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 鉴定和分离功能区的新方法的效用 膜转运蛋白预计这些方法将有助于 将特定功能分配给膜蛋白的特定区域， 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， 随机的、小的、框内插入突变的非常大的文库， 在靶基因中产生的转运蛋白。选择后， 体内的转运功能，突变等位基因的模式， 不能存活的选择显示在凝胶上。基因的某些区域 所选人口中的缺失对应于“足迹”， 所选功能所需的区域。相反， 在凝胶上可见的区域表示允许的区域，即， 容许插入而不损失所选择的功能。第二和 建议工作的第三部分旨在识别和分离稳定的 转运蛋白的区域，其保留参与亚单位的能力， 交互.这些区域可用于生物化学或结构分析。 问题研究截短的转运体分子会干扰 野生型转运蛋白的功能（即显性负性）将是 鉴定这项工作将使用麦芽糖转运系统进行， E.大肠杆菌作为模型。该系统是一个充分表征的细菌ATP结合 磁带，（ABC）运输机。细菌转运系统的研究提供了 利用遗传学和生物化学方法来研究 膜转运蛋白的结构及其活性机制 运输细菌和真核生物之间的显著序列相似性 ATP结合盒（ABC）家族的转运蛋白强调了 从原核系统的研究中获得的知识是 可推广到高等生物的类似系统。许多ABC中的缺陷 转运蛋白与严重的人类疾病有关。其中包括囊性 纤维化（CFTR）;高胰岛素血症低血糖（SUR 1），黄斑变性 肾上腺脑白质营养不良（ALDP）和肿瘤的多药耐药 （P-糖蛋白，MDR 1）。

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).