Structure and Function of Membrane Transport Proteins

描述(由申请人提供)： 拟议研究的长期目标是开发和评估 鉴定和分离人参皂甙功能区的新方法的应用 膜运输蛋白。预计这些方法将有助于 将特定的功能分配给膜蛋白的特定区域和 将有助于确定膜转运蛋白的结构。主要依据 因为这项工作是“基因足迹”技术。在这项技术中， 非常大的随机、小的框内插入突变的文库是 在转运蛋白的靶基因中产生。以下为以下选择 体内转运功能，即突变等位基因的模式 未存活的选择显示在凝胶上。该基因的区域是 从所选群体中缺失对应于“Footprint”并指示 所选函数所需的区域。相比之下， 凝胶上可见的区域表示允许的区域，即可以 允许插入，而不会丢失所选功能。第二个和 拟议工作的第三部分旨在鉴定和分离稳定的 保留参与亚基能力的运输蛋白区域 互动。这些区域可能对生化或结构有用 学习。截短版本的转运蛋白分子，干扰 野生型转运蛋白的功能(即显性-负性)将是 已确认身份。这项工作将使用麦芽糖运输系统进行 以大肠埃希菌为模型。这个系统是一个很好的特征细菌的三磷酸腺苷结合 卡带，(ABC)运输机。细菌运输系统的研究提供了 有机会同时使用遗传和生化方法研究 膜转运蛋白的结构及其活性机制 运输。细菌与真核生物的显著序列相似性 三磷酸腺苷结合盒(ABC)家族的运输蛋白强调 从原核系统研究中获得的知识是 可推广到高等生物体中的类似系统。许多作业成本法中的缺陷 转运蛋白与严重的人类疾病有关。这些疾病包括囊性 纤维化(CFTR)；高胰岛素血症低血糖(SUR1)，黄斑变性 (ABCR)；肾上腺脑白质营养不良(ALDP)；以及肿瘤的多药耐药 (P-糖蛋白，mdr1)。

DESCRIPTION (provided by applicant): The long term goals of the proposed research are to develop and evaluate the utility of new methods for identifying and isolating functional regions of membrane transport proteins. It is anticipated that these methods will aid in assigning specific functions to particular regions of membrane proteins and will aid in determining the structures of membrane transporters. The main basis for the work is the technique of "genetic footprinting". In this technique, very large libraries of random, small, in-frame insertion mutations are generated in the target gene for a transporter. Following selection for transport function in vivo, the patterns of mutant alleles that either do or do not survive selection are displayed on gels. Regions of the gene that are missing from the selected population correspond to a "footprint" and indicate a region that is required for the selected function. In contrast, regions that are visible on the gel indicate the regions that are permissive, that is, can tolerate the insertion without loss of the selected function. The second and third parts of the proposed work are aimed at identifying and isolating stable regions of transport proteins that retain the ability to participate in subunit interactions. These regions may be useful for biochemical or structural studies. Truncated versions of transporter molecules that interfere with the function of the wild-type transporter (i.e. are dominant-negative) will be identified. The work will be carried out using the maltose transport system of E. coli as a model. This system is a well-characterized bacterial ATP binding cassette, (ABC) transporter. The study of bacterial transport systems offers the opportunity to use both genetic and biochemical approaches to investigate the structure of membrane transport proteins and the mechanism of active transport. Significant sequence similarities among bacterial and eukaryotic transport proteins of the ATP binding cassette (ABC) family underscore the concept that knowledge obtained from the study of prokaryotic systems is generalizable to similar systems in higher organisms. Defects in many ABC transporters are associated with severe human diseases. These include cystic fibrosis (CFTR); hyperinsulinemia hypoglycemia (SUR1), macular degeneration (ABCR); adrenoleukodystrophy (ALDP); and multiple drug resistance in tumors (P-glycoprotein, MDR1).