组重组。 这些 属性包括单个通道电导，离子选择性， 饱和，开放和封闭的通道寿命。 通道封锁 将用于筛选潜在阻滞剂。 特定残基在确定通道活性中的重要性 VPU及其对阻滞剂的敏感性将通过设计来评估 和合成的特定地点替换。 贡献 谷氨酸2在N末端，丝氨酸23在C-处 VPU跨膜结构域的末端到阳离子 针对VPU跨膜肽确定的选择性将是 通过替代谷氨酰胺或丙氨酸进行检查。 渠道形成设想是由于寡聚而产生的 VPU。 暴露于低聚物通道管腔的残留物将 确定膜的渗透和阻滞性能 嵌入式VPU通道。 VPU的结构和模型 计算将暗示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 脂质双层中的蛋白质。 这种改进周期将提供 对于孔形成结构的蓝调，应该是关键的 VPU特异性通道阻滞剂的设计。 离子通道活性 因此，VPU为药物干预提供了潜在的目标 基于VPU特定的发展辅助工具管理 通道阻滞剂。