Structural studies of membrane proteins in lipid bilayer

描述(由申请人提供)： 我们打算继续进行重组、膜相关的结构研究。 电子结晶学研究大肠杆菌核糖转运蛋白的组成(目标I) 并为整体膜的二维结晶设计了新的策略 脂双层膜中的蛋白质(AIM II)。我们的具体目标是 如下所述。 I.大肠杆菌核糖转运蛋白组成的结构研究。 核糖极疏水组分RBSC的结构研究 包围溶质运输途径的转运体和嵌合体 核苷酸结合区rBsA和rbsc将被追寻。单粒子图像 分析将提供第四纪组织和总体形状的详细信息 在低分辨率下的蛋白质部分。脂类重建实验 将被执行以产生适合于 高分辨率电子晶体分析。 II.完整膜蛋白结晶的新策略 脂质双分子层。A)我们打算探索一种膜蛋白是否容易 在脂质双层中以二维阵列结晶可以帮助 结晶或提高另一种膜蛋白的结晶度 对两种嵌合体都进行了结晶实验。我们会 目的酵母表达和纯化AQP1-AQP2嵌合体，其中AQP1是 人，红细胞水通道和AQP2是同源的肾脏采集 管道水通道，进行脂质重组的二维结晶实验。b) 作为平面薄膜双层上二维结晶的替代方案，我们将 将螺旋结晶法推广到单层脂管上， 到目前为止，对可溶性蛋白质的研究主要针对膜的整体情况 蛋白质。 起转运体、通道和泵功能的膜蛋白 细胞内外之间的通信，这对 细胞存活，因此是重要的药理靶点。焦点 我们计划进行的针对3维空间的研究 脂双层中的结构可能会对基于结构的 药物设计。

DESCRIPTION (provided by applicant): We intend to pursue structural studies of recombinant, membrane-associated components of the E.coli ribose transporter by electron crystallography (aim I) and devise novel strategies for 2-D crystallization of integral membrane proteins in the lipid bilayer membrane (aim II). Our specific aims are described below. I. Structural studies of the components of the E. coli ribose transporter. Structural studies of the extremely hydrophobic component rbsC of the ribose transporter that encloses the solute transport pathway and a chimera of the nucleotide binding domain rbsA and rbsC will be pursued. Single particle image analysis will provide details of the quaternary organization and overall shape of the protein moieties at low resolution. Lipid reconstitution experiments will be carried out to generate 2-dimensional crystals that are appropriate for high-resolution electron crystallographic analysis. II. Novel strategies for crystallization of integral membrane proteins in the lipid bilayer. a) We intend to explore whether a membrane protein which readily crystallizes in a 2-dimensional array in the lipid bilayer can help in crystallizing or improving the crystallinity of another membrane protein when a chimera of the two are subjected to crystallization experiments. We will subject yeast-expressed and purified AQP1-AQP2 chimera, where AQP1 is the human, erythrocyte water channel and AQP2 is the homologous kidney collecting duct water channel, to lipid-reconstituted 2-D crystallization experiments. b) As an alternative to 2-D crystallization on planar membrane bilayers, we will extend the method of helical crystallization on unilamellar lipid tubules, hitherto carried out for soluble proteins, to the case of integral membrane proteins. Membrane proteins that function as transporters, channels and pumps mediate communication between the inside and outside of the cell, which is crucial for cell survival, and are therefore important pharmacological targets. The focus of our proposed research directed at investigation of the 3-dimensional structure in the lipid bilayer would potentially have impact on structure-based drug design.