STRUCTURE DETERMINATION OF VPU FROM HIV 1

HIV 1 中 VPU 的结构测定

基本信息

批准号：
6107811
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
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 1999-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6107811
关键词：
electrical measurement gene mutation human immunodeficiency virus 1 membrane channels membrane potentials membrane proteins protein structure function recombinant proteins site directed mutagenesis structural biology virus protein

项目摘要

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.

该程序这方面的目的是表征通道重组人免疫缺陷病毒1型Vpu的特性蛋白质以建立寻找所需的功能数据库结构-功能关系。实现此功能的全部威力方法需要高分辨率结构。因此，函数式信息将与获得的结构数据相结合 NMR 光谱、X 射线晶体学、中子衍射和构象能计算。该计划的强度是基于专注于这一单一学科的多学科方法似乎对病毒释放很重要的分子实体 HIV感染的细胞。 Vpu 通道特性的生物物理表征涉及脂质双层中重组蛋白的重建。这些特性包括单通道电导、离子选择性、饱和度以及开放和封闭通道的寿命。通道封锁将用于筛选潜在的阻断剂。特定残基在确定通道活性中的重要性 Vpu 及其对阻断剂的敏感性将通过设计来评估并合成特定位点的替代品。的贡献 N 端有谷氨酸 2，C 端有丝氨酸 23 Vpu 跨膜结构域末端至阳离子 Vpu 跨膜肽的选择性确定为分别通过谷氨酰胺或丙氨酸的替代进行检查。预计通道的形成是由以下物质的寡聚化引起的电压源。暴露于低聚物通道内腔的残留物将确定膜的渗透和阻隔性能嵌入式 Vpu 通道。 Vpu的结构及模型计算将建议用于进一步诱变的候选残基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成孔结构的蓝图对于 Vpu 特定通道阻断器的设计。离子通道活性因此，Vpu 为药物干预提供了潜在的靶点基于 Vpu 特定开发的艾滋病管理通道阻断剂。