MOLECULAR STRUCTURE OF BAND 3 ANION TRANSPORTER

本研究项目的目的是确定分子 红细胞膜负离子转运蛋白Band 3的结构。具体目标 这项建议的主要内容是：(1)继续发展二维(2- D)膜蛋白的结晶方法和(2)获得 电子结晶学研究带3的分子结构信息 方法：研究方法。我们已经成功地重组了带3蛋白 磷脂(DMPC)产生的2-D晶体阵列 高分辨率结构研究的结果。我们将继续我们的 改善带3蛋白结晶质量的努力；这一努力 将对膜的二维结晶有更深入的了解 蛋白质，这对电子的广泛使用至关重要 用于膜蛋白结构测定的结晶学。我们会 首先确定带3的三维(3-D)结构，否定的 用醋酸铀酰染色，以提供有关整体 渠道架构。作为我们研究的总体目标的一部分， 了解Band 3的作用机制，我们也将结晶 遗传性卵圆胞增多症中发现的带3突变，这是一种红血球 一种细胞状态，其特征是坚硬的椭圆形红细胞 抵抗疟疾寄生虫的入侵。这里的目标是获得 关于这个有缺陷的频带3的结构信息，以便 了解抵抗疟疾侵袭的潜在基础 寄生虫。即使在负染样本的相当低的分辨率下， “缺陷”频段3的结构变化预期较大 生化和生物物理研究可以得到证实。我们将致力于 为确定这种蛋白质的详细分子结构所做的努力 包埋在葡萄糖中。对带3的分子结构的了解将 提供有关阴离子结合位点的信息，详细的结构 阴离子通道的设计和结构作用 胞质中可溶部分Band 3具有转运功能。这个 对结构的理解将为我们提供有关 离子跨膜运输的调节。总目标 我们对频带3的结构研究是为了给出一个严格的概念性 合理认识红斑狼疮分子机制的框架 膜转运系统及其转运的特异性 跨膜的溶质和离子，以及理解 卵胞增多症的根本原因及其对侵袭的抵抗力 疟疾寄生虫。

The objective of this research project is to determine the molecular structure of Band 3, the erythrocyte anion transporter. The specific aims of this proposal are (1) to continue the development of 2-dimensional (2- D) crystallization methods for membrane proteins and (2) to obtain molecular structure information of Band 3 by electron crystallographic methods. We have successfully reconstituted Band 3 protein with phospholipids (DMPC) yielding 2-D crystalline arrays that give promising results for high resolution structural studies. We will continue our effort in improving the crystal quality of Band 3 protein; this effort will provide a greater understanding about 2-D crystallization of membrane proteins, which is crucial to the widespread use of electron crystallography for membrane protein structural determination. We will first determine the 3-dimensional (3-D) structure of Band 3, negatively stained with uranyl acetate, to provide information about the overall channel architecture. As a part of the overall goal of our research in understanding the functional mechanism of Band 3, we will also crystallize the Band 3 mutant found in hereditary ovalocytosis, which is a red blood cell condition characterized by a rigid, oval shaped erythrocyte that is resistant to invasion by malaria parasites. The goal here is to obtain the structural information about this "defective" Band 3 in order to understand the underlying basis for the resistance to invasion of malaria parasites. Even at a rather low resolution of negatively stained samples, the expected large structural change of the "defective" Band 3 based on biochemical and biophysical studies can be verified. We will devote major effort toward determining the detailed molecular structure of this protein embedded in glucose. Knowledge of the molecular structures of Band 3 will provide information about anion binding sites, the detailed structural design of the pathways for anions and the structural role of the cytoplasmic soluble portion of Band 3 in the transport function. The structural understanding will provide us with information about the regulation of the transport of ions across the membrane. The overall goal of our structural studies of Band 3 is to give a rigorous conceptual framework for the rational understanding of the molecular mechanism of membrane transport systems and the specificity for the transport of solutes and ions across the membrane, and the basis for understanding the underlying causes of ovalocytosis and its resistance to invasion of malaria parasites.