A Method To Study Membrane Protein Structure

一种研究膜蛋白结构的方法

基本信息

批准号：
6622161
负责人：
GUIDO A ZAMPIGHI
金额：
$ 12.63万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-05-01 至 2004-10-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The long-term objective of this application is to obtain the 3D structure of functional integral membrane proteins in their native environment and without the need for protein purification. We will focus on channels and transporters, membrane proteins that mediate a myriad of crucial cell and tissue functions. To accomplish this objective, we propose to apply freeze-fracture and shadowing, a method that replicates frozen specimens with a layer of metal ions. The immediate objective of the application is to optimize the freeze-fracture and shadowing method to produce replicas representing faithful copies of the outer shell of membrane proteins. The replicas will be studied in the electron microscope and the structure of the membrane proteins reconstructed using single particle computer image processing method. The freeze-fracture and shadowing method will be optimized by imaging purified aquaporin-0 (AQP0) and the Na+/glucose transporter (SGLT) reconstituted in liposomes. The selection of these proteins is based on our familiarity with their purification and reconstitution procedures as well as our ability to modify their structure using biochemical and molecular biology methods. Crucial to the studies proposed in this application is that two proteins of the AQP0 family, the glycerol conducting channel and aquaporin-1 (AQP1), has been solved at atomic resolution using x-ray and electron diffraction methods. These models will guide the optimization freeze-fracture and shadowing method and guarantee accurate 3D-representations of the structure of the channel. The optimized freeze-fracture and shadowing method will be used to determine the 3D structure of AQP0 and SGLT that have been modified using partial proteolysis of the C-terminus, generation of truncated and fusion proteins as well as cysteine mutagenesis and the chemistry of cysteine residues. After optimization of the freeze-fracture and shadowing method in liposomes, AQP0 and SGLT will be expressed in Xenopus laevis oocytes as a first step in determining the structure of membrane proteins directly from their cRNA, without need of protein purification.

描述（由申请人提供）：该应用的长期目标是获得3D结构功能性整合膜蛋白在其本地环境中，没有需要蛋白质纯化。我们将专注于渠道和转运蛋白，介导无数关键细胞和组织功能的膜蛋白。为了实现这一目标，我们建议采用冻结骨折和阴影，一种用一层金属复制冷冻标本的方法离子。应用程序的直接目的是优化冻结骨折和阴影方法生产代表忠实的复制品膜蛋白外壳的副本。复制品将在电子显微镜和膜蛋白的结构使用单粒子计算机图像处理方法重建。冻结和阴影方法将通过成像纯化来优化 Aquaporin-0（AQP0）和Na+/葡萄糖转运蛋白（SGLT）重构脂质体。这些蛋白质的选择是基于我们对他们的纯化和重组程序以及我们的能力使用生化和分子生物学方法修改其结构。至关重要的在此应用中提出的研究是AQP0的两个蛋白家族，甘油传导通道和aquaporin-1（AQP1）已解决在原子分辨率下使用X射线和电子衍射方法。这些模型将指导优化的冻结裂缝和阴影方法，并保证通道结构的精确3D代表。优化冻结裂缝和阴影方法将用于确定3D结构 AQP0和SGLT已通过部分蛋白水解进行了修饰 C末端，截短和融合蛋白的产生以及半胱氨酸诱变和半胱氨酸残基的化学。优化后脂质体，AQP0和SGLT中的冻结骨折和阴影方法将是以爪蟾laevis卵母细胞作为确定的第一步膜蛋白的结构直接从其CRNA中，无需蛋白质纯化。