CRYSTALLIZATION AND STRUCTURE DETERMINATION OF VPU

这个项目的目标是结晶并确定X射线 人类免疫缺陷VPU通道蛋白的结构 1型病毒(HIV-1)。提供了对VPU功能的洞察 结构应该对小说的发展做出实质性的贡献 艾滋病疗法。这项工作是长期努力澄清的一部分 膜结合蛋白的结构及对膜结合蛋白的研究 这些结构的功能意义。 VPU是一种完整的膜蛋白，具有通道活性， 在HIV-1的生命中介导两个重要功能：它控制 新的病毒粒子从感染者的表面萌发和释放 细胞，它诱导宿主体内的CD4蛋白被破坏 牢房。体外感染细胞后，缺乏VPU的HIV-1分离株 与野生型病毒相比，释放到介质中的病毒颗粒要少得多， 支持这样的观点，即这种蛋白质的作用对 病毒行动。除了作为潜在的艾滋病药物的重要性之外 以81个氨基酸为目标，VPU是一个很好的模型系统 发展新的膜蛋白结构方法。 这个项目的具体目标是致力于合理的战略 获得洗涤剂增溶VPU的晶体，并将其用于 晶体来阐明蛋白质的三维结构。一个 将开发基于囊泡的功能VPU通道检测方法，并 用于估计通道的亚单位化学计量比。洗涤剂 将对他们进行筛选，以确定他们是否有能力在 解决方案。然后将使用适当的洗涤剂来显影 用于同时滴定溶解度的结晶分析 VPU洗涤剂的蛋白质和洗涤剂成分 很复杂。有希望的条件将通过基于统计的优化 方法，得到的晶体将用于X射线测定。 蛋白质的晶体结构。

The goal of this project is to crystallize and determine the X-ray structure of the Vpu channel protein of the human immunodeficiency virus type-1 (HIV-1). The insights into Vpu function provided by this structure should contribute materially to the development of novel AIDS therapies. This work is part of a long term effort to elucidate the structures of membrane-bound proteins and to probe the functional significance of these structures. Vpu is an integral membrane protein with channel activity that mediates two important function in the life of HIV-1: It controls budding and release of new virions from the surface of the infected cell, and it induces the destruction of the host's CD4 protein within the cell. After infection of cells in vitro, isolates of HIV-1 lacking Vpu release far fewer virus particles into the medium than wild type virus, supporting the notion that the action of this protein is important to virus action. In addition to its importance as a potential AIDS drug target, at 81 amino acids, Vpu is an excellent model system for the development of new membrane protein structural methods. The specific aims of this project are devoted to a rational strategy for obtaining crystals of detergent-solubilized Vpu, and to using these crystals to elucidate the three-dimensional structure of the protein. A vesicle-based assay for functional Vpu channel will be developed and used to estimate the subunit stoichiometry of the channel. Detergents will be screened for their ability to maintain this stoichiometry in solution. Appropriate detergents will then be used to develop crystallization assays designed to simultaneously titrate the solubilities of the protein and detergent components of the Vpu-detergent complex. Promising conditions will be optimized by statistically-based methods, and the crystals obtained will be used to determine the X-ray crystal structure of the protein.