Structural studies of membrane proteins in lipid bilayer

描述（由申请人提供）： 我们打算进行重组的、膜相关的 电子晶体学分析大肠杆菌核糖转运蛋白组分（aim I） 并设计了用于整体膜的二维结晶的新策略 脂质双层膜中的蛋白质（目的II）。我们的具体目标是 如下所述 I.对E.大肠杆菌核糖转运体。 核糖极疏水组分rbsC的结构研究 封闭溶质转运途径的转运蛋白和封闭溶质转运途径的转运蛋白的嵌合体。 核苷酸结合结构域rbsA和rbsC。单粒子图像 分析将提供四元结构和总体形状的详细信息 蛋白质部分的低分辨率。脂质重建实验 将进行生成二维晶体， 高分辨率电子晶体学分析。 二.膜整合蛋白结晶的新策略 脂质双层a）我们打算探索一种膜蛋白， 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， 对两者的嵌合体进行结晶实验。我们将 受试者酵母表达和纯化的AQP 1-AQP 2嵌合体，其中AQP 1是 人体红细胞水通道与AQP 2是同源肾集合 管道水通道，脂质重组2-D结晶实验。B） 作为平面膜双层上二维结晶的替代方案，我们将 将螺旋结晶的方法扩展到单层脂质小管上， 迄今为止，对于可溶性蛋白质，对于完整膜的情况， proteins. 作为转运体、通道和泵的膜蛋白介导 细胞内外之间的沟通，这是至关重要的， 细胞存活，因此是重要的药理学靶标。重点 我们提出的研究旨在调查三维 脂质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.